diff --git "a/deduped/dedup_0118.jsonl" "b/deduped/dedup_0118.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0118.jsonl" @@ -0,0 +1,44 @@ +{"text": "Each channel has unique biophysical characteristics which determine how it contributes to the generation of action potentials (AP). To better understand how AP amplitude is maintained in nociceptive DRG neurons and their centrally projecting axons, which are subjected to depolarization within the dorsal horn, we investigated the dependence of AP amplitude on membrane potential, and how that dependence is altered by the presence or absence of sodium channel Na1/2 values of -73 and -37 mV. These values are similar to the V1/2 values for steady-state fast inactivation of tetrodotoxin-sensitive (TTX-s) sodium channels, and the tetrodotoxin-resistant (TTX-r) Nav1.8 sodium channel, respectively. Addition of TTX eliminates the more hyperpolarized V1/2 component and leads to increasing AP amplitude for holding potentials of -90 to -60 mV. This increase is substantially reduced by the addition of potassium channel blockers. In neurons from Nav1.8(-/-) mice, the voltage-dependent decrease in AP amplitude is characterized by a single Boltzmann equation with a V1/2 value of -55 mV, suggesting a shift in the steady-state fast inactivation properties of TTX-s sodium channels. Transfection of Nav1.8(-/-) DRG neurons with DNA encoding Nav1.8 results in a membrane potential-dependent decrease in AP amplitude that recapitulates WT properties.In small neurons cultured from wild type (WT) adult mouse DRG, AP amplitude decreases as the membrane potential is depolarized from -90 mV to -30 mV. The decrease in amplitude is best fit by two Boltzmann equations, having Vv1.8 allows AP amplitude to be maintained in DRG neurons and their centrally projecting axons even when depolarized within the dorsal horn.We conclude that the presence of Na Encoded nociceptive information is transmitted via small diameter axons that originate from a population of small cell bodies 20\u201330 \u03bcm) contained within dorsal root ganglia (DRG), which send their central projections to the spinal cord dorsal horn. It is well-established that extracellular potassium levels , and its inactivation is so slow near activation threshold that the current is often referred to as persistent [v1.8 channels in small DRG neurons have activation and steady-state fast inactivation functions that are depolarized compared to those of the TTX-s channels [v1.7 channels have a relatively slow recovery from fast inactivation that presumably reduces its contribution to high frequency firing [v1.7 channels have a relatively slow rate of inactivation from the closed state, which allows them to produce inward current in response to slow depolarizations.Small DRG neurons, which include nociceptors, express five of the nine functional voltage-gated sodium channels that have been sequenced thus far, including three that are selectively expressed within DRG neurons. Three tetrodotoxin (TTX)-sensitive (TTX-s) channels, Na neurons , as are and 1.9 . By comprsistent . With rechannels . In DRG y firing . On the v1.8 and Nav1.9, it was known that DRG neurons were different from other neurons because they could generate sodium-dependent action potentials (AP) in the presence of TTX [v1.8 (-/-) animals by Renganathan et al [v1.8 channels is more depolarized, more Nav1.8 channels are available at RMP to contribute to the AP. Similar conclusions based on voltage and current clamp recordings have been reached by other groups [v1.8 is known to be expressed within the spinal cord dorsal horn, where it is present within the central terminals of primary sensory neurons [v1.8 to AP electrogenesis in DRG neurons from a different perspective, that of the reduction of AP amplitude in current clamp mode. We hypothesized that i) AP amplitude should decrease as a function of membrane potential, because membrane potential determines the degree of inactivation for the various sodium channel subtypes, and that ii)the presence of Nav1.8 should shift the voltage-dependence of AP amplitude in a depolarized direction, permitting relatively large APs to be generated even in depolarized neurons.Before the identification of the TTX-r channels Nae of TTX -20. Inver groups ,22. Nav1 neurons ,24. In th), the cell was stimulated with a positive-going square pulse of current (100 ms) starting just below threshold and proceeding in 10 pA intervals to a level 40\u201350 pA beyond threshold. A plot of AP amplitude (see Methods section for details on measurement procedure) and Vh , while the more depolarized V1/2 is consistent with a contribution from TTX-r channels, which have fast inactivation V1/2's of -30 to -45 mV [We first tested the dependence of AP amplitude on the membrane potential of small (< 25 um) DRG neurons from adult wild type mice. This was achieved in current clamp mode, by using injected current to hold the cell at a series of membrane potentials starting at -90 mV, with successive depolarizing steps of approximately 5 mV. At each holding potential (V current 00 ms sta current 00 ms stao -45 mV .h in DRG from WT mice with the inclusion of TTX (300 nM). As seen in Figure h values from approximately -50 mV to -25 mV, there was a gradual decrease in AP amplitude that was well fit with a single Boltzmann equation. The mean V1/2 characterizing this voltage-dependence was -36 \u00b1 2 mV, which was similar to the depolarized component in absence of TTX and consistent with the elimination of TTX-s currents responsible for the decrease in AP amplitude at more hyperpolarized membrane potentials. An unexpected finding was that AP amplitude actually increased in amplitude as Vh was increased from -90 to about -60 mV. The average AP amplitude at a Vh of -90 mV was 46.2 \u00b1 4.7 mV, representing a decrease of 45 \u00b1 4% compared to the maximum AP amplitude. In the absence TTX-s sodium currents and any other influence on AP amplitude, it would be expected that AP amplitude would remain constant at these hyperpolarized potentials because TTX-r sodium channels would be in closed and activatable states.To investigate the hypothesized contribution of TTX-s currents to AP amplitude, we performed the same analysis of AP amplitude and Vh analysis in the presence of TTX (300 nM), TEA (25 mM) and 4-AP (3 mM) to block a significant portion of outward potassium current. Just before switching to current clamp recording mode, the extent of potassium channel block was determined by holding cells at -100 mV and stepping for 100 ms to membrane voltages between -40 and +25. An example of currents produced by this protocol for two cells, one in the absence (A) and one in the presence (B) of TEA and 4-AP are shown in Figure h values between -60 and -20 from 45 to 9%. The remaining decrease may be attributable to the unblocked outward current or additional factors that can influence AP amplitude.We hypothesized that, in addition to voltage-gated sodium channels, a strong influence on AP amplitude would be from outward potassium currents. To test this hypothesis, we performed the same AP amplitude/Ve of TTX 00 nM, TE1/2, we applied the AP amplitude/Vh analysis to small DRG neurons obtained from Nav1.8(-/-) mice transfected with either GFP alone, or GFP plus a construct containing Nav1.8. As shown in Figure h increased from -90 to -30 mV, and this decrease was best fit with a single Boltzmann equation. The mean V1/2 for 10 cells was -55.0 \u00b1 1.5 mV, a value that is depolarized by about 20 mV compared to the TTX-s component in WT neurons. When Nav1.8 was transfected back into small DRG neurons from Nav1.8(-/-) animals, analysis of the decrease in AP amplitude as a function of Vh two populations revealed two populations: for one group, decrease in AP amplitude was best fit by a single Boltzmann equation . This value was not statistically different from the V1/2 value for the cells from Nav1.8(-/-) animals transfected with GFP alone. Presumably, this group of cells did not express the Nav1.8 construct. In a second group of cells (N = 8), the decrease in AP amplitude was best fit by two Boltzmann equations with mean V1/2's of -71 \u00b1 2.1 mV and -35.6 \u00b1 3.0 mV. These values are not significantly different from the corresponding values for the small DRG neurons from WT animals. In addition, the portion of the decrease in AP amplitude accounted for by each V1/2 was similar to the WT data . These data indicate that the shift in V1/2 observed in the Nav1.8(-/-) neurons, presumably dependent on the fast inactivation of TTX-s sodium currents, is due to the absence of Nav1.8 channels.To further investigate the contribution of TTX-r currents to the decrease in AP amplitude associated with the more depolarized Vn Figure having av1.8 channels are present within the central terminals of primary sensory neurons within the dorsal horn [1/2 values for the two Boltzmann fits (-73 mV and 37 mV)are similar to the V1/2 values for fast steady-state inactivation associated with TTX-s channels (-65 to -75 mV) and Nav1.8 TTX-r channels (-30 to -40 mV), respectively, that are expressed in small DRG neurons [1/2 to AP amplitude (81%), is consistent with previous estimates of the relative contribution of Nav1.8 current to AP amplitude. Using the Goldman-Hodgkin-Katz equation to estimate sodium ion permeability during the AP in DRG from Nav1.8(+/+) and Nav1.8(-/-) animals, Renganathan concluded that Nav1.8 channels contribute 80\u201390% of the current that flows at the peak of the AP [This work was motivated by the question of how AP conduction can be maintained in the central projections of nociceptive DRG neurons which course through the dorsal horn where they are subjected to depolarization ,2 and bysal horn ,24 and t neurons -13. The f the AP . Blair af the AP . They esh values of approximately -60 to -20 mV, AP amplitude decreased gradually, and this voltage-dependence was well fit with a single Boltzmann equation having a V1/2 of -36 mV. The absence of the more hyperpolarized component demonstrates a contribution of TTX-s sodium currents. In place of a decrease in AP amplitude, we observed that AP amplitude increased for Vh values of -90 to -60 mV. It is unlikely that an increasing availability of TTX-r channels could account for increasing AP amplitude in this voltage range because it does not fall with the range for steady-state fast inactivation of these channels. Alternatively, we hypothesized that outward current generated by potassium channel opening might be responsible for the change in AP amplitude in the voltage range. To test this hypothesis, we recorded AP amplitude under conditions known to block the majority of voltage-gated potassium channels (TEA and 4-AP). Estimates of outward current amplitude obtained before switching to current clamp mode (see Methods) indicated that 80\u201390% of the outward current produced by depolarizing voltage steps was blocked. In current clamp mode, analysis of AP amplitude and Vh revealed that the 45% increase in AP amplitude from -90 to -60 mV in the presence of TTX alone decreased to only 9% in the presence of TEA and 4-AP, representing a reduction of 80%. Although we cannot state with certainty the identity of the potassium channels reducing AP amplitude from -90 to -60 mV, possible candidates are the inwardly rectifying current IIR [h [To confirm the contribution of TTX-s channels, we determined the voltage-dependence of AP amplitude in the presence of 300 nM TTX. For Vrent IIR and Ih [t IIR [h , a slowlv1.8 sodium current, we used DRG neurons from Nav1.8(-/-) animals to further investigate the identity of the currents contributing to the depolarized V1/2 characterizing AP amplitude reduction. Previous studies have demonstrated that small DRG neurons from these animals completely lack the slow TTX-r current produced by Nav1.8 channels [1/2 of -55 mV. This value represents a depolarizing shift of 20 mV compared to the V1/2 for the TTX-s component in WT neurons. One possible explanation is that the inactivation properties of the remaining TTX-r sodium channels, Nav1.9, are combining with those of TTX-s sodium channels to produce a single depolarized V1/2. The midpoint of steady-state fast inactivation for Nav1.9 in DRG cells ranges from -44 to -54 mV [v1.9 channel openings indicates that these channels make only a minor contribution to AP amplitude [v1.7 in Nav1.8(-/-) DRG neurons; however, this would not account for the shift in V1/2 because the V1/2 for steady-state fast inactivation of Nav1.7, -71 mV to -78 mV [1/2 associated with the TTX-s component of AP decrease in WT neurons. An alternative explanation for the shift in the V1/2 of the TTX-s component is that the inactivation properties of TTX-s sodium channels are modified in DRG neurons of Nav1.8(-/-) animals. Such a modification has been observed in a previous study [v1.8(+/+) and Nav1.8(-/-) mice, the authors observed a 20 mV depolarizing shift in the voltage dependence of fast inactivation. The mechanism for this shift in TTX-s voltage dependence of inactivation was not determined. Possible mechanisms include G-protein activation, which has been shown to depolarize inactivation V1/2 of Nav1.8 currents by 3\u20134 mV in DRG neurons [1/2 of both TTX-s and TTX-r currents in DRG neurons [1/2 of fast inactivation in cardiac Nav1.5 channels in HEK293 cells, [1/2 of fast inactivation in sodium currents of differentiated PC-12 cells [v1.8(+/+) and Nav1.8(-/-) animals might also account for the observed shift. Irrespective of the modulatory process involved, our additional results with transfection of Nav1.8 into cells from Nav1.8(-/-) animals demonstrates that the process is not permanent and is dependent on the absence of Nav1.8 channels. After transfections, AP amplitude dependence on Vh was again fit best by two Boltzmann equations in half of the cells, and the V1/2 values (-71 and -36 mV) were similar to values for the wild type DRG neurons. These results indicate that the reintroduced Nav1.8 channels led to a shift in the inactivation voltage-dependence of the TTX-s component back to the WT value.In the absence of well-established blockers of the Nachannels . AP amplo -54 mV ,28. Howemplitude ,28. Akopmplitude observedo -78 mV ,29, is s neurons , the pre neurons and tyro3 cells, and hype12 cells . Because12 cells , differe1/2 characterizing the more hyperpolarized component is similar to the V1/2 associated with TTX-s sodium channel steady-state fast inactivation and is eliminated in the presence of TTX. The V1/2 characterizing the more depolarized component is similar to the V1/2 of steady-state fast inactivation for Nav1.8. In small DRG neurons from Nav1.8(-/-) animals, the voltage-dependence of the AP amplitude decrease is characterized by a single Boltzmann component with a V1/2 of -55 mV, a value that is depolarized by about 20 mV compared to the TTX-s component in neurons from WT animals. We believe that this shift is due to a shift in the inactivation properties of TTX-s sodium channels in small DRG neurons from Nav1.8(-/-) animals, but that this shift is not permanent. When these same cells are transfected with Nav1.8, the voltage-dependence of AP amplitude recapitulates the voltage-dependence observed in neurons from WT animals. Taken together, these results suggest that the expression of Nav1.8 within nociceptive DRG neurons permits the central projections of these cells to conduct APs even when depolarized within the dorsal horn.In summary, we have shown that the AP amplitude of small DRG neurons decreases as a function of membrane depolarization, and that this relationship is best fit with two Boltzmann components. The Vv1.8(-/-) mice was raised from a breeding pair of Nav1.8(+/-) mice [All animal care and surgical procedures followed a protocol approved by the Animal Care and Use Committee of Yale University. A colony of Na/-) mice generousv1.8(-/-) mice (in C57/BL6 genetic background). Neurons were isolated as previously described [v1.8 plasmid used for transfections of Nav1.8 into DRG from Nav1.8(-/-) mice was a gift from John Wood . The green fluorescent protein plasmid pEGFP was purchased from Clontech . Sodium channel and GFP constructs (channel:GFP ratio of 5:1) were electroporated into DRG neurons using Nucleofector Solution as previously described [2+- and Mg2+-free culture medium for 5 min. to increase cell viability. The cell suspension was then diluted in culture medium, plated on 12 mm circular coverslips coated with laminin and poly-ornithine, and incubated at 37\u00b0C and 5% CO2.DRGs were harvested from deeply anesthetized adult (4\u20136 weeks) C57BL6 mice or adult Naescribed . Brieflyescribed ,37. Tran2, 2 CaCl2, 10 HEPES . The whole-cell recording configuration was obtained in voltage clamp mode with an Axopatch 200B amplifier . Data was stored on a personal computer via a Digidata 1322a A/D converter (Molecular Devices) at an acquisition rate of 50 kHz with a lowpass Bessel filter setting of 5 kHz. In voltage clamp recording mode, voltage errors were minimized with 85% series resistance compensation, and linear leak currents and capacitance artifacts were subtracted out using the P/N method provided by Clampex (Molecular Devices) acquisition software. Clampfit (Molecular Devices) and Origin were used for data analysis. To obtain an estimate of inward and outward current amplitudes, two activation protocols were applied before proceeding to the current clamp recording mode. Both protocols involved steps to a range of membrane potentials from -45 to 40 mV, in 5 mV increments. For the first protocol, cells were held at -50 mV to eliminate the majority of TTX-s sodium channels. For the second protocol, cells were held at -100 mV to activate both TTX-s and TTX-r channels. A rough estimate of TTX-s currents was obtained by subtraction of the currents obtained from the two protocols. After switching to current clamp recording mode, passive and active cell properties were determined . Cells with RMPs more negative than -35 mV and overshooting action potentials (>85 mV RMP to peak) were used for further data collection. Stimulation threshold at RMP was determined by a applying a series of 100 ms depolarizing current steps starting below threshold and increasing in 5 pA increments. Threshold was defined as the smallest current amplitude that produced an AP. To investigate the voltage dependence of AP amplitude, current injection was used to hold cells at membrane potentials (Vh) starting at -90 mV and depolarizing by 10 mV until an overshooting AP could not be elicited. At each Vh, threshold was determined and AP amplitude for the response at threshold + 10 pA was used for the analysis. AP amplitude was defined as the difference between the peak and the onset of the AP. Onset of the AP was further defined as the point at which dV/dt for the AP was greater than 5 mV/ms . AP amplitude was plotted as a function of Vh and fit with either 1 or 2 Boltzmann equations of the form VAP/VAPmax = 1/(1 + exp((Vh - V1/2)/k)), for which VAP is the AP amplitude, VAPmax is the maximum AP amplitude, Vh is the holding potential, V1/2 is the Vh at which the AP amplitude is 50% of maximum, and k is the slope of the fit. Goodness of fit for 1 and 2 Boltzmann equations was determined by eye. Data is expressed as mean \u00b1 sem, and for all data comparisons, Student's t-test was used to assess statistical differences between means from different groups.Small (20\u201325 \u03bcm) DRG neurons were used for current clamp recording after 24\u201340 hours in culture (with or without transfection). Electrodes had a resistance of 1\u20132 M\u03a9 when filled with the pipette solution, which contained (in mM): 140 KCl, 0.5 EGTA, 5 HEPES and 3 Mg-ATP . The extracellular solution contained (in mM): 140 NaCl, 3 KCl, 2 MgClThe author(s) declare that they have no competing interests.SGW and TPH planned the experiments. TPH conducted all the experiments. Both authors contributed to the writing of the paper."} +{"text": "All theories about the origin and evolution of membrane bound cells necessarily have to cope with the nature of the last common ancestor of cellular life. One of the most important aspect of this ancestor, whether it had a closed biological membrane or not, has recently been intensely debated. Having a consensus about it would be an important step towards an eventual (though probably still remote) synthesis of the best elements of the current multitude of cell evolution models. Here I analyse the structural and functional conservation of the few universally distributed proteins that were undoubtedly present in the last common ancestor and that carry out membrane-associated functions. These include the SecY subunit of the protein-conducting channel, the signal recognition particle, the signal recognition particle receptor, the signal peptidase, and the proton ATPase. The conserved structural and functional aspects of these proteins indicate that the last common ancestor was associated with a hydrophobic layer with two hydrophilic sides (an inside and an outside) that had a full-fledged and asymmetric protein insertion and translocation machinery and served as a permeability barrier for protons and other small molecules. It is difficult to escape the conclusion that the last common ancestor had a closed biological membrane from which all cellular membranes evolved. Yet, beyond these universal features the nature of the last common ancestor of cellular life (or LUCA) is still intensely debated -5. The vund cell . In thisThe idea of a membrane-less, minerally compartmentalised universal ancestor has been proposed because archaebacteria and eubacteria have membrane lipids of different chemical composition and chirality and because these different lipids are synthesized by mostly non-homologous enzymes ,8. If onHere I discuss what properties can we assign to the membranes or hydrophobic layers of the universal ancestor by carefully analysing the structural and function aspects of the universal membrane-associated cellular machineries.In all cells the translocation of proteins across the plasmamembrane (or ER in eukaryotes) and the insertion of most transmembrane proteins are mediated by a transmembrane protein complex, the protein-conducting channel ,11. ProtComparative genomic surveys revealed that the central components of the translation, protein insertion and translocation machineries are present in all forms of cellular life ,13. The The conserved topology and sequence features of SecY reveal that the PCC is ancestrally membrane associated. SecY has an extracellular and an intracellular hydrophilic part, a conserved inner pore and ten conserved transmembrane segments ,15 1). This There is a clear asymmetry in the arrangement of the secretory machinery: signal peptide containing proteins bind to the PCC at one side, are translocated across the PCC, and are cleaved at the other side by the signal peptidase. The asymmetric arrangement of this machinery is self-maintained: the asymmetric SecY channel inserts new copies of itself and translocates new copies of the signal peptidase to the outside. This asymmetric arrangement is universally conserved indicating that it already existed in the universal ancestor. This means that the hydrophobic layers of the universal ancestor were asymmetric with an 'inside' and an 'outside' and this asymmetry was maintained autocatalytically. The active translocation of secreted proteins by the ribosome-signal peptide-SRP-PCC machinery also means that the hydrophobic layers of the universal ancestor must have presented a permeability barrier (i.e. were closed). Otherwise where and why would signal-peptide containing proteins have been translocated?0F1-ATPase. F0F1-ATPases generate a transmembrane electrochemical gradient at the expense of ATP or catalyze the synthesis of ATP using an electrochemical gradient [0 c subunit of the complex and the conserved function of the proton ATPase indicate that the last common ancestor was associated with a hydrophobic layer able to maintain a proton gradient, i.e. it was necessarily closed. A hydrophobic layer by itself, if not closed, is not enough for proton ATPase function. Importantly, the PCC also uses a proton gradient to drive the insertion of transmembrane proteins [There is additional evidence that the universal ancestor had a closed membrane that presented a permeability barrier to small molecules. As shown by comparative genomics, the last common ancestor carried in its membrane the ancestor of the universal membrane protein complex, the Fgradient . Althougproteins highlighBy the comparative genomic and functional reconstruction of membrane-associated functions the following characteristics can be attributed to the hydrophobic layers of the last universal ancestor. These layers had a (i) hydrophobic core with two hydrophilic sides (ii) had a protein insertion and translocation machinery (iii) had a clear asymmetry (iiii) represented a permeability barrier to proteins and small molecules numbers for the gene content of the universal ancestor . If thisNot accepting the cellular nature of the last common ancestor would mean that membrane-bound cells originated twice independently. The origin of cellular life where genes, membranes and catalysts are integrated in a replicating whole is probably the most difficult problem in cell evolution. The theory of the independent origin of eu- and archaebacterial cells has to solve this problem twice and therefore is clearly most unparsimonious. Given the evidence summarised here it is hard not to conclude that the universal ancestor was membrane bound. In this case it is time to start seriously considering different scenarios of lipid segregation or replacement ,8,9 to aThe author(s) declares that he has no competing interests."} +{"text": "In the title compound, fluorite-like large rods are recognized, built up from a group of 16 Sr atoms of which 6 are substituted by V atoms, leading to [Sr10V6]\u221e units. These rods extend infinitely along the b axis and are inter\u00adconnected by the three water mol\u00adecules. Each of the water mol\u00adecules is shared by two different Sr atoms belonging to two different rods. The rods are also inter\u00adconnected by an \u2018independent\u2019 F atom in a distorted triangular [FSr3] coordination and by hydrogen-bonding inter\u00adactions via donor water mol\u00adecules. The acceptors are either F atoms or the O atoms of the vanadyl ion, VO2+, that is part of the [VOF5] isolated octa\u00adhedron.The title compound, penta\u00adstrontium tris\u00ad[penta\u00adfluorido\u00adoxido\u00advanadate(IV)] fluoride trihydrate, was obtained under hydro\u00adthermal conditions. Its crystal structure has been refined from intensity data of a non-merohedrally twinned crystal. Two domains in almost equal proportions are related by a \u2212180\u00b0 rotation along the reciprocal [101]* vector. The structure may be considered as a derivative of the fluorite structure type, adopted here by SrF IV in [VOF5] coordination, see: Crosnier-Lopez et al. 3F(H2O)3 SrM r = 996.97 Monoclinic, a = 11.217 (2) \u00c5 b = 8.1775 (15) \u00c5 c = 19.887 (4) \u00c5 \u03b2 = 105.999 (4)\u00b0V = 1753.5 (6) \u00c53 Z = 4 K\u03b1 radiationMo \u22121 \u03bc = 16.79 mmT = 298 K 0.36 \u00d7 0.08 \u00d7 0.04 mm Bruker SMART APEX CCD area-detector diffractometerTWINABS; Bruker, 2003T min = 0.065, T max = 0.553 (expected range = 0.060\u20130.511)Absorption correction: multi-scan (6596 measured reflections6596 independent reflectionsI > 2\u03c3(I)4299 reflections with R[F 2 > 2\u03c3(F 2)] = 0.038 wR(F 2) = 0.083 S = 0.93 6596 reflections290 parameters9 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 1.28 e \u00c5\u22123 \u0394\u03c1min = \u22121.11 e \u00c5\u22123 \u0394\u03c1 SMART used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008DIAMOND (Brandenburg, 1999ORTEP-3 (Farrugia, 1997publCIF (Westrip, 2009Data collection: 10.1107/S1600536809019126/wm2236sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809019126/wm2236Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The incidence of nonsyndromic supernumerary premolars is rare. Supernumerary premolars are likely to undergo pathological changes. The most commonly encountered complications with these teeth are dentigerous cyst and root resorption of the adjacent tooth. This paper is about impacted double fused supernumerary premolars in the right mandiblular body associated with an impacted first premolar in a 17-year-old male. Under local anesthesia, the supernumerary premolars and the impacted permanent first premolar were surgically removed. Early diagnosis followed by an appropriate treatment at the right time will result in favorable prognosis in such cases. Supernumerary teeth (ST) occur in addition to the normal complement of teeth in permanent or deciduous dentitions . These tST is considered a developmental anomaly and has been argued to arise from multiple etiologies. Different factors give rise to different types of supernumeraries and combined etiological factors responsible for same. Some of the theories for the formation of ST include atavism, splitting of the tooth bud, local, independent conditioned hyperactivity of the dental lamina, and a combination of genetic and environmental factors , 6, 7. HST are classified according to their morphology as rudimentary or supplemental. Rudimentary teeth are smaller and tuberculate in shape , whereasThe presence of ST may be part of developmental disorders such as cleft lip and palate, cleidocranial dysostosis, Gardner's syndrome, Fabry Anderson's syndrome, Ellis-Van Creveld syndrome , Ehlers Danlos syndrome, incontinentia pigmenti, and Tricho-rhino-phalangeal syndrome .This paper aims to document a rare case of nonsyndromic fusion of two supernumerary supplemental premolars associated with an impacted permanent first premolar.A 17-year-old Saudi male visited the Dental Diagnosis Clinic of King Khalid University, Abha, Saudi Arabia, with a chief complaint of pain in lower right jaw region. No extra oral abnormality was observed. Intraoral examination revealed a missing premolar in the lower right quadrant and tilted permanent right mandibular canine. Orthopantomograph revealed unilateral impacted partially formed and fused double premolars associated with, but separate from an impacted premolar . After tThe prevalence of SP has been reported differently in various studies due to the differences in patient population samples, age groups, ethnicity, and applied radiographic techniques . SP are ST may occur with or without more than 20 syndromes and developmental conditions; however, nonsyndromic multiple supernumerary teeth are rarely encountered prevalence of this being 0.08% , 16, 19.SP are usually asymptomatic and most cases are diagnosed by chance during inspection of radiographs prior to the commencement of orthodontic treatment , 7. SupeCompression of SP on the adjacent teeth and their closeness to the mental and inferior dental nerves may lead to pain . Our patTooth fusion is defined as union between the dentin and/or enamel of two or more separate developing teeth . Shafer In our paper a short root and one root canal were clearly visible on the radiograph of the SPs. Due to the crown form and short root, the authors of this paper diagnosed it as fusion of the two supernumerary teeth. This represents a rare combination of fusion of two supernumerary supplemental premolars. These fused SPs have also caused impaction of the neighbouring second premolar. Treatment options available in such cases include (1) Maintenance in situ with regular followup; (2) extraction if accompanied by pathologic changes; (3) orthodontic treatment.Treatment decision should involve judicious assessment of the case considering the potential risks of leaving ST in situ or the hazards of surgical removal. The consequences of surgical removal of impacted ST, especially in the mandibular premolar region should be evaluated, where the teeth are in close proximity to the inferior dental and mental nerves . The timIn cases when these teeth are associated with pathological formation or when they hinder the eruption of, or give rise to malpositioning of permanent teeth, they should be removed at the earliest . In our Early diagnosis and proper selection of treatment method is likely to result in favorable prognosis. In order to provide symptomatic relief and avoid pathological complications, we deduce that surgical intervention of impacted fused SPs is the most appropriate treatment method."} +{"text": "The surgical removal of supernumerary teeth is necessary in some cases, especially before the commencement of any orthodontic or implant treatment procedure. In the mandibular supernumerary premolar, a more conservative approach is required because of the presence of complications associated with conventional surgery due to the close proximity of the said premolar to the alveolar inferior and mental nerves, and the need for bone conservation for implant placement. The endoscopic surgical approach has been used for the removal of the maxillary supernumerary tooth, impacted third molar, and implants. In this case report, we present an endoscopically assisted surgical technique for the removal of an unerupted supernumerary premolar in the mandible associated with a dental implant placement procedure. A supernumerary tooth is defined as any tooth or odontogenic structure formed from a tooth germ in excess of the usual number in any region of the dental arc . SupernuSupernumerary teeth are the result of changes occurring in the process of normal epithelial-mesenchymal interactions of tooth development. However, the etiology of supernumerary teeth is unknown. Some of the most accepted theories suggest the dichotomy of the tooth germ, other overgrowth, or hyperactivity of the dental lamina, where the proliferations of epithelial rests of dental lamina induced by pressure from the rest of the dentition are outbreaks of supplemental supernumerary teeth .The diagnosis is easy when supernumerary teeth are erupted. However, many do not erupt and may remain asymptomatic throughout life. Some cases are responsible for disorders such as delayed tooth eruption, tooth malposition, or associated pathologies such as dentigerous cyst, requiring surgical intervention . The finThe use of support endoscope makes a minimally invasive and more predictable procedure possible, in terms of greater conservation of bone tissue, less tissue damage, and minimization of blood loss , 5. SomeA 34-years-old man, systemically healthy, was underwent surgery at the Oral Microsurgery Center, Dental School, Universidad de La Frontera. The patient has given their informed consent for the case to be published. We planned the placement of one titanium osseointegrated-type implant in the edentulous left posterior mandible, in the premolar (3.5) area. In the review of the preoperative panoramic radiograph, no supernumerary tooth in the surgical area was observed . NeverthFor support endoscopy (SE), an endoscope of 2.7\u2009mm diameter and a 30-degree view angle, with support and irrigation, was used for drilling of the tooth. Later for immersion endoscopy (IE) , an endoFinally, the bone defect was filled with particulate bone graft , with thThe use of endoscopy in oral and maxillofacial surgery has improved the visualization of the surgical field through the magnification of the operative field, with lighting and additional irrigation attached to the support, making possible minimally invasive and conservative approaches with precise dissection. These characteristics make it an ideal alternative for the elimination of unerupted supernumerary teeth , 9.The occurrence of supernumerary teeth is not unusual. It is associated with more than 20 syndromes and different growing conditions and nonsyndromic conditions. Premolars are often asymptomatic, and most cases are diagnosed as imaging test findings before orthodontic or implant placement treatments. Only 2% of premolars are likely to generate pathological conditions as a dentigerous cyst or the adjacent tooth root resorption. In many cases, surgical removal of these teeth is the only treatment option, with the complex vision of the surgical site requiring the surgeon to remove a large amount of bone tissue, which can cause neurosensorineural complications when the teeth are very close to the inferior alveolar and mental nerves. Moreover, due to the complex morphology of these teeth, there are potential risks of leaving remnants of dental tissues and dental sac. In addition, supernumerary teeth are not always implemented in the direction of normal eruption; they can appear upside down or cross an ectopic or abnormal eruption path, making their removal even more complex.It has been reported that the presence of unerupted supernumerary in a potential implant site may compromise the placement of implants and bone blocks, thus requiring their prior removal. If the unerupted supernumerary is removed at the same time of implant placement, the use of bone grafts may be necessary in the defect created , or it mSeveral cases have demonstrated the advantages of endoscopic surgery. Hasbini et al. reportedAnother benefit observed in our case, aside from the optimal viewing at any time of the surgery with SE, was the minimal and controlled removal of bone tissue, with the immediate postoperative control of clean bedding without remaining tooth structure (with IE). Therefore, we obtained a four-wall defect without communication to the implant and preservation of the buccal cortex, thus improving the prognosis of the implant, especially when a tooth ankylosed, it is very difficult to define its margin, where SE and IE show superiority ensuring complete dental tissue removal.Nevertheless, some considerations are necessary for this procedure. First, the technique requires a core team of endoscopic and specially instructed surgeons. The endoscope provides a magnified image of two dimensions on a video monitor at a distance, thus requiring the development of specific hand-eye coordination, with a broad understanding of the three-dimensional concept of oral and maxillofacial surgical anatomy . Second,We suggest the use of an endoscopic surgical approach as a first-line treatment for the removal of supernumerary teeth adjacent to the implant area, to preserve bone and prevent possible damage to neurovascular structures or implant viability. This endoscopic removal technique can be applied for unerupted supernumerary mesiodens in the maxilla that is the most common type."} +{"text": "Alocasia species have been used in India as folk remedy for the treatment of various inflammatory ailments including rheumatism and bruise. The ethanolic extract of leaves of Alocasia indica Schott. was evaluated by using different in vitro antioxidant models of screening like scavenging of 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical, nitric oxide radical, superoxide anion radical, and hydroxyl radical. The antinociceptive activity was tested by acetic acid-induced writhing response, hot plate method, and tail flick method in albino rats. The anti-inflammatory potential of gels of ethanolic extract has been determined by using carrageenan-induced paw edema assay, formalin-induced paw edema assay, arachidonic acid-induced ear edema assay, and xylene-induced ear edema assay. The extract showed remarkable antioxidant activity in all models, comparable to the standard reference drug ascorbic acid. The ethanolic extract of Alocasia indica and its gels produced dose-dependent antinociceptive and anti-inflammatory activity, respectively. This finding suggests that ethanolic extract of A. indica possess potent antinociceptive and anti-inflammatory activity possibly due to its free radical scavenging properties.Extracts obtained from the leaves of various Alocasia indica Schott. is an indigenous herb belonging to family Araceae. Different parts of this plant are traditionally used in inflammation and in diseases of abdomen and spleen.[d spleen. The juicd spleen. It has hd spleen. and antid spleen. This plad spleen. Since noO-phosphoric acid, napthyl ethylene diamine dihydrochloride, trichloroacetic acid (TCA), glacial acetic acid, nitroblue tetrazolium (NBT), ethylene diamine tetra acetic acid (EDTA), riboflavin, and Fe-EDTA. Carrageenan was obtained from Sigma-Aldrich, USA. Volini gel (Diclofenac sodium) was a gift sample from Ranbaxy Laboratories Ltd, Punjab. UV-Visible spectrophotometer (Shimadzu 1700) was used for recording the spectra.All chemicals and solvents were of analytical grade and obtained from Loba Chemicals Ltd, Mumbai. 1, 1-diphenyl, 2-picryl hydrazyl (DPPH) was obtained from Sigma Chemicals, USA. The other chemicals used were sodium nitroprusside, sulphanilamide, Wistar albino rats weighing 175-225 g of either sex were obtained from Krishna Institute of Medical Sciences, Karad, Dist- Satara (Maharashtra) and were acclimatized for 10 days under standard housing conditions . The animals had free access to rat food and water. The animals were habituated to laboratory conditions for 48 h prior to the experimental protocol to minimize any nonspecific stress. The experimental protocol was approved by the Institutional Animal Ethics Committee by Government College of Pharmacy, Karad, India, and animals were maintained under standard conditions in the animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).A. indica Schott. (Araceae) collected from different places at Karad were authenticated at Botany Dept., Yashwantrao Chavan College of Science, Karad, and a voucher specimen was deposited at the Herbarium of institute.Fresh leaves of A. indica was prepared by soxhletion. The powdered plant material (750 g) was repeatedly extracted in a round bottom flask with 2250 ml ethanol. The reflux time was 40 cycles for complete extraction. The extract was cooled at room temperature, filtered, and evaporated to dryness under reduced pressure in a rotary evaporator. The percentage yield was found to be 2.79 g. The ethanolic extract of A. indica was referred as AI.The ethanolic extract of leaves of Chemical tests were carried out on ethanolic extract of the plant for qualitative determination of phytochemical constituents.DPPH assay: To 1 ml extract of different concentrations, 1 ml solution of DPPH (0.1 mM) was added. An equal amount of methanol and DPPH solution served as control. After 20 min of incubation in the dark, absorbance was measured at 517 nm.[7 517 nm.7 AscorbicScavenging of nitric oxide radical: Nitric oxide was generated from sodium nitroprusside and measured by Griss reaction.[O-phosphoric acid, and 2 g of 0.1% napthyl ethylene diamine dihydrochloride). The absorbance of chromophore formed during diazotization of nitrite with sulphanilamide and its subsequent coupling with napthyl ethylene diamine was read at 546 nm. Ascorbic acid was used as standard. The experiment was performed in triplicate.reaction.10 SodiumHydroxyl radical scavenging activity: Extracts of different concentrations (200-1000 \u00b5g/ml) were taken in different test tubes and evaporated on water bath. To these, 1 ml of Fe-EDTA, 0.5 ml of EDTA, and 1 ml DMSO were added and the reaction was initiated by adding 0.5 ml ascorbic acid to each of the test tubes. Test tubes were capped tightly and heated in water bath at 80-90 \u00b0C for 15 m. Then the reaction was terminated by addition of 1 ml of ice-cold TCA to all the test tubes and kept aside for 5 min. The formaldehyde formed was determined by adding 3 ml Nash reagent . This reaction mixture was kept aside for 15 min for color development.[elopment. Intensitelopment.Scavenging of superoxide radical by riboflavin--NBT system: The assay was based on the capacity of the sample to inhibit blue formazon formation by scavenging the superoxide radicals generated in the riboflavin--NBT system. The reaction mixture contains 50 mM phosphate buffer pH 7.6, 20 g riboflavin, 12 mM NBT. Reaction was started by illuminating the test samples of the extract (200-1000 \u00b5g/ml). The absorbance was measured at 590 nm. Ascorbic acid was used as positive control.[ control.14in vitro models are calculated by following formula:The percentage scavenging of each extract in different concentrations for different 50 was calculated by using formulae:ICb = \u2211 x.y / \u22112xa = y \u2212 xb50 = a + b (50)ICb = regression coefficient of x on y; a = intercept of the line; x = concentration; and y = % scavenging; x = mean of the concentration; y = mean of % scavenging.Where, Writhing test: Writhing was induced in rat (n = 6) by intraperitoneal injection (10 ml/kg) of 0.6% acetic acid. The number of writhings was counted over a 20 min period as previously reported.[reported.16 AnimalTail immersion test: Six rats were administered orally with vehicle (3 ml/kg), pentazocine (30 mg/kg), and ethanolic extract of AI (200 and 400 mg/kg). The distal part of the tails of the animals was immersed in hot water maintained at 55.0 \u00b1 1.0 \u00b0C. The time taken to withdraw the tail was noted as reaction time.[ion time. A cut-ofHot plate test: Rats were placed on an aluminum hot plate kept at 55 \u00b1 0.5 \u00b0C for a maximum time of 30 s.[ of 30 s. ReactionPreparation of gels: The gels of AI were prepared with carbopol-940 and PEG by cold method.[d method. Differend method.. The weiCarrageenan-induced paw edema assay: The animals (n = 6) were divided into five groups. 0.5 g of AI gels and marketed formulation of diclofenac sodium (2%) were applied to the planter surface of the left hind paw by gently rubbing 50 times with the index finger.[V \u2013 Vi/Vi) \u00d7100, where V is the paw volume 3 h after the carrageenan injection and Vi is the initial paw volume.Percentage anti-inflammatory activity = into the subplanter area of right hind paw of rat.[w of rat.Arachidonic acid-induced ear edema assay: The animals (n = 6) were divided into five group. Samples were given 1 h prior to application of arachidonic acid.[nic acid. One hourXylene-induced ear edema assay: The same procedure as mentioned above in arachidonic acid-induced ear edema assay was followed except that acute inflammation was produced by applying xylene to anterior and posterior surfaces of right ear of rat.[r of rat.P < 0.05 was considered significant.The statistical significance was assessed using one way of variance (ANOVA) followed by Bonferrini\u2019s multiple comparison tests. The values are expressed as mean \u00b1 SE and A. indica showed that it contains flavonoids, cynogenetic glycosides, citric acid, ascorbic acid, and polyphenolic compounds.The preliminary phytochemical investigation of the ethanolic extract of A. indica were tested for their antioxidant potential using different in vitro models. It was observed that free radicals were scavenged by test compounds at different concentrations. The maximum inhibitory concentration (IC50) in all models viz., DPPH, nitric oxide radical, superoxide, hydroxyl radical scavenging activity, were found to be 7.30, 10.97, 9.8, and 7.86 \u00b5g/ml, respectively. The antioxidant model and percentage scavenging of each concentration of extract and standard are shown in Five concentrations ranging from 200 to 1000 \u00b5g/ml of the ethanolic extract of P < 0.05) reduced the number of writhings induced by acetic acid in rat [P < 0.001).Oral administration of the ethanolic extract of AI (200 and 400 mg/kg) significantly [P < 0.05) [Gels containing AI and marketed formulation of diclofenac sodium produced dose-dependent inhibition of carrageenan and formalin-induced paw edema as compared to the control ( < 0.05) . In xyle < 0.05) .in vitro antioxidant activity of AI was in a concentration-dependent manner.Recent studies suggest that the inflammatory tissue damages are due to the liberation of reactive oxygen species from phagocytes invading the inflammation sites.\u201326 In adThe ethanolic extract of AI at the doses of 200-400 mg/kg protected rat against both chemical- and thermal-induced noxious stimuli, which were evidenced from the acetic acid-induced writhing, tail immersion, and hot plate tests. Acetic acid induces writhing syndromes16 and caCarrageenan-induced edema is a biphasic response in which the involvement of the cyclo-oxygenase products of arachidonic acid metabolism and the production of reactive oxygen species are well established. The firsAnother model of inflammation used is the ear edema induced by arachidonic acid and xylene, which serves to identify the mediators of inflammation of the lipoxygenase pathway.34 TopicaA. indica contains flavonoid compounds. Flavonoids have been shown to possess various biological properties related to antioxidant, antinociceptive, and anti-inflammatory mechanisms by targeting reactive oxygen species and prostaglandins which are involved in the late phase of acute inflammation and pain perception.[A. indica possess antioxidant, antinociceptive, and anti-inflammatory activities may be due to the presence of flavonoids and other polyphenolic moieties present in it, which seems to support the use of this plant in traditional medicine. Finally, the precise mechanism(s) and site(s) of this activity and the active constituent(s) involved are still to be determined in addition to toxicological studies.Preliminary phytochemical screening showed that ethanolic extract of the leaves of rception.\u201337 It ca"} +{"text": "Triticum aestivum), durum wheat (T. durum), and barley (Hordeum vulgare). Natural genetic variants at these loci result in major phenotypic changes which have adapted crops to the novel environments encountered during the spread of agriculture. The polyploid nature of bread and durum wheat means that major flowering time loci in which recessive alleles confer adaptive advantage in related diploid species have not been readily identified. One such example is the PPD-H2 flowering time locus encoded by FLOWERING LOCUS T 3 (HvFT3) in the diploid crop barley, for which recessive mutant alleles confer delayed flowering under short day (SD) photoperiods. In autumn-sown barley, such alleles aid the repression of flowering over the winter, which help prevent the development of cold-sensitive floral organs until the onset of inductive long day (LD) photoperiods the following spring. While the identification of orthologous loci in wheat could provide breeders with alternative mechanisms to fine tune flowering time, systematic identification of wheat orthologs of HvFT3 has not been reported. Here, we characterize the FT gene families in six Poaceae species, identifying novel members in all taxa investigated, as well as FT3 homoeologs from the A, B and D genomes of hexaploid (TaFT3) and tetraploid wheat. Sequence analysis shows TaFT3 homoeologs display high similarity to the HvFT3 coding region (95\u201396%) and predicted protein (96\u201397%), with conservation of intron/exon structure across the five cereal species investigated. Genetic mapping and comparative analyses in hexaploid and tetraploid wheat find TaFT3 homoeologs map to the long arms of the group 1 chromosomes, collinear to HvFT3 in barley and FT3 orthologs in rice, foxtail millet and brachypodium. Genome-specific expression analyses show FT3 homoeologs in tetraploid and hexaploid wheat are upregulated under SD photoperiods, but not under LDs, analogous to the expression of HvFT3. Collectively, these results indicate that functional wheat orthologs of HvFT3 have been identified. The molecular resources generated here provide the foundation for engineering a novel major flowering time locus in wheat using forward or reverse genetics approaches.To date, a small number of major flowering time loci have been identified in the related Triticeae crops, bread wheat ( The rising global human population requires increases in agricultural productivity in order to meet food demand. However, the effects of climate change on crop harvests have the potential to cause wide-scale food shortage and fluctuations in supply. Accordingly, there is a need to develop crop varieties adapted to maintain and increase yields in the face of future climatic change. Given the central role of flowering time in the adaption of crops to local agricultural environments, the efficient manipulation of this trait is of particular interest under future climate change scenarios.Hordeum vulgare; diploid, 2n = 2x = 14), durum wheat and bread wheat . A subset of these loci are located at collinear chromosomal locations in multiple species, and are encoded by orthologous genes , rye , durum wheat , and bread wheat , encoded by orthologous PSEUDO RESPONSE REGULATOR (PRR) genes loci result in the abolishment of vernalization requirement, resulting in rapid-cycling accessions which can proceed to flowering without the need for vernalization treatment (prolonged periods of low non-freezing temperature) abolish vernalization requirement -like gene HvFT3 in wheat, the identification and consolidation of recessive alleles at all three homoeologous genes into one genetic background may provide a novel source of flowering time variation of particular relevance to a warming climate, allowing floral repression in the winter without the need for a strong vernalization requirement. Indeed, \u201calternative\u201d barley varieties appear to possess flowering time gene haplotypes that invariably include recessive ppd-H2 alleles, thus helping to prevent premature flowering databases (and only six barley ESTs were present prior to specific investigation of HvFT3). However, the recent availability of survey sequence of the bread wheat genome offers new opportunities for the investigation of wheat gene content. This provides a timely opportunity to use reverse genetic approaches to create a wheat accession possessing a SD floral delay analogous to that deployed in winter barley, and of particular relevance to climate change scenarios.In barley, the major flowering time locus HvFT3 in the A, B, and D genomes of hexaploid and tetraploid wheat, show that they (i) group phylogenetically with FT3 genes from barley and other cereals, (ii) map to collinear positions in the related cereal species wheat, barley, rice (Oryza sativa) and Brachypodium distachyon, and (iii) are upregulated under SD photoperiods. Collectively, these resources provide the foundation from which forward and reverse genetic approaches can be deployed toward the creation of novel SD responsive flowering time loci in polyploid wheat, aiding adaptation to future climate change scenarios.Here, we systematically identify orthologs of HvFT3 coding regions were used for BLASTn analysis -value threshold of 1.0e-30) to identify putative orthologs in the genomic survey sequence of bread wheat cv. Chinese Spring . The previously determined rice FTL gene family is as described by Higgins et al. .Barley s et al. , as are d Devos, . Wheat Fhttp://prosite.expasy.org/). Predicted protein sequences were aligned using Clustal Omega . The resulting alignments were used for phylogenetic analysis, conducted using the PHYLIP package v3.5 , with pairs selected based on the number of homoeolog-specific nucleotides incorporated. Polymerase chain reaction (PCR) amplification (10 \u03bcl reactions) were performed using the reagents shipped in the FastStart Taq DNA polymerase kit (Roche). PCR cycling was carried out using a Veriti 96 well Thermo Cycler (Applied Biosystems) with the parameters: 5 min at 96\u00b0C, followed by 35 cycles of 50 sec at 96\u00b0C, 50 sec annealing temperature, 90 sec at 72\u00b0C, final extension of 7 min at 72\u00b0C. Primer sequences and annealing temperatures are listed in Table Genomic DNA was extracted from wheat leaf material using the DNeasy 96 Plant Kit (Qiagen). Primers were designed from genomic DNA sequence contigs using Primer3 v0.4.0 , following previously described protocols lines and one tetraploid (Kronos) spring wheat varieties were used for gene expression analyses. Varieties were selected for the following reasons: (1) Chinese Spring: worldwide wheat reference variety for genome sequencing. (2) Cadenza: parent of the UK reference Avalon \u00d7 Cadenza genetic mapping population; Ethyl methanesulfonate (EMS) mutated Targeting Induced Local Lesions in Genomes (TILLING) population available are listed in Supplementary Table \u22121. Three technical repeats were undertaken for each primer pair. Primer efficiencies were calculated using the formula: EFFICIENCY = \u22121+10(\u22121/Gradient), where Gradient = LINEST . The relative expression level of TaFT3 compared to the four reference genes was calculated using the Pfaffl method , following the manufacturer's instructions. RNA concentrations were determined using a Nanodrop 2000 spectrometer (Thermo Fischer Scientific), standardized to 250 ng/\u03bcl using nuclease-free water (Promega), and RNA samples (1.75 \u03bcg each) were DNAse treated using a RQ1 RNase-Free DNAse kit (Promega). Prior to cDNA synthesis, genomic DNA was digested by addition of RNase-free DNase (Promega) to each sample, and reactions terminated using RQ1 DNase Stop Solution (Promega), following the manufacturer's instructions. cDNA synthesis performed using a M-MLV Reverse Transcriptase kit (Promega), according to the manufacturer's instructions. All qRT-PCR reactions were undertaken using SYBR MM (Roche), and carried out in total reaction volumes of 10 \u03bcl using a Light Cycler 480 (Roche) with the following cycling conditions: 95\u00b0C 5 mins; 45 cycles of 95\u00b0C for 10 s, 60\u00b0C for 15 s, 72\u00b0C for 30 s, cooling 60\u00b0C; melt curve was carried out by ramping 0.11\u00b0C/s from 60 to 95\u00b0C, with 5 readings taken per second. RT-PCR primers for the three Pfaffl, , where t Pfaffl, .HvFT3 gene thought to underlie the barley PPD-H2 locus, the barley HvFT3 CDS (HM133572) was used for BLASTn searches of hexaploid wheat sequence contigs in the flow sorted wheat genome survey sequence, identifying 21 significant (8x-fold increase in expression). As in the hexaploid varieties, TaFT3-B1 expression under LDs was consistently low, reaching a peak at week 4 (1.16). Mean expression at all time-points was higher under SDs compared to LDs, and while significant differences were only observed at week 3, comparison of SD expression between week 1 and all subsequent time points found transcription to be significantly higher in all cases.In the tetraploid wheat variety Kronos Figures , expressFT is a key component in the floral signaling pathway , Komiya et al., CENTRORADIALIS 8 (ZCN8), Meng et al., VRN-H3), and wheat (VRN-B3) . While Peng et al. , lineage-specific members, as well as species specific CNV. FT members identified as lineage specific, or as exhibiting inter-specific CNV represent good candidates for further investigation, given the prominent role of CNV underlying known Poaceae flowering time loci. It may also be possible that FT members involved in inter-specific CNV are also prone to intra-specific CNV, thus highlighting a possible route toward identifying genetic variation of importance to the control of flowering. Similar examples have previously been raised, such as the lack of genes orthologous to the wheat and barley VRN-2 loci in brachypodium, which may be due to the brachypodium accession sequenced being a mutant rapid cycling vernalization insensitive spring type of flowering pathway genes within Poaceae species is known to play a prominent role in the control of flowering locus have found it to map to the long arm of chromosome 1D, co-segregating with microsatellite marker Xcfd63 and closely linked to Xgdm111, XBarc62, and TaBradi2g14790 . Similarly, investigation of micro-colinearity around the FT3 physical regions in six Poaceae species confirmed (i) high conservation of gene order despite the evolutionary divergence of the grasses between 55 to 70 million years ago in support of her participation on the University of East Anglia Plant Genetics and Crop Improvement MSc course. MP was supported by an Erasmus grant under the LLP Erasmus Placement Programme. JC and AB were partially funded by BBSRC projects BB/M011666/1 and BB/I002561/1, respectively.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Bismuth (Bi) electrodeposition was studied on Si/Ti/Au, FTO-, and ITO-coated glasses from acidic nitrate solutions with and without gluconate within a narrow potential window (\u0394E = 80 mV). This potential range was sufficient to observe a change in particle shape, from polyhedrons (including hexagons) to dendrites, the trend being slightly different depending on substrate activity. In all cases, though, the formation of dendrites was favoured as the applied potential was made more negative. Bi particles were more uniformly distributed over the substrate when sodium gluconate was added to the electrolyte. X-ray diffraction analyses of dendrites grown at \u22120.28 V indicated that they exhibit the rhombohedral phase of Bi and are predominantly oriented along the (003) plane. This orientation is exacerbated at the lowest applied potential (\u22120.20 V vs. Ag|AgCl) on glass/ITO substrate, for which completed and truncated hexagons are observed from the top view scanning electron microscopy images. Electrodeposition is a surface treatment method which allows the growth of films, micro- and nanostructures with a simple and relatively low-cost set-up. By changing the parameters, such as applied potential or current density, composition of the electrolyte, and the substrate type, it is possible to obtain deposits with different properties and functions. Electrodeposition of semi-metal bismuth has attracted much attention due to its unique physical and chemical properties, which make it a choice for a wide range of applications in different areas, including magnetoresistance ,2, therm3)3\u00b75H2O dissolved in diluted nitric acid could be tuned, to some extent, through the addition of ethylenediaminetetraacetic acid (EDTA) to the bath and the substrate type ) [3 + 5\u201320 mM Bi(NO3)3\u00b75H2O and produced small crystallites over GCE at cathodic potentials between \u2212100 and \u2212300 mV vs. Ag|AgCl (3 M KCl) and deposition times ranging from 10 to 350 s [3)3 dissolved in 1 M HNO3 [3+ in acidic nitrate solutions since electro-reduction of nitrate is very slow and marginally detectable. Bulk deposition of Bi was observed ca. \u22120.5 V vs. SSE ) over a pre-deposited Bi grown by underpotential deposition (UPD). Compact, relatively thick films (1.0\u201315 \u03bcm thick) were characterized morphologically and structurally. More recently, fern-shaped Bi dendrites were grown on GCE using a two-step potentiostatic deposition scheme consisting of applying \u22120.5 V for 200 s followed by \u22120.8 V for 1000 s. In the second deposition step, hydrogen co-evolution took place [It has been shown that bismuth can be electrodeposited in different configurations, ranging from dense or particulated films to sparse (ITO)) . A few yto 350 s . Sandnes1 M HNO3 . The autok place .Compared to compact films, submicron- and nanostructures are known to possess larger surface-to-volume ratios and, therefore, their production attracts a great deal of interest for surface-sensitive applications like heterogeneous catalysis, gas storage, and sensing. Most frequently, low populated surfaces consisting of small nuclei/crystals relatively far away from each other are reported in the literature . HoweverIn this work, micrometer- and submicrometer-sized bismuth particles are electrodeposited onto Si/Ti/Au, ITO-coated, and fluorine-doped tin oxide (FTO)-coated glass substrates. The beneficial effect of gluconate additives on electrode coverage uniformity is reported. We show that particle shape and size can be tuned within a very narrow potential window. The potential at which the transition from polyhedral particles to dendrites occurs is substrate-dependent.3)3\u00b75H2O + 1.5 M HNO3 electrolyte onto Si/Ti/Au, glass/FTO, and glass/ITO substrates are shown in The CV responses from 5 mM Bi at the screened potentials in spite of the low pH, CVs including initiation of Ar purging at the positive sweep were carried out and compared to those obtained under stagnant conditions . In order to confirm that Hnditions b. The oxThe addition of gluconate to the electrolyte did not delay the onset of deposition but caused a shift toward more positive potentials c. This eMicrometer- and submicrometer-sized Bi particles were grown by applying constant potentials between \u22120.20 and \u22120.28 V, i.e., within 80 mV interval, for deposition times ranging from 120 to 800 s. Noisy j\u2013t curves were recorded as argon2 in both FTO and ITO gets dissolved in the solution at high cathodic potentials in the low pH media, the bath cannot be reused further [The cathodic potential could not be extended beyond \u22120.3 V, especially on glass/ITO, as ITO suffered from instabilities at such acidic pH during cathodic polarization, mostly causing an abrupt increase of the resistance . Moreove further . 2 flux was not fully efficient to dry the deposits, i.e., in many cases the particles displayed an intricate morphology and remained wet. Meanwhile, if the samples were left to dry spontaneously, the remaining water droplets were capable of fully oxidizing the Bi film. Immediately after deposition, deposits were rinsed in deionized water and dried in hot air. It was noticed that a NThe substrate type had an obvious impact on the particles\u2019 shape. For example, sharper dendrites were observed on glass/FTO than on glass/ITO under identical deposition conditions . DepositThe addition of gluconate to the electrolyte enabled the production of more uniformly covered substrates a,b. As aGood adhesion was noticed on all substrates for deposition times up to 250 s. Applying longer durations caused detachments from the substrate, similar to the gluconate-free bath. Polyhedral particles and incipient dendrites were observed on glass/FTO at \u22120.20 V . DendritThe nature of the substrate influences the coating structure within the initial electrodeposition stages. Surface characteristics including surface finish (roughness), conductivity, and structure , have a decisive impact on the nucleation rate. In our case, Au is a metal whilst both ITO and FTO are semiconductors. Therefore, island nucleation and growth might be affected by substrate conductivity to a great extent . This isAt \u22120.20 V, the polyhedrons formed on Si/Ti/Au had sizes varying from 1 to 4.5 \u00b5m. On glass/ITO, they had lengths between 1.4 and 7 \u00b5m and width of 1 \u00b5m. Meanwhile, polyhedrons with a width up to 2 \u00b5m formed on glass/FTO, accompanied with dendrites up to 4 \u00b5m in length and 2 \u00b5m in width. As aforementioned, well-shaped hexagons were only observed on glass/ITO. In this case, their size distribution became narrower as the potential was made more negative; namely, at \u22120.20 V they exhibited sizes ranging from 600 nm to 3 \u00b5m, whereas at \u22120.24 V 1 \u00b5m-sized hexagons were obtained. Concomitantly, dendrites with lengths up to 7.2 \u00b5m and widths up to 2 \u00b5m were seen on this substrate. Compared to glass/ITO, smaller dendrites formed on glass/FTO at \u22120.24 V, with lengths reaching up to 4 \u00b5m and widths of 600 nm. As the absolute value of the cathodic potential increased from \u22120.24 to \u22120.28 V, the lengths of the dendrites showed an increase on each substrate up to 10 \u00b5m and a decrease in their widths down to 600 nm.(hkl)/I(012) ratios obtained, Bi dendrites grown on Si/Ti/Au, glass/FTO, and glass/ITO substrates show a predominant orientation along the (003) plane . This orientation is exacerbated in the dendrites grown on the glass/ITO substrates. The collected X-ray diffraction (XRD) patterns indicated that single-phase deposits consisting of pure Bi crystallizing in the rhombohedral lattice were obtained irrespective of the applied potential and substrate type. In order to study the influence of the substrate on the microstructure, XRD patterns of Bi electrodeposited at \u22120.28 V on Si/Ti/Au, glass/FTO, and glass/ITO substrates are presented in a = 4.546 \u00c5 and c = 11.862 \u00c5) but slightly lower, indicating possible lattice distortion. For each diffraction pattern, the full width at half maximum (FWHM) intensity of each peak was measured and the crystallite size was determined using the Williamson-Hall plot. At \u22120.28 V, slightly larger crystallite sizes are obtained for Bi grown on glass/FTO substrate (~49 nm), while smaller crystallite sizes are obtained for that grown on Si/Ti/Au substrate (~37 nm). To evaluate the contribution of the deposition potential on the microstructural features of Bi, XRD of deposits grown on glass/ITO at the three potentials studied in this work was carried out. The obtained diffraction patterns are presented in (hkl)/I(012) ratios ratios the more (hkl)/I0 ratios (3)3\u00b75H2O and 1.5 M HNO3 and the other with 0.05 M NaC6H11O7 (sodium gluconate), in addition to the components mentioned. Argon was purged into the electrolyte both before and during the electrodeposition to avoid possible oxidation and favour mass transport. Prior to electrodeposition, Si/Ti/Au substrates were conditioned by cleaning with acetone, isopropanol and Milli-Q water and dried with nitrogen, whereas glass/FTO and glass/ITO substrates were cleaned with isopropanol and dried. The working area was between 0.25 and 0.56 cm2. After the electrodeposition step, the samples were rinsed in Milli-Q water and hot dried. All samples were stored in a desiccator.CV studies and potentiostatic electrodeposition of bismuth at room temperature on Si/Ti (10 nm)/Au (90 nm), FTO-coated glass , and ITO-coated glass substrates were implemented in a three-electrode cell with a Pt wire as the counter electrode and a Ag|AgCl (3 M KCl) reference electrode (E = +0.210 NHE). Electrode arrangement and separation were the same in all experiments. Two different baths were used, one of which consisted of 5 mM Bi(NO\u03b1 radiation. Rietveld refinement using MAUD software [Scanning electron microscopy (SEM) observations were done using a Merlin Zeiss microscope operated at 2 kV. Energy dispersive X-ray (EDX) analyses were carried out at 15 kV using a detector coupled to the SEM microscope. Wide-angle XRD patterns were collected on a Philips X\u2019Pert diffractometer in the 20\u00b0\u201375\u00b0 2\u03b8 range using CuKsoftware was carrFinely-distributed micrometer- and submicrometer-sized Bi particles were obtained by electrodeposition from an aqueous solution containing nitric acid and sodium gluconate. A narrow window of deposition potentials and relatively short deposition time (250 s) to prevent the material from detachment was examined. The substrate type greatly impacted both the shape and the size of the particles obtained at each applied potential. Differences in substrate conductivity likely play a key role in Bi particles\u2019 shape. A transition from polyhedral-shaped particles, including hexagons towards dendrites, was most clearly observed on glass/ITO. Rhombohedral Bi with (003) predominant orientation and crystallite size below 50 nm was obtained at \u22120.28 V on all electrodes. The (003) orientation further increased when completed and truncated hexagons formed on the substrate, as was the case for the glass/ITO electrode."} +{"text": "Mexico is an important global reservoir of biological and cultural richness and traditional knowledge of wild mushrooms. However, there is a high risk of loss of this knowledge due to the erosion of traditional human cultures which is related with the rapid acculturation linked to high migration of rural populations to cities and the U.S.A., and the loss of natural ecosystems. The Mixtec people, the third largest native group in Mexico only after the Nahua and the Maya, maintain ancient traditions in the use and knowledge of wild mushrooms. Paradoxically, there are few studies of the Mixtec ethnomycology. This study shows our ethnomycological research, mainly focused on knowledge and use of wild mushrooms in communities of the Mixteca Alta, in southeastern Mexico. We hypothesized that among the studied communities those with a combination of higher vegetation cover of natural pine and oak forests, lower soil erosion and higher economic margination had a greater richness and knowledge of wild mushrooms. Our study therefore aimed to record traditional knowledge, use, nomenclature and classification of wild mushrooms in four Mixtec communities and to analyze how these aspects vary according to environmental and cultural conditions among the studied communities.In order to analyze the cultural significance of wild mushrooms for the Mixtec people, 116 non-structured and semi-structured interviews were performed from 2009 to 2014. Information about the identified species, particularly the regional nomenclature and classification, their edibility, toxicity and ludic uses, the habitat of useful mushrooms, traditional recipes and criteria to differentiate between toxic and edible species, and mechanisms of knowledge transmission were studied. The research had the important particularity that the first author is Mixtec, native of the study area. A comparative qualitative analysis between the richness of fungal species used locally and the official information of the natural vegetation cover, soil erosion and economic marginalization in each of the studied communities was conducted.A total of 106 species of mushrooms were identified growing in pine and oak forest, deciduous tropical forest and grassland; among the identified mushrooms we recorded 26 species locally consumed, 18 considered toxic, 6 having ludic uses and the remaining 56 species not being used in the studied areas but some of them having potential as food (56 species) or medicine (28 species). We recorded that 80, 22 and 4 species are ectomycorrhizal, saprotrophic and parasites, respectively. Our study shows that a complex and accurate knowledge related with the use, nomenclature, classification, ecology, gastronomy of wild mushrooms has been developed by Mixtecs; and that there is a relation between natural vegetation cover, lower soil erosion and higher economic marginalization and richness, knowledge and use of mushrooms in the studied communites.Our study showed that conservation and adaptation of ancestral mycological knowledge survives mainly through oral transmition, maintenance of cultural identity, forest protection, preservation native language and also paradoxically through the current socieconomical marginality among the Mixtec people. We also found that those studied communities with a combination of higher vegetation cover of natural pine and oak forests, lower soil erosion and higher economic marginalization showed a greater richness and knowledge of wild mushrooms. Use and sustainable management of wild mushrooms can be an alternative for local integrated development, but local knowledge and traditional worldview should be included into the regional programs of Mixtec biocultural conservation. Mexico is considered a megadiverse country since it harbours approximately 10\u00a0% of the terrestrial biodiversity of the planet (1.8 million species) estimateMixtec comes from the Nahuatl language, meaning \u201cpeople of Mixtlan or place of the clouds\u201d. However, the Mixtec people call themselves \u00f1uu savi, meaning \u201cpeople of the rain\u201d is considered by the Mixtec group as a \"bioclimatic indicator\". It is believed that it only emerges when there is a thunder and announces the rainy season. If the fungus develops in the months of February and March, that indicates that the rainy season will begin early and continue for a long period. Conversely, if it appears in the months of April or May, the rains will be delayed and the season will be short. In observations made in this study during the years 2009 to 2014, the accuracy of this belief was recorded. For the Mixtec peoples, rain (savi or dau) is the predominant meteorological phenomenon, and other phenomena are linked to it.Lactarius indigo (xi\u00b4i kuilu) is associated with the plumage colour of the \u201cchara\u201d bird (Aphelocoma woodhouseii) (t\u2009+\u2009daa kuilu), which produces an alarming chant that the Mixtec peoples consider to mean that an attack is coming or something bad will happen.Some species have names in the region because they: i) were consumed in the past, ii) are similar to another species that is currently used; or iii) are associated with an important familiar entity in the known universe of the interviewees. For example, Lycoperdon in their immature or mature state because of their form of \"small balls\", and they also like to squeeze them because of the \"dust\" (consisting of spores) they emit. Similarly, they collect sporocarps of Astraeus hygrometricus , which, because of their star shape, are collectibles . An advantage of this mushroom for ludic use is its durability. Also, informants reported that species of the genus Calvatia or Pisolithus , when ripe, are used in children's games as projectiles to throw at friends, siblings or grazing livestock. Undoubtedly, the natural curiosity of children represents potential and hope for the preservation and care of invaluable mycological resources for their ludic importance.In the community of Santa Catarina Estetla, the youngest children collect species of The use of hallucinogenic mushrooms in the communities under study was not reported. However, the informants mentioned that in the San Antonio Huitepec Municipality south of the Santa Catarina Estetla and San Juan Yuta communities, healers and shamans use mushrooms for divination or healing purposes.A. bisporigera, A. verna and A. virosa, which are abundant in the region and are recognised locally as deadly mushrooms. Inhabitants of Santa Catarina Estetla and San Juan Yuta classify deadly or poisonous mushrooms into three groups: i) \u201cfrog mushrooms\u201d (xi\u2019i la\u2019va) that include Amanita species with a smooth, slimy and wet pileus such as A. verna, A. phalloides and A. virosa; ii) \u201ctoad mushrooms\u201d (xi\u2019i la\u2019va nd\u0268\u2019y\u0268) that include species with remains of volva or flakes (\u201cpimples\u201d) in the pileus, such as Amanita muscaria and Lepiota spp.; and iii) \u201cbad or crazy mushrooms\u201d (xi\u00b4i kue\u00b4e) or \u201cnot edible mushrooms\u201d, which comprise all species not included within the two former groups that have either no known use or no known name. The term \u201ctoad mushrooms\u201d is used to scare people, especially children, to prevent intoxication. People in the communities hold the belief that when someone approaches a toad, the animal expels urine at the observer\u2019s eyes and that this can cause blindness.Knowledge of toxic mushrooms has an ancestral origin in the studied communities, and it is based on local mycetismus derived from the probable consumption of species such as Within the universe of fungi present in the communities under study, some identified species have been reported to contain compounds with pharmacological and nutraceutical potential Table\u00a0.Table 4SN. lepideus presents a phenological pattern of early fruiting; it can be collected in the months from February to April, whereas A. campestris can be found in May and early June with a prolonged early pattern.The timing of the onset of the reproductive phase of mushroom species is related to the rainy period. In general, it starts in February and ends in September. A. sect. caesarea and Ustilago maydis develop, exhibiting a short phenology. In September, prolonged late-maturing species such as C. cibarius s. l. can be found.In the mid-season months of highest precipitation, July and August, species such as H. lactifluorum and H. macrosporus parasitise R. brevipes, providing a leathery, hard consistency and orange or coffee colouring to the fungus, respectively. N. lepideus and S. commune are saprophytic species that, once dried, can be stored without losing their flavour, to be consumed during the months in which no mushroom species develop. P. radicata is a species that develops in the dead roots of the guach\u00e9pil tree (Diphysa robinioides Benth.), whose development is encouraged on cropland because its flowers and young pods are also used as food.Of the 116 mushroom species hitherto recorded in the study areas, 99 93\u00a0%) species are Basidiomycetes and 7 (7\u00a0%) are Ascomycetes. Eigthy species (75\u00a0%) grow on soil/litter substrates, whereas the remaining 25\u00a0% develop on wood or other substrates, 80 species (75\u00a0%) are ectomycorrhizal, 22 species (21\u00a0%) are saprobionts and four are parasites 4\u00a0%) . Some species are found in microhabitats where pine (Pinus spp.) or oaks (Quercus spp.) predominate. The exceptions are H. lactifluorum, which prefers sites with a predominance of \u201cyellow oak\u201d (Quercus magnoliifolia N\u00e9e) and pointleaf manzanita (Arctostaphylos pungens Kunth); species such as A. campestris, A. pampeanus and M. oreades grow in sites with secondary vegetation or pasture; and species such as H. petaloides, P. aff. eryngii, P. aff. dryinus and S. commune develop in the tropical deciduous forest where species of Copal (Bursera spp.), Tepehuaje tree (Lysiloma acapulcensis (Kunth) Benth) and round oak can be found.With respect to the vegetation where fungi grow, most occur in places with pine-oak litter (Sciurus aureogaster) and deer (Odocoileus virginianus oaxacensis), consume and disseminate A. sect. caesarea, Boletus edulis s. l. and Russula mexicana. This can be considered a very advanced ecological notion based on strong observational skills, linked to both fungal mycophagy and subsequent dispersion caused by mammals.Interviewees mentioned that some wild animals, such as squirrels when they can go to the field. The main instruction is in the places where fungal species can be found and the identification of species that are edible, toxic and have ludic uses. At the time of collecting fungi, people cut the stipe, leaving the volva or stipe base. With this, fungal growth the next year is guaranteed. People know with certainty where each species develops and visit the same sites to collect useful wild mushrooms, constituting an initial notion of the growth of \"something\" under the earth that produces sporomes.The main factors that influenced the richness, knowledge and use of edible mushrooms in the four studied communities were: i) the natural vegetation cover, for example in the communities of Santa Catarina Estetla and San Juan Yuta, which have 72 and 50\u00a0% of natural pine and oak forests, respectively, there was a greater richness and therefore, knowledge and use of wild edible fungi compared with the communities of San Miguel Tulancingo and San Andr\u00e9s Yutat\u00edo in which there is no coniferous forest and the oak forest occupies only 40\u00a0% . In this municipality, until recently, agricultural, livestock or harvest products such as wild mushrooms were used as currency in the regional tianguis or \"open air market\". Currently, during the rainy season, species such as A. sect. caesarea, C. cibarius s. l. and H. repandum are sold in the regional market of Zaachila, in the Central Valley and in the \u201cMercado de Abastos\u201d [Food Market] of the city of Oaxaca. These species are collected and kept by farmers who bring their products to market, who originate in the Mixtec communities of San Miguel Peras and Santiago Tlazoyaltepec.There is no information on the commercialization of wild mushrooms in the studied communities. However, it is now common for people who live in the communities centre or other surrounding communities to ask farmers to obtain specific species for purchase; this occurs, for example, in the community of Santiago Huaxolotipac located in the Municipality of San Antonio Huitepec with the species siye\" for fungus first appears in writing. In the ethno-linguistic variants of the communities studied, there is a classification for plants, animals and fungi very similar to that recorded in 1593 in Teposcolula refers to various edible species of Pleurotus or to Hohenbuehelia petaloides; xiti is Ustilago maydis, jih\u00ec naa corresponds to Amanita sect. caesarea; jih\u00ec landia is Lactarius indigo; jih\u00ec leyu refers to various edible species of Agaricus including A. campestris or A. pampeanus; jih\u00ec tak\u00e0 corresponds to edible species of Ramaria including R. botrytis or R. aff. flava; jih\u00ec yaha or ji\u00b4i ya\u00b4a refers to edible species of the genus Russula including R. mexicana or R. aff. cyanoxantha, jih\u00ec k\u00f3hl\u00f3 corresponds to Neolentinus lepideus; and ji\u00b4i t\u0268ndaku refers to Hydnum repandum.Various names for wild mushrooms are registered in the Mixtec-Spanish dictionaries published by the Summer Institute of Linguistics in Mexico without specifying the fungal species involved \u201348. HoweSchizophyllum commune. Some authors have reported the importance of S. commune in the tropical region, indicating a geographical range of consumption and sale extending from the coast of the Gulf of Mexico to the tropical zone of Guatemala. However, the records of its consumption appear to be restricted to the tropical zones with the exception of the town of Huautla de Jim\u00e9nez, Oaxaca, where the climate is rather temperate humid [Neolentinus lepideus it has been consider that only are produced at the beginning of rainy season with the first thunders. The relationship between thunders and sporocarp production has long been considered a \"belief\"; however, it is necessary to take into account the deep mycological knowledge that different cultures have of the ecological relationships in the environments they inhabit. Several experimental studies have shown that electrical shocks have a positive response on sporocarp formation in edible mushrooms such as matsutake (Tricholoma matsutake) [Laccaria laccata [Amanita crocea, A. fulva, A. rubescens, Austroboletus betula, Boletus frostii, Clitocybe gibba, Helvella lacunosa s. l., Helvella crispa s. l., Hypomyces macrosporus, Hygrophorus russula, Laccaria laccata s. l., Lactarius indigo, Lycoperdon spp., Lyophyllum decastes, Macrolepiota procera, Russula brevipes, R. delica, R. aff. cyanoxantha, Sparassis crispa and Suillus collinitus and Lacc laccata . Fifty-s laccata , 61\u201363. us Table\u00a0.Auricularia as a \"small bag\" or \"small balloon\" by separating the membranes and making a small hole [Chak ach (Cookeina sulcipes and C. tricholoma) to hear sounds by blowing into the cup and then putting it on the ear [Ganoderma applanatum for making prints of animals, plants and/or landscapes [Ludic uses occur in other ethnic groups in Mexico, for example: i) the Chinantec people of the state of Oaxaca use all hole ; ii) the the ear ; and iiindscapes . UndoubtCodex Yuta Tnoho , given that the locations were established in the Post classic period (950\u20131520\u00a0A.D.) before the arrival of the Spaniards, when the Mixtec culture reached its peak [Psilocybe for sacred or divination purposes in the Mixtec group contrasts with: i) the great diversity of these species in the state of Oaxaca, which contains 27 of the 53 hallucinogenic species of Psilocybe known in Mexico [Codex Yuta Tnoho, which describes a sacred ceremony in which various Mixtec deities consumed sacred mushrooms prior to the first sunrise [This could be related to rituals of pre-Hispanic heritage mentioned in the its peak . In thatits peak mentionen Mexico , as well sunrise , 15. One sunrise .Amanita crocea and A. rubescens, are included in the \u201cfrog\u201d or \u201ctoad\u201d mushrooms groups and are considered toxic in the region despite being widely consumed in other regions of Mexico [A. muscaria has the erroneous reputation in the study locations of being a very poisonous mushroom; in fact, it only produces mycetismus of the gastrointestinal type [Amanita and to any species to be wary of. In 1953, in a journey through Mayan lands, Wasson [lava that relates to female genitalia, the mention of which causes hilarity or disgust among people.Despite the fact that in general the criteria used by Mixtecs, to distinguish edible and toxic mushrooms are in general accurate, some general principles have limitations given that some edible species, such as f Mexico , 62, 63.nal type , causingnal type . Wasson nal type mentione, Wasson discoverAmanita caesarea (Scop.) Pers. [Albatrellus ovinus (Schaeff.) Kotl. et Pouzar [Boletus edulis Bull. [Cantharellus cibarius Fr. [Helvella lacunosa Afzel [Hydnum repandum L. [Laccaria laccata (Scop.) Cooke [Ramaria botrytis (Pers.) Ricken [Ramaria flava (Schaeff.) Qu\u00e9l. [Hydnum repandum L. [Laccaria laccata (Scop.) Cooke [Lyophyllum decastes (Fr.) Singer [Ramaria flava (Schaeff.) Qu\u00e9l. [Russula cyanoxantha (Schaeff.) Fr. [Cantharellus cibarius Fr. [Russula cyanoxantha (Schaeff.) Fr. [Ramaria botrytis (Pers.) Ricken [Ramaria flava (Schaeff.) Qu\u00e9l. [Hygrophorus russula (Schaeff.) Kauffman [Today, mushrooms are valued for their nutritional value, which is attributed to their high levels of protein, fibre, carbohydrates, vitamins and minerals and low levels of fat. Some species have also been used to increase human longevity and quality of life via their medicinal and nutraceutical properties . Within .) Pers. , Albatret Pouzar , Boletusis Bull. , Cantharrius Fr. , Helvellsa Afzel , Hydnum andum L. , Laccari.) Cooke , Ramaria) Ricken y Ramari.) Qu\u00e9l. . In otheandum L. , Laccari.) Cooke , Lyophyl) Singer , Ramaria.) Qu\u00e9l. y Russulff.) Fr. , antitumrius Fr. and Russff.) Fr. ; antibac) Ricken and Rama.) Qu\u00e9l. and antiKauffman .Dasyprocta mexicana) [Peromyscus alstoni, Reithrodontomys megalotis and Microtus mexicanus) [Neotomodon alstoni, Peromyscus maniculatus and P. alstoni) consume fungi such as Laccaria trichodermophora, Suillus tomentosus and Russula cuprea and found that spore viability is not affected by consumption, suggesting that these may be effective dispersers of spores of fungal species.Mixtec people know that some wild animals consume and disseminate some edible mushrooms. Previously, the consumption of some species of hypogeal fungi by animals such as the agouti (exicana) and wildxicanus) has beenxicanus) showed tA preservation of tradicional knowledge among Mixtecs was recorded in the studied communities. Caballero mentioneCantharellus cibarius s. l. and Boletus edulis s. l., which have estimated annual values according to the retail market of $1.67 billion and more than $250 million U.S. dollars, respectively. Additionally, Amanita sect. caesarea also has an international market. The main countries that demand these species are Canada, France, Italy, Spain, USA, China and Germany [In the community under study there are species with potential to be exploited in a sustainable way as a non-timber forest product for export to international markets, which could be an alternative for the conservation of mycological knowledge and resources. International trade in wild mushrooms is valued at billions of dollars annually. One reason for this high cost is that most species cannot be cultivated and are Germany , 92.Sustainable management of wild mushrooms among the Mixtecs would be enhanced by implementing the following strategies: i) transmission of technologies to local people in order to give an added value to the commercialization of mushrooms in the region, by methods such as dehydration, preparation of brines and vinaigrettes; ii) dissemination of existing knowledge of mushrooms growing in local communities with value in international markets, which can be an important source of economic resources locally; iii) promotion of use of species that are not consumed in the region and have edible potential; iv) development of mycotourism involving the local population, including activities such as mycological tours and mycogastronomy; v) biotechnological applications of mycological resources, including cultivation of saprophytic species of biocultural importance regionally, inoculation of edible ectomycorrhizal fungi in native tree species tending to reforestation of degraded areas and development of mycosilviculture including forest management practices tending to increase natural production of wild mushrooms, mainly ectomycorrhizal species; and vi) upgrading and strengthening of ecological, cultural and socioeconomic importance of mycological resources, through activities such as fairs, exhibitions and local culinary samples, with the participation of members of the Mixtec communities, local and national government and non-governmental organizations. Some of these strategies have been successfully developed and applied in several European and Asian regions, contributing to forest conservation and sustainable management of mycological resources and natural ecosystems \u201395.This ethnomycological study is the first to focus on the Mixtec group, which is the third-largest in Mexico after the Nahua and the Maya.Traditional knowledge of fungi in the communities of Santa Catarina Estetla and San Juan Yuta is of high accuracy from the western taxonomic and ecological perspective. Currently there is an important preservation and oral transmission of mycological knowledge to new generations of Mixtec people particularly in these later communities. The inhabitants of these localities can distinguish and name the parts of the fungi with high precision; group and assign one or more common names to edible or poisonous mushrooms; and pinpoint exactly the habitat and phenology of the species they use.Differencial environmental, soeconomic and cultural factors among the four studied communities affect the richness, knowledge and use of edible wild mushrooms.Despite the strong existing acculturation processes and migration in the region, the ability to recover local, traditional ethnomycological knowledge is dynamic and survives among the Mixtec peoples of Santa Catarina Estetla and San Juan Yuta. The mechanisms through which traditional mycological knowledge is maintained and adapted include manteinance of cultural identity, forest protection, preservation native language and also paradoxically through the current socieconomical marginality among the Mixtec people. In this framework, the use and sustainable management of wild mushrooms could be an alternative for local integrated development, but only if the Mixtec worldview is incorporated into regional programs."} +{"text": "Fungi have multiple uses in temperate areas of M\u00e9xico, but an important decrease in the traditional knowledge of uses and customs of mushrooms becomes a fundamental issue for fungi conservation. However, only few studies quantify the traditional ethnomycological knowledge in M\u00e9xico, and this study is the first quantitative report for Quer\u00e9taro, a central state with both Otom\u00ed and Mestizo communities and a high fungi diversity.H\u00f1\u00e4h\u00f1u (Otom\u00ed) communities in Amealco de Bonfil, Quer\u00e9taro, M\u00e9xico, between August 2013 and November 2014. We conducted a stratified sampling, where uses common H\u00f1\u00e4h\u00f1u and Spanish names, and eight quantitative variables that conform the \u201cEdible Mushrooms Cultural Significant Index\u201d (EMCI) were recorded from 100 informants. For the classification and ordination analysis of species and uses, we used multivariate techniques such as cluster, multidimensional scaling, and principal components (PC).The present study was conducted registering traditional knowledge on the use and consumption of mushrooms in three Amanita basii, Fistulinella wolfeana, and Lactarius indigo. Edibility was the main use detected in the survey, and people harvested mushrooms provided by the forest mainly during the rainy season. We observed that mushroom searching and collection are activities that strengthen the family ties and are crucial for the transfer of this knowledge through generations. Cluster analysis separates groups according to different values in EMCSI variables, and principal components ordinate the species by frequencies (PC1) and traditions (PC2).Thirty-three mushrooms species were registered, most of them used for consumption by households, few aimed for commercial purposes, one species is medicinal, another has veterinary, and other ludic uses (as a toy). The three species with the highest EMCSI were The current state of knowledge in the studied communities is strong, especially among women, but with a tendency to disappear due to migration and lack of interest among new generations. Future quantitative studies are important to analyze tendencies of the traditional ethnomycological knowledge transferred to new generations. The first ethnomycological studies in M\u00e9xico were conducted with emphasis on mushrooms used in rituals, but indigenous knowledge goes beyond this use, such as species used for food, medicine, and recreation \u20134.With the arrival of the Spaniards, many of these uses and customs were banned, because these traditions were considered as inappropriate . RespondData from different sources shows that indigenous people collect fungi mainly in the rainy seasons for direct consumption by households, while mestizo people in M\u00e9xico use them to obtain monetary benefits by selling them in municipal markets or houseTraditional knowledge on the recognition of edible wild mushrooms is very important for the implementation of adequate management strategies and for the transfer of the mycological knowledge to the new generations. Therefore, inadequate practice or ignorance about such species can lead to the use of toxic fungi and then health consequences such as irreversible damage of the vital organs, such as liver and kidneys .The traditional knowledge on forest resources can be measured based on how many times it is mentioned by people , 16\u201318 oH\u00f1\u00e4h\u00f1u communities have been carried out mainly in two federal estates \u201cEstado de M\u00e9xico\u201d and \u201cHidalgo\u201d [H\u00f1\u00e4h\u00f1u settlements in other states such as Guanajuato, Jalisco, Michoac\u00e1n, Puebla, Quer\u00e9taro, Tlaxcala, and Veracruz [H\u00f1\u00e4h\u00f1u population settlements, with important interactions with the H\u00f1\u00e4h\u00f1u from the neighbor state \u201cEstado de M\u00e9xico\u201d where N\u00fa\u00f1ez-L\u00f3pez [Mycological studies in M\u00e9xico with Hidalgo\u201d , 24\u201329. Veracruz . The staez-L\u00f3pez reportedez-L\u00f3pez .H\u00f1\u00e4h\u00f1u, such as the recognition of mushrooms as a biological group, the recognition of different species and its edibility, medicinal or dangerous properties, and finally how and who are the responsible for the transference of mycological knowledge to the next generations, using interviews and participant observation. A technique that helps us to register and quantify different assets of the traditional knowledge is the Cultural Significant Index because it provides an objective numerical scale, eliminating the subjectivity of giving arbitrary values to each variable. This scale was initially proposed by Pieroni et al. [The aim of this study is to answer some fundamental questions about the real knowledge of fungi by the i et al. as the \u201ci et al. that comH\u00f1\u00e4h\u00f1u communities in Quer\u00e9taro, M\u00e9xico, and (b) to make the first quantitative analysis of ethnomycological knowledge in Quer\u00e9taro, using the \u201cCultural Food Significant Index.\u201dThe two main objectives of this study are (a) to conduct the first ethnomycological study in three Arbutus (\u201cmadro\u00f1o\u201d), Arctostaphylos (\u201cping\u00fcica\u201d), and pine [Amealco is one of the 18 municipalities in the state of Quer\u00e9taro, located southeast of the capital, between the coordinates 20\u00b0 11\u2032 17\u2033 N and 100\u00b0 8\u2032 38\u2033 W, with an altitude between 2500 and 3150\u00a0m above sea level , and witand pine , 35.H\u00f1\u00e4h\u00f1u speaking Otom\u00ed in M\u00e9xico: 18,933 inhabitants, of whom 11,740 (62%) live in Amealco [Quer\u00e9taro has the third biggest community Amealco . The thr Amealco , and dueTo determine the used species of mushrooms, we conducted a stratified sampling . The peoThe Garibay-Orijel et al. methodolQI, the quotation or citation index is a sub-index that relates the number of times a fungus is mentioned, divided between the number of informants, and by tenfold .PAI, the perceived abundance index, is the abundance of each species of mushroom that people can perceive during the rainy season, how many of these species they can find, and how many they can collect.FUI, Frequency of Use Index, is how often people consume each species of mushrooms during the rainy season.TSAI, taste score appreciation index, using the scale to evaluate the taste of mushroom species consumed by people.MFFI, multifunctional food index, to evaluate how people cook mushrooms and whether they eat them alone or combined.KTI, knowledge transmission index, to determine how knowledge is transmitted or how people learn how to use the mushrooms.HI, Health Index, indicates how safe people feel to consume these mushrooms, and how beneficial is for their health.EI, Economic Index, indicates if people sell mushroomsWhere:For the classification analysis of species (rows) and sub-indexes (columns) in a multivariate matrix, a cluster analysis was conducted with standardized Euclidean distances (SED) and the Ward clustering method using JMP 8 for Mac . For the ordination methods (MDS and PCA), a matrix containing the 33 species mentioned in the interviews and the averages of each sub-index were constructed and analyzed with PC-ORD 6.08 methods.Quercus spp. and Pinus spp. according to the methods proposed by Guzm\u00e1n [Specimens were collected from April 2013 to November 2014. The sampling was conducted in forests dominated by y Guzm\u00e1n . Photogry Guzm\u00e1n \u201347 and sPeople from the three communities still conserve the tradition of collecting mushrooms, mostly for self-consumption and some for sale, depending on the abundance. We recorded 155 Spanish common names and 21 Otom\u00ed common names Table\u00a0, mainly Amanita basii Guzm\u00e1n & Ram.-Guill., Lactarius indigo (Schwein.) Fr. and Fistulinella wolfeana Singer & J. Garc\u00eda, which was the first record of edibility [A. basii is the most important mushroom in San Mateo Huexoyucan, Tlaxcala, M\u00e9xico, and Montoya et al. [Ramaria Fr. ex Bonord is considered the most important to people of La Laguna de F\u00faquene, Andes Nororientales [Helvella crispa (Scop.) Fr. was mentioned to be consumed by only one person. Lyophyllum P. Karst had few mentions, which might be because only few people know where to find it [Citation Index (QI). dibility , were thdibility where A.a et al. found itientales . Here, H find it Fig.\u00a01)Amanita bLactarius indigo, Russula aff. brevipes Peck, R. aff. cyanoxantha (Schaeff.) Fr., and Fistulinella wolfeana had the highest values, while Amanita novinupta Tulloss & J.E. Lindgren, Boletus auripes Peck, Gymnopus dryophilus (Bull.) Murrill, Helvella crispa, and Suillus granulatus (L.) Roussel had the lowest values. It is important to point out that Russula and Agaricus campestris L. were the most abundant according to with Pe\u00f1a-Ca\u00f1\u00f3n & Enao-Mej\u00eda [A. campestris is the most abundant for Alonso-Aguilar et al. [Russula spp. or S. granulatus is common in the forest, people preferred to collect other mushrooms because they do not consider these palatable. Sometimes, when Ramaria spp. is not common, people prefer to walk long distances to find it. In this case, as mentioned by Bautista [A. basii are more difficult to find due to the condition of forests. Some informants mentioned that the number of mushrooms they could find is decreasing, arguing that the abundance and uses were higher in the past.perceived abundance index (PAI). ao-Mej\u00eda , while Ar et al. ; this reBautista , some spAmanita basii, Boletus spp., Fistulinella wolfeana, Lactarius indigo, Ramaria spp., and Ustilago maydis (DC.) Corda are consumed four times, or more, during the rainy season, and sometimes even four times a week, depending on their abundance. Pe\u00f1a-Ca\u00f1\u00f3n and Enao-Mej\u00eda [Russula sp. and A. basii, and Garibay-Orijel et al. [Cantharellus Juss and Pleurotus (Fr.) P. Kumm. are the most abundant mushrooms in Ixtl\u00e1n de Ju\u00e1rez, Oaxaca. Hypomyces lactifluorum (Schwein.) Tul. & Tul. has a minor index, probably because people have to scrape the ground to find it. Less-used mushrooms were Helvella crispa, Gymnopus dryophilus, and Lycoperdon marginatum Vittad.; the first is due to its low abundance, the second because only some people consider it as non-edible, and the third is due to the need of finding a considerable number of individual mushrooms to prepare a hearty meal. It is important to note that the frequency of use of determined species is determined by the access and the amount available.Frequency of Use Index (FUI). ao-Mej\u00eda and Alonao-Mej\u00eda mentionel et al. said thaAmanita basii was the most palatable species according to many informants as we could see with Alonso-Aguilar et al. [Ramaria were the most appreciated according to Pe\u00f1a-Ca\u00f1\u00f3n and Enao-Mej\u00eda [Gomphus clavatus (Pers.) Gray had the highest value of the overall species reported by Garibay-Orijel et al. [Harrya chromapes (Frost) Halling, Russula aff. lepida Fr., Suillus granulatus, and Xerocomus illudens (Peck) Singer, mainly because some Russula species have a spicy flavor, and some have a sour taste, such as the case of these boletes.Taste score appreciation index (TSAI). In general, all mushrooms were well accepted by people who consume them; r et al. . In contao-Mej\u00eda , and Goml et al. . Only feAmanita basii, Ustilago maydis, Boletus auripes Peck, and Hypomyces lactifluorum for being very palatable. Fistulinella wolfeana and Lactarius indigo can only be eaten roasted and have fewer larvae inside. Some people like to mix mushrooms, and the most frequent combination include Cantharellus cibarius Fr., Boletus variipes Peck, and Lactarius indigo, referring this as a very delicious combination. Calvatia cyathiformis (Bosc) Morgan and Agaricus campestris could also be consumed mixed together because they have a meat-like taste. Lycoperdon perlatum Pers. and L. marginatum were also consumed alone, as U. maydis in \u201cquesadillas,\u201d but the latter could be eaten mixed with squash blossoms. Ramaria spp. could be prepared alone too, and in many cases with eggs. Some mushrooms can be preserved for long periods, such as L. indigo and F. wolfeana, which are dried on rocks in the sun, or hanged on a chain, as reported by Estrada-Torres and Aroche [Ramaria spp. is shredded into fine strands, and Hypomyces lacrifluorum is cut into thin slices and left in the sun, sometimes preserved frozen for up to 3\u00a0months. Our results on the MFFI are consistent with those of Garibay-Orijel et al. [Multifunctional food index (MFFI). The main form of mushroom consumption was by roasting, only by cooking on a griddle , with a little salt. Many people cook them with green sauce or \u201cpasilla\u201d chili sauce. The only mushrooms consumed alone were d Aroche . Ramarial et al. , Alonso-l et al. , and Pe\u00f1l et al. where weExudoporus frostii (J.L. Rusell) Vizzini, Simonini & Gelardi or Boletus cf. speciosus Frost, were considered poisonous by some informants, because they have blue spots (like other boletes that change color); however, a woman (and her family) who learned that E. frostii is edible, has shared that knowledge with their neighbors, situation previously observed by Garibay-Orijel et al. [Knowledge transmission index (KTI). Perhaps, this is the main index of the survey because it allowed to detect how traditional knowledge has been transferred from one generation to another. M\u00e9ndez et al. and Garil et al. and Pe\u00f1al et al. too. Forl et al. mentionel et al. .Russula species, which do not have a spicy taste, or Ramaria that have a mild flavor. Regarding the names of the mushrooms, most of them are similar to those reported elsewhere and are consistent with recent studies in Otom\u00ed areas [Mushroom collection is commonly a family activity, in which people share and strengthen their family ties, and it is where children (from 5 to 10\u00a0years old) learn how to recognize edible and inedible mushrooms, and where mushrooms grow (main collection points). People have their techniques to recognize edible specimens from inedible ones, but it has been mentioned that some inedible species were similar to edible mushrooms. People commonly collect only red m\u00ed areas \u201329.Russula aff. lepida and Suillus granulatus were consumed with caution. The \u201cskin hat\u201d is commonly removed because it is considered as the main factor causing diarrhea or stomach pain. Xecomus illudens and Harrya chromapes are reported to have a sour taste but without consequences. Besides, only some people mentioned feeling healthier by eating mushrooms in general [Health Index (HI). People indicate that when they eat mushrooms, they cannot drink milk or alcohol, eat avocado, or consume medicine because they have side effects. Only general , and not general reportedAmanita basii has the highest price ($60\u2013$80 Mexican pesos per four basidiomata), but only a few people sell it. A 15\u00a0l bucket full of Fistulinella wolfeana was priced at $35. Other species, such as Lactarius indigo, Ramaria spp., Leccinum aff. aurantiacum (Bull.) Gray, Hypomyces lactifluorum, Boletus variipes, B. auripes, Agaricus campestris, Calvatia cyathiformis, Cantharellus cibarius, and Armillaria mellea (Vahl) P. Kumm. have a lower value, between $15 and $50 for about ten basidiomata. Calvatia cyathiformis is priced at $70, and A. campestris costs $50, but this can only be collected on plains at the beginning of the rainy season. To compare prices, at Acambay market (State of M\u00e9xico), fungi prices range from $40 to $80 per 200 to 400\u00a0g for species like B. variipes and A. caesarea (Scop.) Pers. complex, while in the municipal market of Amealco (Quer\u00e9taro), a plate of mushrooms, which contains about 100\u00a0g of C. cibarius and Ramaria spp. has a price of about $20 or 200\u00a0g for $35. Many respondents either have a store, work as laborers, have a piece of land to grow corn, or have relatives who send them money to help cover expenses, thus collecting mushrooms is not their main economic source. Only some people trade mushrooms for basic supplies like corn seeds, oil, beans, rise, etc. Until now, there is no consensus about this sub-index because it is in function of almost all the sub-indexes that compound the EMCSI and could be disregarded [Economic Index (EI). regarded . In thisregarded , people L. indigo (63.24), A. basii (61.95), and F. wolfeana (61.23). It is probably because L. indigo and F. wolfeana are available after the rainy season, which gives them a high value, even over A. basii. Some people consume them for up to 1\u00a0year after they have been collected; they put dry basidiomata in hot water and then cook them. This contrasts with QI and PAI values, where A. basii had a higher value in the QI and a lower value in the PAI, compared to L. indigo and F. wolfeana. In many cases, before we start talking about mushrooms, people mention A. basii immediately, so we might consider these three species with similar ethnomycological importance values, and this matches with the results of other studies [A. caesarea complex are the most priced mushrooms, although it is possible to find other more valuable species in other places [The information obtained in the three communities and the value of the indexes and total EMCSI are shown in Table\u00a0 studies , 23, wher places , 16, 28.Ustilago maydis as a remedy for burns and to combat vomiting. Mature basidiocarps are used by placing them directly on burn injuries, where informants argue a faster healing. This coincides with reports from other cultures that use U. maydis as a remedy for burns [Edibility was the main use found for mushrooms. Almost all respondents mentioned that they consume fungi, only three people said they quitted eating them or using them for reasons discussed below. Regarding medicinal use, only three people mentioned or burns . Other iE. frostii to control diabetes. Basidiomes of Leccinum spp., which are not consumed, are used by some people as toys during harvest time, throwing these ones to others as a game. Mushrooms that they do not consume are considered poisonous, even if others eat them. A recent record of mushroom poisoning occurred in a community near Amealco, where four people confused the Omphalotus mexicanus Guzm\u00e1n & V. Mora with a Lactarius indigo, and the symptoms consisted mainly of vomit and diarrhea; they were treated at the health center only with activated carbon and antihistamines, and people mentioned that one woman and her daughter died about 10\u00a0years ago due to the consumption of poisonous mushrooms; however, they do not know which species. According to Pe\u00f1a-Ca\u00f1\u00f3n and Enao-Mej\u00edar [Some informants mentioned the use of o-Mej\u00edar , people Russula spp. which is called \u201cHongo de Borrego (a)\u201d (lamb mushroom), because when people take their animals out to the pasture, lambs eat these mushrooms. C. cibarius is called \u201cPeric\u00f3n\u201d or \u201cSanta Mar\u00eda\u201d due to the similar yellow color with Tagetes lucida Cav. Other example included L. indigo, which has names related with its bright blue color \u201cpant mushroom,\u201d because the blue color is a reminder of a pair of blue jeans. As mentioned, people only named mainly edible mushrooms and considered the remaining non-used as venomous. It is interesting how they recognize the good ones, and this knowledge is transmitted generationally mainly by women, being the principal characteristics color, smell, maturity, and in some cases, the taste (they avoid spicy and bitter flavors). Alonso-Aguilar et al. [A. basii, among other mushrooms, is the most appreciated species. In the present work, we found F. wolfeana, L. indigo, Ramaria spp., and B. variipes (species close to B. pinophilus Pil\u00e1t & Dermek) as having the highest values of EMCSI, and the species had highest sub-indexes values. These values are in function of the QI sub-index because not all the interviewed people mentioned other species with significant abundances, such as Russula aff. brevipes, E. frostii, Lyophyllum sp., G. dryophilus, A. novinupta, or H. crispa, which were mentioned by less than 20% of the interviewed people. In this case, those positions in the EMCSI value are relative to the mentioned above and could be modified along the time by the factors mentioned in KTI sub-index discussion.People harvest mushrooms provided by the forest as part of the natural resources available during the rainy season. On the other hand, many of them used to grow vegetables a couple of months before the rainy season began, so they could get benefits from mushroom collection, by adding diversity and enrichment of the daily diet and helping them to save money. Mushroom recollection was restricted mainly to August and September, the months where we conducted the ethnographic work and where we found most of the species. All mushrooms have a utilitarian category . We suspr et al. found A.Finally, we have noted that some people begin to get upset with the intervention of people who do not belong to their community; for this reason, it is important to consider as necessary a previous diagnose, and then get previous consent, considering and giving all the people the opportunity to participate and being interviewed, as observed by Garibay-Orijel et al. , and accL. indigo, Ramaria spp., F. wolfeana, A. basii, B. variipes, B. cf. speciosus, C. cyathiformis, U. maydis, C. cibarius, L. aff. aurantiacum, H. lactifluorum, A. mellea, and Clitocybe gibba (Pers.) P. Kumm., and includes B. auripes and C. cibarius. U. maydis has no EI value but has a high TSAI value because it is one of the most palatable mushrooms. In this group, the closer species were B. cf. speciosus and C. cyathiformis (with a linkage SED\u2009=\u20090.65). The second group (B) is formed by Retiboletus aff. griseus (Frost) Manfr., Leccinum sp., H. chromapes, X. illudens, Leccinum rugosiceps (Peck) Singer, A. novinupta, S. granulatus, R. aff. lepida, Boletus sp., R. aff. brevipes, and E. frostii, species that show medium to low QI values; and the third group (C) is composed of R. aff. cyanoxantha, L. marginatum, L. perlatum, Nolanea (Fr.) P. Kumm., Lyophyllum sp., G. dryophilus, and H. crispa, the last is the one with the lowest EMCSI value. This group had a linkage SED of 3.96.The dendrogram of similarities Fig.\u00a0 with theP\u2009=\u20090.0196). According to the cluster shown in Fig.\u00a0i is directly related to the abundance of spi, and this is influenced by the number of mentions of spi, as we can see in PCA. The last components, which conform PC3, have both positive and negative values, which makes sense due to only a few species that cause stomach ache, such as Suillus spp. or red spicy cap Russula spp., but these species are considerably abundant.An analysis of the stress in the MDS suggested a 3D configuration language. On the other hand, people refer that forest does not have the same shape they remember, and many of them mentioned that the amount of mushrooms has decreased because of environmental degradation and deforestation.Alonso et al. reported species , 22, 23.Bautista refers tU. maydis as a veterinary medicine could open the possibility for research into animal welfare products. The uses of quantitative methods such as the Edible Mushrooms Cultural Significant Index (EMCSI) prove an easy and important method for future studies. The socio-politic conditions have been changing, and the economy has shown modifications over the last years. Therefore, we consider that future studies must be conducted using compatible methods in order to compare future results and then analyze the tendencies of traditional knowledge over time.The current state of knowledge in the studied communities is strong but with a tendency to disappear due to migration and less interest among new generations. Mushroom consumption is part of integral and seasonal cultural knowledge of the resources in the Otom\u00ed communities at Amealco municipality in Quer\u00e9taro, M\u00e9xico. Mainly women transmit traditional mushroom knowledge. Food, medicine, and venom were the three principal categories we identified, and only few people use them for trade or sale within the same communities. The use of"} +{"text": "The aim of this study was to use three-dimensional datasets to identify associations between treatment for adult crowding, using Invisalign aligner and interproximal enamel reduction (IER), and changes in the volume of interradicular bone.A total of 60 cone-beam computed tomography (CBCT) scans from 30 adult patients were examined retrospectively in order to measure bone volume three-dimensionally. The patients\u2019 average age was 36.03\u2009\u00b1\u20099.7\u00a0years. The interradicular bone volume was measured with OsiriX at four levels in the anterior tooth areas of the maxilla and mandible. Differences in bone between T0 and T1 were analyzed with IBM SPSS 21.0 using the Wilcoxon test for paired samples.P\u2009<\u2009\u00a00.001), while in the maxilla there was a slight loss of bone, which was highly significant in the apical third .Overall, a slight increase in the quantity of bone was found (0.12\u2009\u00b1\u20090.73\u00a0mm). There was a highly significant increase in bone in the mandible . This effect is apparently independent of IER. This is extremely important with regard to the treatment outcome, since IER and root proximity have been matters of debate in the literature and teeth should remain firmly embedded in their alveolar sockets. Among adult patients there is growing interest in having a functionally healthy and aesthetically attractive dentition . PatientOne possible treatment for relieving crowding consists of expanding the dental arch in the labial direction in order to provide space for normal positioning of the affected teeth. Another method of creating space is IER. Potential periodontal changes in the anterior tooth area during orthodontic treatment with IER for adult crowding have been a topic of discussion in the literature \u20136. In adHow is the interradicular bone volume altered by aligner therapy?What effects on interradicular root distances are associated with interproximal enamel reduction (IER)?In what ways does the interradicular bone volume change after initial findings corresponding to a root proximity ?There have to date been no three-dimensional investigations of changes in interradicular bone volume in relation to treatment for adult crowding. The present study was carried out in order to investigate whether orthodontic treatment and resolution of crowding may even lead to an improvement in the bone situation. As a result of the use of conventional two-dimensional imaging to date, only limited quantification of the pre-therapeutic and post-therapeutic interradicular bone situation has been possible . It is oFor periodontal assessment . These patients had a fragile gingival type, with less bone in the anterior tooth area. Pre-treatment CBCT was therefore intended to visualize root proximity and resorption and help with therapeutic decision-making on whether to carry out IER or extraction of one anterior tooth. The following CBCT was intended to visualize root proximity and resorption after the completion of possible treatment, to check whether IER was still appropriate for creating space, since the teeth need to be covered by bone in order to avoid recession.For temporomandibular joint assessment . Some patients had craniomandibular disorders (CMD) with rheumatoid arthritis. In this small number of selected cases, pre-treatment CBCT was used to identify bone degenerative deformity, condylar positions, and bony structures in the temporomandibular joint. After successful CMD therapy and orthodontic treatment, CBCT scans were taken again to view the condylar positions and bony structures in the temporomandibular joint due to recurrent CMD problems and in order to adjust anti-inflammatory therapy.Changes in the interradicular distance were measured at a total of 720 measurement points in the present study. Pre-therapeutic and post-therapeutic cone-beam computed tomography (CBCT) scans from a total of 30 patients were examined retrospectively. In accordance with the SEDENTEXCT guidelines, the CBCT scans were taken for two reasons:The patients\u2019 average age was 36.03\u2009\u00b1\u20099.7\u00a0years. The use of the data was approved by the ethics committee of Marburg University Hospital (ref. no. 34/15).Presence of adult crowding capable of being adjusted using conservative orthodontic space-gaining measures such as protrusion, proclination, expansion and IERPermanent dentitionSuccessfully completed treatment with Invisalign alignersAvailability of one CBCT each from before and after treatmentThe following inclusion and exclusion criteria were applied. Inclusion criteria:Extraction of anterior teeth during the course of treatmentmacrodontia/ hypoplasiaabnormal change in tooth morphologyProsthetic treatmentSkeletal anomaliesGeneral medical findings relevant to bone metabolism Periodontal disease and previous periodontal surgery proceduresThe following parameters represented exclusion criteria:All of the images were taken with a KaVo 3D eXam DVT system using a scan with 360\u00b0 revolution, a duration of 26.9\u00a0s and a voxel size of 0.25\u00a0mm. The datasets were collected and evaluated using OsiriX with an Apple OS X operating system.All of the patients had provided written consent to the use of their data in the study (in accordance with the Helsinki Declaration). The data were all analyzed on a semi-blinded basis.Measurements of the mesiodistal interradicular distance Fig.\u00a0 were modThe shorter of the roots in the two teeth adjoining the interradicular space was set as the reference tooth. A connecting line was drawn in the sagittal view from the buccal enamel\u2013cement boundary (ECB) to the palatal or lingual enamel\u2013cement boundary Fig.\u00a0. From thThe interradicular distance was measured in the axial view. For this purpose, two auxiliary lines were drawn parallel to the sagittal plane and were shifted in parallel as far as the root surfaces of the neighboring teeth Fig.\u00a0. In the Contemporary Orthodontics. St Louis, Mo: Mosby; 2000:224.Whether and to what extent IER or expanding the dental arch in the labial direction influenced the interradicular space was investigated using ClinCheck software with the exact IER protocol. For each interradicular space we analyzed the change of the distance between the roots in comparison to the amount of IER. The total amount of IER was between 0.0\u00a0mm and 0.5\u00a0mm. In our study crowding was defined as the difference in millimeter between the arch perimeter and the mesial to distal tooth size total form S1-S3 (upper anterior jaw) and S4-S6 (lower anterior jaw) Fig.\u00a0. MaxillaAll of the digital volume tomograms were analyzed a second time by the same investigator (N.S.) one month later, to allow assessment of the reproducibility of the measurements. The means of the two evaluations were used for statistical analysis. All patients were treated by one operator (T.D.) in the same office.P\u2009=\u20090.05.Statistical analyses were carried out using IBM SPSS for Mac, version 21.0 . The intraoperator correlation for each examination was initially calculated. For further analysis, the normal distribution of the values was checked. The values were tested for significant differences using the Wilcoxon test. The significance level was set at P\u2009<\u2009\u00a00.001, two-sided) intraoperator correlation (r\u2009=\u20090.837) for the interradicular distance measurements.Kendall\u2019s tau-b test showed a highly significant (P\u2009\u2264\u20090.001) changes between T0 and T1 at all levels in the mandible, highly significant (P\u2009\u2264\u20090.01) changes at the apex measurement level, and significant (P\u2009\u2264\u20090.05) changes at the three-quarter level in the maxilla benefited more from aligner treatment than a periodontally healthy dentition (>\u20090.8\u00a0mm). Of the 124 measurement points that had a root distance \u22640.8\u00a0mm in the initial findings, 88.71% had increased space after treatment. An interradicular space increase of more than 0.8\u00a0mm was even observed in 71.77% (Table\u00a0P\u2009\u2264\u20090.001).By comparison, periodontally high-risk dentitions showed much larger increases in the interradicular bone volume Table\u00a0. As TablIn the group of patients investigated in the present study, treatment for adult crowding was associated with an overall increase in interradicular space. The increased space was gained particularly in the mandible, as the increase was larger than the slight loss of space in the maxilla. One possible explanation for this might be the varying severity of the crowding. In this group of patients, adult crowding usually appeared earlier and with greater severity in the mandible than in the maxilla. More extensive measures to create space were therefore needed with a smaller bone volume. Another explanation might be the different methods used to create space.One possible treatment for relieving crowding consists of expanding the dental arch in the labial direction in order to allow space for normal positioning of these teeth . This meAnother method of creating space is IER . AlthougDuring treatment for adult crowding, interproximal enamel reduction (IER) was only carried out supportively in a few interdental spaces. When only the subtopic of IER is considered, it is notable that IER did not have any significant effect on the bone volume between the anterior dental roots. The distribution pattern of changes in the interradicular distance was almost identical with and without IER.In general, the advantage of interproximal enamel reduction is that the extent of the expansion in the labial direction can be reduced, thereby reducing the risk of bone dehiscence occurring. In addition, the widened approximal contacts stabilize the treatment result . These fThe most noticeable positive effect was seen when teeth with root proximity were treated. Vermylen et al. defined Another advantage of the present study is the three-dimensional imaging of the interradicular spaces. Other studies on measurement of bone volume have only been carried out using two-dimensional images, and have always noted the difficulty of depicting the interradicular distance precisely . Two-dimUnfortunaly this study has a retrospective design with a risk of bias. A prospective randomized controlled trial would be interesting, but could currently not be carried out because of the ALARA principle. In view of the principle that radiation exposure should be \u201cas low as reasonably achievable\u201d (ALARA), CBCT is not indicated as a routine method for the imaging of bone support , 25. A COverall, treatment of adult crowding using Invisalign and IER, particularly in patients with severe conditions , appears to have a positive effect on the interradicular bone volume, at least in adult female patients. The effect is also apparently independent of IER."} +{"text": "The paper proposes a sensors platform to control a barrier that is installed for vehicles entrance. This platform is automatized by image-based license plate recognition of the vehicle. However, in situations where standardized license plates are not used, such image-based recognition becomes non-trivial and challenging due to the variations in license plate background, fonts and deformations. The proposed method first detects the approaching vehicle via ultrasonic sensors and, at the same time, captures its image via a camera installed along with the barrier. From this image, the license plate is automatically extracted and further processed to segment the license plate characters. Finally, these characters are recognized with the help of a standard optical character recognition (OCR) pipeline. The evaluation of the proposed system shows an accuracy of 98% for license plates extraction, 96% for character segmentation and 93% for character recognition. The security sensitive areas of a country, such as classified defense areas, government buildings and military installations, are under constant surveillance to avoid potential threats. Such surveillance also extends to the vehicles that constantly access these areas. A vast majority of the currently installed systems use barrier gates that are either manually operated or use vAn automatic license plate recognition (ALPR) system is instrumental in identifying a vehicle from the image of its LP. As a common rule in various parts of the world, the government issues LPs with fixed aspect ratios, fonts and backgrounds. However, arbitrarily designed LPs is an ever growing problem in countries like Pakistan, where the ALPR becomes a challenging task for a few reasons. First, the position of an LP is not fixed on the front side of the vehicle. Second, there exists a huge variation in the aspect ratios of the LPs. Third, the backgrounds of the LPs vary from on to the other. Finally, variations are also found due to non-uniform font styles and font sizes. Some of these variations are depicted in Image capturing device acquires an image or extract an image from a videoLocalization and extraction of license plate in the acquired imageCharacter segmentation and recognition OCR in the extracted LPA typical ALPR system mainly consists of the following steps ,4:ImageDevelopment of a prototype for barrier access control and then deploying it in a real world scenario.An image dataset of challenging number plates commonly used in Pakistan with variations in background, position on vehicle, fonts and font styles.Development of an algorithm for character extraction and segmentation of LPs having different background, position on vehicle, fonts and font styles.An extensive performance evaluation of classifiers for optical character recognition.A performance evaluation of the proposed system on two different hardware environments to select the one which is favorable for real-time application.The ALPR process begins with LP localization and extraction from the vehicle image. LP localization techniques extract the rectangular bounding box or the text regions directly from the image . WithoutThe rest of this paper is structured as follows: In this section, we briefly introduce the related work about LP Localization in vehicle images, characters segmentation and characters recognition.In an ALPR system, the starting step is LP detection and extraction. If the LP is not properly extracted, then the LP segmentation will be severely affected . As a coCharacter segmentation divides the LP into individual characters and digits. Character segmentation becomes challenging due to multi-color background and foreground of an LP. Tabrizi et al. proposedOne of the main components of ALPR is the automatic recognition of characters. Chen et al. proposedR engine with modR engine for LP cR engine proposedR engine comparedR engine proposedR engine proposedVehicle arrival detection and image acquisitionImage pre-processing and edge image generationImage segmentation based on detected edgesLP extraction via the count of connected componentsCharacter segmentation and features calculationOptical character recognitionVehicle authorization and barrier control systemThis section explains the proposed architecture including the main functions from vehicle detection to the barrier control mechanism. These steps are further explained in the following subsections.S is the distance, V is the speed of sound: .034 m/t is the time in In the proposed ALPR method, we convert the captured image into grayscale. It reduces the processing complexity and processing time and is robust to color changes due to different lighting conditions. A canny edge detector is applied to this image to detect all the edges. The Canny edge detector is a combination of a Gaussian filter for smoothing and a Sobel filter for edge detection. Equation shows thAfter the Gaussian filter, we apply the Sobel masks to detecOn the generated edge image, we perform various morphological operations such as dilation, horizontal erosion, vertical erosion and hole filling. Dilation adds the pixels to the boundary of edges to complete the boundary and increases the efficiency of LP extraction. Mathematically, Equation shows thEquation .(6)IholWe use vertical and horizontal erosion to remove those pixels, which makes it difficult to extract the LP. All the unnecessary lines and parts connected to the LP area create problems for the LP extraction. Equation shows thWe find the 8-connectivity components based rectangular bounding box objects in the eroded image. In addition to the LP, there are other rectangular objects such as headlights, radiators, grille and bumper. Therefore, it is likely that these objects are also segmented along with the LP. Due to this reason, we use the count of connected components in each segment as a clue to differentiate between the LP and other rectangular objects. To this end, for a segment to be considered an LP, the number of objects inside that segment should be more than five. This is due to the fact that the Pakistani LP consists of at least five characters as shown in Once the LP region is extracted, character segmentation is employed to extract the LP characters. For this purpose, as a first step, the LP region is binarized using the algorithm shown in Zoning: It divides the character image into various sub-images. Mathematical it can be calculated by Equation .(8)zoniPerimeter: The set of interior boundary pixels of a connected component (character image (C)) [age (C)) . We consExtent: It is the ratio of white pixels in an image to the total number of pixel in the binary image. Equation sumEccentricity: Finds how close an object is to being a circle. It is ratio of the linear eccentricity to the semi-major axis.Orientation: The major axis of an ellipse around the object and then finding the angle which the major axis made with the x-axis.In this paper, we focus on the features-based approach for character recognition. We extract the following features of a character.In this paper, we evaluated various supervised learning algorithms (classifiers) to recognize characters on the LP. The real-time system for vehicle detection and authorization is implemented on a Raspberry Pi. The ultrasonic sensors interfaced to the Raspberry Pi detect the approaching vehicle on entrance. The LP of this vehicle is then verified using its image. If the vehicle is permitted then the Raspberry Pi sends a command to open the barrier. PC(Intel(R), Core(TM) i3-4010U CPU 1.70GHz,RAM: 4.00GB) running Matlab running Python with OpenCV. The Raspberry Pi-based system had low computational time, a smaller size, and low power consumption, due to which it was used in the real-time application. In future, we are working to increase the dataset of handwritten LP characters to improve accuracy and laser beam-based vehicle detection to increase the detection range."} +{"text": "The independent variable was the response to a work environment (WE) question as to whether their supervisor always creates an open and trusting environment. Regression models were adjusted for demographic characteristics with each of the LS7 CVD risk factors as dependent variables. Twenty-one percent of workers reported that their supervisor did not create an open and trusting environment. Trust was associated with increased adjusted odds of having many of the LS7 CVD risk factors. Among those workers whose supervisor created a mistrustful environment, the odds ratios were greatest (>20%) for having four or more of the LS7 CVD risk factors.This study examined associations between trust, an important aspect of workplace social capital, with seven cardiovascular disease (CVD) risk factors (American Heart Association Life\u2019s Simple 7 (LS7)): smoking, obesity, low physical activity, poor diet, diabetes, high cholesterol, and high blood pressure. Data are from the U.S. Gallup-Sharecare Well-Being Index (2010\u20132012), a nationally representative telephone survey of U.S. workers ( Cardiovascular disease (CVD) continues to be a costly and significant problem and is the leading cause of death in the United States . MoreoveNumerous studies have examined associations between work stress and CVD ,14,15,16\u00ae Life\u2019s Simple 7 (LS7) [Cardiovascular health can be assessed by AHA\u2019s My Life Check 7 (LS7) ,5,6. The 7 (LS7) ,5. Recent literature has focused on the theory of social capital as important in explaining these health behaviors. While there are many definitions of social capital ,36,37,38In the past, literature on associations between social capital and health focused mainly on community, residential, or geographic areas ,41,42,43Some findings of associations between social capital and health behaviors have been mixed ,50. A re\u00ae Workshop [The report of the 2017 Total Worker HealthWorkshop identifiWorkshop includinWorkshop . MoreoveWorkshop reminds Workshop . For truIt is often difficult and expensive to collect data on work environment and workplace psychosocial factors across multiple worksites and regions. In the U.S., a number of ongoing national surveys, such as the Quality of Worklife , the HeaThe current study examines whether trust, an important aspects of social capital, is associated with the seven CVD risk factors identified in the AHA LS7 screening tool. Increasing social capital may improve health behaviors and outcomes directly, or in conjunction with workplace prevention and intervention programs. Due to the gender differences in the prevalence, progression, and underlying mechanisms in CVD, results are presented separately for women and men .Data for this study are based on the Gallup-Sharecare Well-Being Index (WBI) survey conducted between 2010 and 2012 in the United States. Every day, the Gallup Organization conducts computer-assisted telephone interviews (in English or Spanish) with 1000 randomly sampled U.S. adults (\u226518 years of age) on political, economic, and well-being topics. Random-digit-dial (RDD) to landlines and cell phones was used to reach wireless-only and wireless-mostly households. Although the response rate is low, 9\u201311%, it is estimated that the Gallup sample covers more than 95% of the U.S. adult population. Gallup weights data daily to account for disproportionate selection in age, sex, geographic region, gender, education level, ethnicity, race, self-reported location, and phone use status .The sample consists of survey participants interviewed between 2010 and 2012 who reported being currently employed by an employer for at least 30 h per week. Only workers who were employed by an employer were selected because this study focuses on supervisor behavior. In addition, full-time workers were selected because results from this study would be more relevant to those who spend a greater proportion of their time at work, than those working part time.2; the LS7 definition for at-risk BMI is \u226525 kg/m2. For diet, the WBI asks respondents the number of days per week they consumed five or more servings of fruits or vegetables. For this study, a healthy diet was defined as consuming five or more servings of fruits or vegetables seven days per week; adequate consumption on less than seven days was defined as a poor diet. The LS7\u2032s dietary assessment requires additional information such as the amount of fish, sodium, whole grains, and sugary beverages consumed daily or weekly. Similarly, for physical activity, the WBI asks about the number of days of exercise for at least 30 min during the past week, but does not ask about the intensity of physical activity. The LS7 definition includes the amount of vigorous and moderate activities per week. In the current study, meeting physical activity guidelines was defined as exercising for at least 30 min five days per week. Performing less than this was defined as insufficient exercise. In the WBI, smoking is defined as a yes response to a question about whether the respondent smokes. In the LS7, smoking is based on whether the person is a current smoker or quit <12 months ago.Life\u2019s Simple 7: The WBI included questions that address the seven cardiovascular health components of the LS7, but are not a complete match to the AHA definition. Work environment: Gallup scientists created a work environment question related to social capital based on their understanding of the literature. Work environment was coded in a negative direction and measured by the question: \u2018Does your supervisor always create an environment that is trusting and open, or not?\u2019 (No = mistrustful environment).Z-tests for the difference between women and men were calculated. Logistic regression models were run with each of the LS7 CVD risk factors as dependent variables in separate regression models. Odds ratios and 95% confidence intervals were calculated. Confidence intervals excluding 1.0 indicate significance at p < 0.05. In addition, a sensitivity analysis was conducted to examine the impact on associations using four or more CVD risk factors as a dependent variable in the regression model. Trust was entered into separate logistic regression models as an independent variable. All models were adjusted for potential confounders including demographic factors: age (years); race/ethnicity ; education ; marital status ; family income and any health insurance. Due to the large sample size in the current study, we focused on effect sizes (odds ratios) rather than p-values. According to a recent policy statement by the American Statistical Association on p-values and statistical significance, any effect, no matter how tiny, can produce a small p-value if the sample size is large enough [All analyses were stratified by gender and calculated using SAS 9.3 , using weights provided by Gallup to represent the U.S. population and to account for the complex survey design. Gallup surveyed 1,059,894 individuals between 2010 and 2012, of which 412,884 were full-time employees (working 30 or more hours per week). Responses of \u2018don\u2019t know\u2019 or \u2018refused\u2019 were set to missing. Because of a skip pattern, 23,554 (6%) were not asked the open and trusting environment question. Approximately 14% of the sample were missing income data, and 5% or fewer were missing data for the LS7 risk factors. Descriptive statistics were calculated for demographic, LS7, and the trust question. e enough .Demographic characteristics: Forty-two percent of the sample were women. Descriptive statistics for workers stratified by gender and trust are presented in Mistrustful environment: Approximately 22% of women and 20.3% of men indicated that their supervisor did not always create an open and trusting environment . As showp < 0.05 for each outcome. Trust was associated with the LS7 CVD risk factors in both men and women after adjustment for covariates. Due to the large sample size, we report effect sizes with a focus on those \u226510%. We found that workers who did not work in an open and trusting environment had greater odds of having high blood pressure , high cholesterol , and diabetes compared to those who reported having an open and trusting environment with their supervisor. Both women and men workers had greater odds of being a current smoker (both 15%), having a poor diet , and being obese (both women and men = 18%). Women reporting a mistrustful environment also had greater odds of having a low physical activity level (10%). Odds ratios for having four or more LS7 CVD risk factors were elevated for those working in a mistrustful environment .Multivariate results for trust with LS7 CVD risk factors, after adjustment for demographic factors and having any health insurance, are shown in The findings of this study suggest that lower workplace social capital, as measured by the WBI, is associated with higher odds of having one or more of the LS7 CVD risk factors.Our findings are consistent with others who have found associations between social capital and health ,81,82,83Furthermore, workplace stress can indirectly affect CVD risk through at-risk health behaviors ,87. ThesAlthough we adjusted for potential demographic confounders in our models, we examined each of the LS7 CVD risk factors separately. Health behaviors are often interrelated and can affect the presence or absence of other health behaviors. For example, smoking was found to increase caloric intake , while aAnalyses were presented separately by gender, not only due to the differences in CVD risk between women and men, but also due to the importance of gender in the social capital and managerial psychology literature . Odds ra\u00ae framework meets the goals of illness prevention to advance worker well-being. Efforts can also be made to target the health behaviors themselves. There are a range of possible strategies for addressing the LS7 risk factors in the workplace. For example, physical modification to the work environment, such as installing sit/stand desk stations and walking workstations, can reduce sedentary behavior and may increase physical activity. Additionally, increased access to nutritious food in the workplace may improve diet. Supervisors who support workplace wellness may help in reducing CVD risk factor in workers [Improvements to the work environment are needed to reduce CVD risk among workers. Social modification to the work environment, such as adjusting managerial style to create an open and trusting environment, can decrease work stress. Considering managerial trust from a Total Worker Health workers .This study has several strengths. The WBI is a large, nationally representative survey. Skopec et al. found thThe work environment question included in this study allows us to examine an important workplace psychosocial factor that is often difficult or expensive to study. It is unclear where the work environment question originated. Documentation provided by Gallup indicated that it was based upon findings from leading scientists in the areas of survey research, behavioral economics, and health. No information on the validity or reliability of the Gallup question is available.This study also has several limitations. The survey is cross-sectional and therefore no conclusions can be made regarding causality. Data were collected via a telephone survey that has a low response rate, potentially affecting the representativeness of our findings. For each regression model, observations with missing values for included covariates were dropped. All data were self-reported at one point in time and are subject to response biases, such as recall and social desirability. Social desirability bias is the tp-values. Despite these limitations, results show that more than 20% of workers report that their supervisor does not always create an open and trusting environment. This is associated with a 20% increase in odds for having four or more CVD risk factors, suggesting that this is an important factor when designing interventions to address worker cardiovascular health. Therefore, these results show support for the usefulness of this aspect of social capital to understand the work environment, supervisory behavior, and their association with worker cardiovascular health.Health behavior questions in the WBI were different from AHA\u2019s LS7 definitions, particularly diet that included only one of the five diet variables. The health factor metrics are markedly different in the WBI compared to the AHA\u2019s LS7 definitions, noticeably absent are the clinical measurements of blood pressure, blood cholesterol, fasting glucose, and medication used to treat these health factors. Lastly, the study\u2019s large sample size increases the probability of finding statistically significant associations; therefore, we focused on effect size rather than \u00ae framework.This study found that a negative work-environment characteristic representing an aspect of workplace social capital contributed to greater odds of having important CVD risk factors among full-time workers. Results suggest that supervisor behavior can play an important role in improving worker health. Workplace intervention programs for CVD and other chronic health conditions should consider addressing this aspect of workplace social capital, and supervisor competencies and behavior in particular, with proper training as a potential means to improve worker health. Thus, our results reinforce the notion voiced elsewhere ,100 that"} +{"text": "Health Information Technologies (HITs) can be an effective way to advance medical research and health services provision. The two-fold objective of this work is to: (i) identify and review state-of-the-art HITs that facilitate the aims of evidence-based medicine and (ii) propose a methodology for HIT assessment. Methods: The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Furthermore, we consolidated existing knowledge in the field and proposed a Synthesis Framework for the Assessment of Health Information Technology (SF/HIT) in order to evaluate the joint use of Randomized Controlled Trials (RCTs) along with HITs in the field of evidence-based medicine. Results: 55 articles met the inclusion criteria and refer to 51 (RCTs) published between 2008 and 2016. Significant improvements in healthcare through the use of HITs were observed in the findings of 31 out of 51 trials\u201460.8%. We also confirmed that RCTs are valuable tools for assessing the effectiveness, acceptability, safety, privacy, appropriateness, satisfaction, performance, usefulness and adherence. Conclusions: To improve health service delivery, RCTs apply and exhibit formalization by providing measurable outputs. Towards this direction, we propose the SF/HIT as a framework which may help researchers to carry out appropriate evaluations and extend their studies.Background: The application of Health Information Technology (HIT) has been proposed as a promising solution for the improvement of efficiency, effectiveness, quality of healthcare delivery. Some of the most important benefits of HITs are the reduction of medical errors and costs, the improvement of patients\u2019 quality of life and the enhancement of medical decision making.The application of HIT to transform the delivery of health care. Despite the fact that interoperable and multipurpose HIT systems have been developed, their widespread adoption in the course of care has been limited by the lack of knowledge about their types, the existence of standardized assessment frameworks/methodologies and classification methods of their outcomes that will improve health care provision. The assessment and reporting of HIT systems features requires standardized methods and classification procedures.Recent research efforts have focused on the potential of Evidence-Based Health Informatics (EBHI) can be defined as the conscientious, explicit, and judicious use of current best evidence to support a health care decision that employs Information Technologies (ITs) [Randomized Controlled Trials (RCTs) are considered to be a well-established experimental clinical tool, suitable not only for evaluating the efficacy of interventions, but also for supporting the conduct of an adequately designed systematic review [es (ITs) . Towardsc review .EBHI and RCTs are currently at the forefront of physicians\u2019 support for clinical decision-making. Thus, this work focuses on recent research efforts that examine whether and to what extent HITs are employed for evidence-based medicine purposes and, moreover, to propose a methodology for HIT assessment in health care delivery.Both HITs, and to what extent? It remains unclear whether the advances of HITs in recent years take into account or suggest such evaluation models or standards.RQ1. Are there any classification methods for the detailed description of the results associated with HITs in order to evaluate, compare or extend results from other studies and systematic reviews? It is uncertain whether comprehensive and standardized assessment frameworks/methodologies have been proposed, or if further investigation and study is required in the field.RQ2. Is there any framework or methodology to follow for the assessment of HITs that support evidence-based medicine and are integrated in medical and nursing practices?RQ3. Which are the most established HIT interventions of the included studies in this research, and their classification in accordance with the medical/health domain?RQ4. What are the features, the outcomes and the types of Due to the key role played by information technology in every aspect of health care, the aforementioned benefits have to be validated. Thus, we identified four broad Research Questions (RQs) that will guide the rest of our work:HITs with respect to the value and functionalities designed to work with them that facilitate the aims of evidence-based medicine, and (ii) propose a framework for considering the HIT assessment in health care delivery. As a result of these objectives, this work consists of a literature and a systematic review.The main goal of this study is to map out and propose a common and strict research method so that it leads to comparable findings among many studies. To achieve this, we used a previous systematic review of ours as a model of this study. More analytically, the two-fold objective of this article is to: (i) identify and review the state-of-the-art HITs emerged. Based on that work, the potential indicators of the proposed framework were developed and validated by a Delphi study [Initially, in the literature review, we examined step by step the research methodology of the articles included in our previous systematic review and an uhi study .Delphi method was used to collect experts\u2019 opinions regarding key indicators . Within this framework, two key domains were identified. The collection methodology of the appropriate articles was designed accordingly and the literature review .More analytically, the d items) for the eview in was condThus, as depicted in SF/HIT framework statement [Moreover, in order to respond to RQ3 and RQ4, we used data collected in a previous work of ours [medicine ,7,8. Theent Tool , while itatement .CONSORT-EHEALTH Checklists [HITs-based healthcare provision. Finally, we considered RCTs studies with at least one published article in order to compile the basic material of our study. Details about the applied methods, the electronic databases and the queries involved, as well as the data collection process are provided in our previous review [In addition, we applied the ecklists to furths review .HIT applications spanning from short message services in medical environments and high-performing healthcare systems [Electronic Health Record (EHR) systems [A short literature survey depicts systems ,12 to te systems ,14, e-me systems , Electro systems and sens systems .HITs, the so-called Health Technology Assessment (HTA), is a crucial multidisciplinary activity in research and medical practice that systematically examines and evaluates the properties, effects, and/or impacts of health technology [The evaluation of chnology .HTA, we identified several methods and taxonomies (addressing RQ1 and RQ2) that classify the assessment and properties of the evaluations of HITs.During this literature review on HTA.The International Network of Health Technology Assessment Agencies developed a 14-item health technology assessment checklist as further support for a consistent and transparent approach of EUR-ASSESS project proposed a framework for conducting and reporting HTA that describes a good practice in both undertaking and reporting HTA, while it also identifies the needs for methodologic development. This framework is complemented by specific recommendations and implementation tools, e.g., by providing the structure of the scientific summary reports and a checklist for the evaluation of methodology and the quality of HTA reports [Moreover, the reports .EUnetHTA project developed the HTA Core Model. This is a novel approach that enables effective national and transnational production and sharing of HTA results in a common, structured format and represents a wide range of perspectives [Also, the pectives .HITs on behalf of the AHRQ\u2019s National Resource Center for HIT in the USA. It is organized into 6 major and 28 minor categories with two additional sub-levels, forming a hierarchical and interrelated classification schema for the development, implementation, and evaluation of HITs. A review conducted by Jamal et al. [Electronic Health Records, Computerized Provider Order-Entries, and Decision Support Systems in medical care. Additionally, the same work examined the level of compliance in respect to evidence-based guidelines among clinicians.In addition, Dixon et al., in , proposel et al. studied HIT for improving quality and efficiency while total development and implementation costs cannot be accurately calculated.Furthermore, the authors in systematHITs, during 2009, the U.S. government introduced the Health Information Technology for Economic and Clinical Health (HITECH) Act [Act posed new requirements on health care organizations and professionals in terms of meaningful-use criteria, which drive reimbursements from the U.S. government for patient-centered care. The meaningful-use criteria are a set of requirements that health care organizations and professionals must meet for the adoption of HITs.To accelerate the use of ECH) Act . This AcHIT functionalities with regard to quality, safety, and efficiency metrics. The authors confirm that the most important improvement incurred in HIT evaluations is increased measurement, analysis, and reporting of the effects of contextual and implementation factors.In the same direction, the authors of examinedEHR systems, 44 articles that studied order entry systems and 44 articles that evaluated clinical support decision systems. The authors observed that the literature was not clear in determining which criteria that strongly impact the efficient use of HITs had been met. However, the review revealed that the HITs systems improved several aspects of healthcare provision services, without noticing deterioration in any case. The study also determined that articles dealing with more than one substantial operation of HITs had slightly increased positive findings compared to articles that did not consider them at all. This finding is attributed to the fact that including substantial and measurable criteria makes the measurements more accurate. Consequently, the benefits of HITs can be determined more accurately.The review conducted by Buntin et al. includedHITs.Defining a common set of criteria and a strict framework , the conduct of reviews and meta-analyses will provide sufficient, comparable and reliable results , minimizing in parallel the risk of bias. Towards this direction, the next section proposes the framework of a classification system that enables the integration of results from multiple clinical trials and draws conclusions on the usage of health IT evaluation.In this work (addressing RQ1 and RQ3), we aim at identifying the functionalities that can be used to prototype health care delivery and to promote HITs in evidence-based medicine (addressing RQ2). Thus, we extend a previous work of ours and we pEHR systems on the basis of some core functionalities , as well as by the supported administrative processes . The classification criteria posed by the Committee include: patient safety, delivery of effective patient care, facilitate management of chronic conditions, efficiency and feasibility of implementation.The Committee on Data Standards for Patient Safety classifiHITs are classified by study design, care setting, health IT components, functions included in the meaningful-use criteria, and finally addressed outcomes. Outcomes are classified by their type and their rate .In another work, described in , HITs arSynthesis Framework for the Assessment of Health Information Technology (SF/HIT) the Health Core Domains category category . SF/HIT Health Core Domains category includes the findings associated with health care provision, and is divided into three sub-categories, namely (a) design; (b) annotation; and (c) evaluation.According to this methodology, the design sub-category, we consider (i) the PRISMA 2009 methodological tool [RCTs [RCT type [annotation sub-category, the trials are annotated under the ICD-10 medical terminology [evaluation sub-category we consider (i) the CONSORT statement and checklist [RCTs, while the second and third are used in order to evaluate bias type and its level respectively.In the ol [RCTs ,29; as wRCT type , e.g., ominology . Finallyhecklist ; (ii) thhecklist ; and development, (b) functionality and (c) evaluation.The development sub-category, uses a taxonomy based on the Medical Subject Headings (MeSH) classification system [The n system , CPOE (Computerized Physician Order Entry), DSS (Decision Support Systems), EHR , and other types\u2014that describe the functional capabilities of HITs.The functionality sub-category consists of 5 evaluation sub-category includes (i) the CONSORT-EHEALTH Checklists [CONSORT-EHEALTH Checklists stands as a standard evaluation report, while the impacts in the reviewed studies are recorded according to the suggestions described in [Finally, the ecklists ; (ii) thribed in .PRISMA 2009 flow diagram , Health Level 7, International Clinical Trials Registry Platform, Iranian Registry of Clinical Trials, International Standardised Randomised Controlled Trial Number, Japan Primary Registries Network, The Netherlands National Trial Register, Pan African Clinical Trials Registry, United States Trials Registry, European Clinical Trials Database, Linked Clinical Trials. Also, the search strategy was performed over the following academic digital libraries: Medline/PubMed, IEEE Xplore, World Health Organization library databases, Google Scholar, Wiley Online Library and Scopus.HIT interventions, we performed single as well as multi-term queries combined with Boolean operators. In order not to overlook critical synonyms in our queries, we firstly included all related MeSH terms . After having collected a significant number of articles from our queried sources, we excluded keywords and terms which appeared rarely, while we added synonyms that appeared with high frequency and were not included in our initial term set. We finally ended on the following keywords and subjects: \u201ccomputer-interpretable, computer-based, measurement, assessment, evaluation, scale, rating, inventory, monitoring, tool, e-health, telemedicine, tele-health, telecare, care plan, clinical guideline.\u201dFor compiling a solid basis of articles that fall within the concept of RCT and (ii) employed ICT (Information and Communications Technology)\u2014based methods or uses computer-based equipment.Then, we applied a two-fold eligibility criterion. We considered an article in our review if (i) it referred to at least one RCTs) published between 2008 and 2016;Interventional completed trials ;Results of the trials are published in at least one article that belongs to well-known and established scholar databases. Therefore, every article is related to one or more trials and vice-versa;Publication language is English.PRISMA checklist item 8), are provided in our preliminary work [Due to the heterogeneity of study designs, no meta-analyses were considered. Further details on our search strategy, so as to be replicated by a third person (according to ary work .Sub-category 1.3.2. and 1.3.3. of SF/HIT). More precisely, all the elements are classified using the rationale of the proposed SF/HIT Framework .Category of disease/health domain .RCT Experimental type .HITs Functional capabilities . To define values for this output, we based on the suggestions made by the authors of [thors of ,34.ICT category\u2014under the MeSH classification system .Sub-categories 2.3.2. and 2.3.3. of SF/HIT).Outcomes/impacts by type and rating experimental type; (iii) HITs functional capabilities; and (iv) ICT category.Moreover, studies/trials were further examined with respect to their: (i) In a similar way, HIT-based interventions of the included studies according to the ICD-10 classification system.As far as RQ4 is concerned, we systematically present the descriptive elements of the studies and the types of 5 studies/5 trials):A00\u2013B99: Certain infectious and parasitic diseases and its related publication year.4 studies/4 trials):C00\u2013D48: Neoplasms study the impact of a web-based intervention that utilizes text messages to improve cancer prevention behaviors among adolescents [\u25cf\u2003lescents , (ii) evlescents , (iii) ilescents , and and the publication of the related results was approximately 5.3 years.7 studies/7 trials):E00\u2013E90: Endocrine, nutritional and metabolic diseases . Between the initiation of a clinical trial (registration year) and its related publication, the average time was approximately 1.3 years.18 studies/18 trials):F01\u2013F99: Mental, behavioral and neurodevelopmental disorders :I00\u2013I99: Diseases of the circulatory system and the respective publication year was 4.3 years.3 studies/2 trials):J00\u2013J99: Diseases of the respiratory system :Z00\u2013Z99: Factors influencing health status and contact with health services : In , the aut studies ,79 assesy living , as welly living . We alsoy living , the auty living comparedy living examinedAll clinical trials were conducted over samples varying from 10 to 504 participants. The mean time between the initiation of the clinical trial and its related publication was approximately 2.9 years.4 studies/3 trials):Other subjects , there is still a significant time period required for applying ICTs in clinical medicine .Health Information Technology category is concerned : 14 trials support all types of Computer Communication Networks including Internet (MeSH term: L01.224.230.110 (MeSH term: L01.178.847.652) offer positive results (66.7%). 26 trials support the use of Smartphones and Text Messaging technologies of information science (MeSH terms: L01.178.847.698.300.250 and L01.178.847.698.300.500), describing 50% positive outputs. 1 of 3 trials of User-Computer Interface or Virtual Systems (MeSH term: L01.224.900.910) offer positive results (33.3%). Finally, 14 trials support all types of Medical Informatics Applications (MeSH term: L01.313.500.750), of which 9 offer positive results (64.3%) (.230.110 ), of whi (64.3%) .RCTs fall within the treatment type, 7.8% refer to screening type while the rest of them belong to prevention (15.7%), supportive care (15.7 %), health services research (13.7%) and none in the diagnostic type (0%) .Functionality sub-category : The majority of trials (27) were related to CBA. 14 of them (51.9%) were assessed to have positive outputs. 6 trials support DSS with 100% positive results. The rest of them, i.e., CPOE, EHR and Other types of systems, were assessed with 50%, 50% and 68.4% positive values, respectively : In this sub-category we examine the following three parameters:Standard evaluation report: Despite the fact that CONSORT-EHEALTH improves and standardizes the evaluation reports of Web-based and mobile health interventions [(a) ventions , yet we ventions ,60,61 thDimension of impacts by type : Similar to the outcomes of [RCTs remain the gold standard for assessing the effectiveness, preventive care, safety/privacy, appropriateness, satisfaction, service delivery/performance, usefulness and adherence/attendance. In our study, we identified the above-mentioned outcomes as well as some more . Furthermore, in accordance with [(b) comes of , our resnce with , we clasMoreover, with respect to RQ4, we observed positive results in: (i) preventive care ; (ii) acceptability ; (iii) safety/privacy/security ; (iv) appropriateness (2 out of 3\u201466.7%); (v) effectiveness (26 out of 45\u201457.8%); (vi) satisfaction (3 out of 5\u201460%); (vii) service delivery/performance (3 out of 5\u201460%); (viii) usefulness (4 out of 7\u201457.1%); and (ix) adherence/attendance (5 out of 10\u201450%). Lower levels of positive results appeared in cost effectiveness and efficiency .Dimension of impacts by rating : Although in our study we classified the effects into two main groups influence on the results (impacts)) and neutral/negative ), we noticed that our findings are in line with [HITs ((c) ine with , where tine with , we idens\u201460.8%) . The majs\u201460.8%) .HIT and computer-based tools are applied to support evidence-based medicine.This review outlines the environment in which MeSH, functional capabilities of HITs, etc.) and similar metrics . To assist other researchers in taking the correct actions and producing tangible and reusable results, we ensured that all relevant studies employed the same types of coding and classifications are concerned, the framework is already quite strict, thus several elements described in RCT type and experimental type). However, we believe that it would be useful in future studies to include: (i) the functional capabilities of HITs; (ii) the information science domains; and (iii) categorization of the impact under the ICD-10 classification scheme adequate and analytical assessment outputs; (b) a common framework for the evaluation of HITs to enhance evaluation and comparability issues; and (c) a standardized reporting mechanism on the extensive application of HITs in evidence-based medicine.Therefore, for the improvement of health service delivery through the successful integration of SF/HIT framework and the relative guide were presented and validated through a Delphi consultation and adopted as a valuable methodology. This framework, if it is taken into account both in the design of RCTs and systematic reviews, will help researchers to better evaluate HITs and expand their reviews and/or meta-analyses.Towards this direction, the To ensure the highest possible quality of our study, we used the following methodologies.Delphi method by means of the Practical Guidance described in [Delphi procedures that are performed in order to select healthcare quality indicators and the construction of the final SF/HIT.We have applied the literature review in accordance with the ribed in for the We conduct a systematic review, since this kind of review is originally used in the medical sciences to examine the effectiveness of health-care interventions and to support the practice of evidence-based medicine. Systematic reviews involve identifying, synthesizing and assessing all available evidence, quantitative and/or qualitative, in order to generate a robust, empirically derived answer to a focused research question. They are highly valuable, improve transparency and emphasize the importance of empirical evidence over preconceived knowledge .PRISMA review methodology to ensure the transparent and complete reporting of our systematic review In line with the above-mentioned, we follow a sufficiently clear, systematic and thorough search strategy over multiple clinical trial registries and academic digital libraries. We also adopt the Cochrane Risk of Bias Assessment Tool [Cochrane risk of bias items, with each of them being identified in one of the following three bias risk categories: high, low or uncertain that support measurements by type and by rate.Finally, as Buntin et al. identifiCBA, CPOE and EHR systems. In addition, RCTs study mental, behavioral and neurodevelopmental disorders, nutritional, metabolic and circulatory diseases, as well as factors that are related to health status communication with health services.According to our findings, it appears that research surrounding Internet, telemedicine, smartphones and text messaging technologies, user-computer interfaces and virtual systems, as well as medical informatics applications, dominates the literature. Most of them are related to the development of HIT-based approaches, namely wearable biosensors, artificial intelligence and machine learning systems for health monitoring and delivery that are underrepresented in this work, mainly due to their recent appearance in the field and the necessary time before the publication of the results of an RCT.Nevertheless, there are some state-of-the-art RCTs apply and exhibit a clear formalization by providing measurable outputs that can be compared and evaluated in respect to several HIT-based categories and domains. Towards this direction, we propose the SF/HIT framework in order to classify the findings of our study in two categories and subcategories. The use SF/HIT provides unique insights with respect to HITs and health care delivery assessment in evidence-based medicine by (i) revealing the required healthcare quality indicators for the implementation of an RCT study over HITs, and (ii) helping the researchers to carry out the appropriate studies and evaluations and/or meta-analyses and extend their systematic reviews.Finally, to improve health service delivery,"} +{"text": "The Autonomous Sensory Meridian Response (ASMR) is a tingling, almost euphoric, sensation often elicited following certain visual or auditory stimulations . Despite Despite these similarities, ASMR is associated with several physiological experiences that set it apart from other sensory phenomena. For example, although frisson and ASMR can both be described as \u201cpleasant tingling,\u201d their time courses differ: frisson typically occurs and spreads quickly, while ASMR has idiosyncratic durations, coupled with sensations that often spread to other bodily areas with increasing intensity (The Autonomous Sensory Meridian Response (ASMR) is a sensory phenomenon that is typically elicited in response to visual or audio stimuli that cause static, tingling sensations that originate in the head and often spread to the neck and sometimes other regions of the body . In addintensity . Similarntensity .autonomous, in that individuals are assumed to have no control over initiating it, and sensory, in that it is a physical response that occurs along the body\u2019s meridian, or center (www.reddit.com/r/ASMR) with over 131,735 subscribers. Moreover, when ASMR is featured in popular media, it is often described using attention-grabbing terms, such as \u201cbrain orgasms\u201d and \u201cwhisper porn\u201d , recruited from Psychology courses, who received partial course credit in exchange for participating. The remaining 102 participants were recruited from an Internet forum dedicated to the ASMR phenomenon (https://www.reddit.com/r/asmr/). These participants volunteered their time and were not compensated. Participants were randomly assigned to Instruction conditions (described below), such that 103 received encouraging instructions and 106 received discouraging instructions . This research was conducted with Institutional Review Board approval granted by Louisiana State University . All participants provided informed consent prior to beginning the study.A total of 209 volunteers completed the experiment. Of these, 107 were college students (The Autonomous Sensory Meridian Response (ASMR) is a sensory phenomenon in which people experience a pleasant tingling, static-like sensation in response to listening to specific audio or visual stimuli.After providing informed consent, participants received written instructions. All participants read the following:Over the course of this experiment, you will hear audio from 3 different video presentations. All of the audio files that you are about to hear have been shown to produce the ASMR effect, and we are interested in the causal mechanism causing the response. Please listen to the clips, because you will be asked to rate the degree to which you experienced the tingling, static sensation of the ASMR.Those in the encouraging instructions condition then read:On the following pages, you will hear 3 different audio clips. None of the clips that you are about to hear have been shown to produce the ASMR effect, and we are interested in determining which audio-visual characteristics prevent ASMR effects from occurring. Please listen to the clips, because you will be asked to rate the degree to which you experienced the tingling, static sensation of the ASMR.Those in the discouraging instructions condition instead read:Prior to playing the first clip, participants read that they would listen to each clip for 5 minutes, before rating their ASMR experience on a Likert scale from one to seven . Participants then indicated whether they understood the instructions and wished to proceed.In addition to randomly assigning participants to Instruction conditions, they were also randomly assigned to one of the five ASMR category conditions. Although participants would have ideally listened to all categories, we manipulated this between-subjects to keep the experiment a reasonable length, as each category contained three 5-minute clips see , and parPrior to analyzing the data, we ensured that the student sample was na\u00efve and the Reddit sample was familiar with ASMR by examining their post-experimental answers about previous experience with the phenomenon. Whereas none of the student sample reported having watched ASMR videos, 99 participants from the Reddit sample reported regular viewing of ASMR media . The Reddit participants\u2019 ASMR sensations are summarized in Because we were interested in whether participants\u2019 expectations played a role in their experience with ASMR and ASMR-like (which would not be expected to induce ASMR) videos, we analyzed the data with and without 12 participants who either guessed the study hypothesis in the post-experiment questionnaire, or revealed a familiarity with more than one of the video clips (11 of these participants were from the Reddit group). The patterns in the data were the same regardless of inclusion, so we present the full analyses. Degrees of freedom are Greenhouse-Geisser corrected, when needed.F = 5.16, p = 0.007, \u03b7p2 = 0.03, it was qualified by an interaction with Group, F = 21.69, p < 0.05, \u03b7p2 = 0.10. As shown in t-tests comparing these ratings. With the exception of participants in the encouraging instructions condition who were rating the ASMR clips (t(101) = \u22120.13, p = 0.90), all other differences in ratings were significant. For participants in the encouraging condition, na\u00efve participants were significantly more likely to provide higher ratings for both the foil, t(94.74) = 4.77, p < 0.001, d = 0.94, and music clips, t(74.32) = 6.06, p < 0.001, d = 1.18, than the Reddit group. With regard to the discouraging instructions, the Reddit group provided higher ratings to the ASMR clips than the na\u00efve group, t(104) = \u22122.29, p = 0.02, d = 0.44. However, for the foil, t(98.29) = 2.33, p = 0.02, d = 0.45, and music, t(76.25) = 4.5, p < 0.001, d = 0.87, clips, Reddit participants provided significantly lower ratings, relative to the na\u00efve group. Similarly, the main effects of Instruction, F = 18.89, p < 0.001, \u03b7p2 = 0.09, and Group, F = 7.93, p = 0.005, \u03b7p2 = 0.04, reliably interacted, F = 6.82, p = 0.01, \u03b7p2 = 0.03. As shown in To determine whether ASMR effects can be elicited by suggestion, and whether this differs based on existing ASMR experience, we analyzed participants\u2019 post-video ratings of their \u201cASMR tingling, static-like sensations\u201d in a 2 \u00d7 3 \u00d7 2 mixed model ANCOVA, with Instruction and Group serving as between subjects measures, and Age and Gender included as covariates. Although there was a main effect of Clip Type, F = 100.6, p < 0.001, \u03b7p2 = 0.51; Category: F = 3.84, p = 0.006, \u03b7p2 = 0.14) were qualified by an interaction, F = 7.81, p < 0.001, \u03b7p2 = 0.24. As shown in F = 2.77, p = 0.007, \u03b7p2 = 0.10. Unlike the Reddit group, only the personal attention clips yielded reliable differences: both the ASMR and music clips produced higher ASMR ratings, relative to the foil clips . Although we did not predict expectancy effects from this source, the finding is nevertheless consistent with an explanation of ASMR based on individuals\u2019 expectations.Although experienced ASMR participants were unaffected by the expectation manipulation (via the suggestive instructions), their ASMR ratings indicate that they were affected by their own expectations, driven by their history of ASMR viewing or participation in ASMR discussion boards. As shown in Overall, our results suggest that ASMR may be explained by expectations, and any stress reduction or improved psychological function resulting from ASMR may be placebo effects. This, however, does not diminish the utility of ASMR for those who report experiencing it. Given the similar outcomes produced by both mindfulness exercises and the ASMR experience, part of ASMR\u2019s utility may lie in its resemblance to guided meditation or mindfWhile our findings support expectancy effects from both experienced ASMR users and na\u00efve participants, we note that our research has several limitations. First, these results do not explicitly rule out the possibility that there are preexisting physiological differences between the two groups that contribute to susceptibility to ASMR experiences. Despite the limited experimental literature investigating ASMR, there is evidence that individuals who experience ASMR may have reduced functional connectivity between the frontal lobes and sensory brain regions , as well as decreased DMN connectivity . These dfrisson, and not ASMR effects. If ASMR experiences are indeed based in underlying physiology, it is possible that the na\u00efve participants in our study were incapable of experiencing ASMR at all and instead mistook any pleasant tingling or static-like sensations as the relatively more common experience of frisson experience ASMR, and that they did not regularly seek out ASMR content beyond the bounds of our experiment. Instead, we were limited to self-reported items that relied on their response indicating that they were unfamiliar with the phenomenon. This constitutes a limitation to our design, but does not negate our findings: even if participants were familiar with ASMR content but indicated they were not, na\u00efve participants\u2019 data support the conclusion that expectancy effects contribute to experiences consistent with ASMR. Finally, our results are limited by the possibility that na\u00efve observers may have experienced sson see . We thinsson see . Only onsee also . Future Although ASMR effects may represent placebo effects resulting from individuals\u2019 expectations, they remain useful for many people. In recent years, relaxation (or \u201cmindfulness\u201d) techniques have been gaining popularity . Major cGiven the physical and psychological consequences of chronic stress to achieve it? If yes, please describe. If no please write N/A.Do you feel a tingling sensation when watching ASMR videos?Are these tingling sounds triggered by specific stimuli?Crisp sounds WhisperingWater pouringPersonal attention VacuumingAirplane noiseLaughing a lot and doing things that make you happySmilingWatching repetitive tasks Slow movementsNot applicable to meIf you answered yes to the previous question, please select the items that trigger your tingling sensations while viewing ASMR videos. If you answered no, please select the N/A option.If you have any other triggers, please list them below. If this does not apply, please write N/A.Do any stimuli stop or prevent this tingling sensation from continuing? If yes, please describe. If no, please write N/A.HeadShouldersChestBackArmsStomach/lower abdomenGenitalsHipsLegsFeetThis does not apply to me because I do not experience ASMRPlease indicate where your tingles originate.Prior to this study, had you heard any of the clips that we used in the study? If you have heard one or more of the clips that were used, please write which one(s) in the box below. If you did not hear any of the clips previously, please write \u201cN/A.\u201dWhat do you think the purpose of this study was?10.7717/peerj.5229/supp-1Supplemental Information 1Click here for additional data file."} +{"text": "Autonomous Sensory Meridian Response (ASMR) is a sensory experience elicited by auditory and visual triggers, which so far received little attention from the scientific community. This self-reported phenomenon is described as a relaxing tingling sensation, which typically originates on scalp and spreads through a person\u2019s body. Recently it has been suggested that ASMR shares common characteristics with another underreported condition known as misophonia, where sounds trigger negative physiological, emotional and behavioural responses. The purpose of this study was to elucidate whether ASMR is associated with heightened levels of misophonia.The Misophonia Questionnaire (MQ) was administered to individuals reporting to experience ASMR and to age and gender matched controls.Compared to controls ASMR group scored higher on all subscales of MQ including the Misophonia Symptom Scale, the Misophonia Emotions and Behaviors Scale and the Misophonia Severity Scale.Individuals reporting ASMR experience have elevated levels of misophonia. Sensitivity to sound, especially sound produced by humans, is termed misophonia, which literally means \u2018hatred of sound\u2019 . AlthougInterestingly, anecdotal reports and recent findings suggest that there may be an increased incidence of misophonia among individuals who experience Autonomous Sensory Meridian Response (ASMR) . ASMR isWhile ASMR sensations are described as pleasurable and relaxing , many viFurthermore, it has been suggested that misophonia and ASMR might represent two ends of the same spectrum of sound sensitivity and that they may be linked to synaesthesia, which has been reported to be more common in ASMR-responders compared to general population . All theThe aim of this study was to establish whether ASMR is associated with heightened levels of misophonia. To do so the Misphonia Questionnaire , which cThis study was conducted online and was approved by the ethical committee of Goldsmiths University of London. Participants were first presented with information about the study. They were also informed that the study is anonymous and that their participation is voluntary and that they may withdraw at any point without any consequences. Participants were requested to give electronic consent prior to commencing the study.M\u00a0=\u00a028.81, SD = 6.05, range: 18\u201338) and sixty eight controls . The two groups did not significantly differ in terms of age or gender with ASMR-responders scoring overall higher on both subscales compared to controls . In addition, 36% of ASMR-responders compared to 22% non-responders scored 14 and above on the Misophonia Symptom Scale, which is treated as clinically significant misophonia symptoms. However, this difference in proportions between the two groups was statistically non-significant . Performance on the MQ Total was analysed using an independent samples t-test which revealed a significant group difference with ASMR-responders scoring overall higher on both subscales compared to controls . As with the primary analyses described above, performance on the Misophonia Severity Scale was analysed using an independent-samples Mann\u2013Whitney U test. This analysis revealed a significant group difference (U(92)\u00a0=\u00a0768.000, z\u00a0=\u00a0\u00a0\u2212\u00a02.178, p\u00a0=\u00a0.029) due to ASMR-responders scoring higher (Mdn\u00a0=\u00a04.00) than controls (Mdn\u00a0=\u00a03.00). Thus, qualitatively similar results were found when analysing female participants only to the analysis performed on both male and female participants.As there were more females in the control group a control analysis was also conducted in order to establish whether gender impacted the results. This entailed running the same analysis on female participants only. The two groups did not differ in terms of age [\u03c7N=92)2. Additionally, while the percentage of participants who reported 7 or above on the Misophonia Severity Scale was exactly the same as for the entire ASMR group (12.5%) only 3.8% of the control sample scored 7 or above on this scale. This result was also statistically non-significant [\u03c7N=92)2.There were, however, differences in the proportion of female participants who scored 14 and above on the Misophonia Symptom Scale compared to the proportion reported by groups including both male and female participants. Specifically, 35% of ASMR-responders compared to 26% non-responders scored 14 and above on this scale. However, similar to the primary findings involving data from males and females, this difference in proportions of participants scoring in the clinically relevant range was non-significant [In addition to these primary analyses, we also conducted analyses on a subset of control participants who reported ASMR . We observed a qualitatively similar pattern of results see .The aim of this study was to examine whether ASMR is linked to increased levels of misophonia. The suggestion that these two phenomena may be linked was originally put forward by In order to establish whether ASMR is linked with elevated levels of misophonia a group of ASMR-responders and controls were administered the online Misophonia Questionnaire . The resASMR-Responders also scored higher on the Misophonia Emotions and Behaviors Scale, which measures emotional and behavioural responses to certain sounds that include actively avoiding certain situations in anticipation of the sound, having violent thoughts or becoming physically aggressive. This suggests that heightened sound sensitivity present in ASMR as indicated by elevated scores on the Misophonia Symptom Scale in this group is associated with more severe negative behavioural and emotional outcomes relatively to control participants. In addition, ASMR-responders had increased scores on the Misophonia Questionnaire Total scale, which is calculated by adding the two individual subscales (MSS and MEBS) together.Further, ASMR-responders had increased scores on the Misophonia Severity Scale, which suggests greater level of severity of sound sensitivity in this population compared to controls. 12.5% of ASMR-responders compared to 6% of the control sample reported 7 or above on this scale, which is interpreted as clinically significant misophonia symptoms that can interfere with normal functioning. Although twice as many ASMR-responders reported clinically significant misophonia symptoms relative to control participants in this study, this difference was statistically non-significant and overall, these percentages are much lower than previously reported prevalence rates in the general population which is quite perplexing 19.9%\u2014. This maA large proportion of participants recruited outside of the ASMR Facebook site (47%) also indicated to experience ASMR based on the given description of the phenomenon. These participants were not included in the primary analysis as it was not possible to firmly exclude the possibility of them providing false positive answers as no additional questions regarding ASMR were asked. Nevertheless, it is possible that these individuals also genuinely experience this phenomenon. In order to establish whether this group showed a similar pattern of results to ASMR Social-Media recruited from the aforementioned Facebook site dedicated to ASMR an additional analysis was conducted see . As the Therefore, similarly to participants recruited through ASMR Facebook site individuals recruited in a more systematic way who self-identified as experiencing ASMR also show increased levels of misophonia compared to controls. Moreover, their misophonia appears to be even more pronounced compared to ASMR-Responders recruited through ASMR community sites as evidenced by increased scores on all variables and a much larger proportion of them scoring within the clinically significant range on the MSS.As the overall pattern of results with respect to misophonia was similar between ASMR Social-Media and ASMR Non-Social Media it appears that the latter group may have consistent experiences to those reporting ASMR from the social media community. However, in order to eliminate the possibility of a third variable accounting for this result a more thorough investigation controlling for such potentially confounding factors will need to be carried out in the future. The findings also suggest that when recruiting participants in a more systematic way almost half of the sample reported ASMR experiences, suggesting that this phenomenon may be highly prevalent in the general population. This could account for the huge popularity of the YouTube channels producing ASMR content. However, as mentioned earlier, stricter verification methods will need to be employed in the future in order to assess this claim.Current results may also suggest that while individuals who experience ASMR may have increased overall levels of misophonia compared to controls, those who seek membership in online communities dedicated to ASMR may have lower levels of misophonia compared to individuals who experience ASMR but are either not aware of the phenomenon or do not actively engage in such online communities. It is feasible that only those individuals with moderate levels of misophonia would seek ASMR videos as they often include sounds which could also trigger misophonia in some ASMR-responders. However, as no questions regarding viewing habits were included in the questionnaire it is not possible to establish how many of ASMR Non-Social Media group were previously aware of this phenomenon and whether they engage in watching ASMR videos. The latter was also not established in case of the ASMR Social-Media group either. This shortcoming will need to be addressed in future research to gain better understanding of the obtained results.Taken together, the findings reported here of heightened misophonia symptomatology among individuals who report ASMR relative to controls are in line with previous suggestions of a potential link between the two conditions . This fuThese findings are also interesting in light of the suggestion that ASMR and misophonia appear to rely on similar inducer-concurrent mechanisms as synaesthesia . Indeed,Current results are also interesting in light of the reports of an association between misophonia, obsessive-compulsive disorder, anxiety and depressive symptoms and findAdditionally, as the current study uses a binary classification with respect to ASMR, examining the degrees of relationship between ASMR and misophonia is limited. Employing rating scales measuring the intensity of ASMR would be an important next step as it would allow for more comprehensive examination of the relationship between ASMR and misophonia and other conditions which also exist on a continuum.In summary, the current findings suggest increased levels of misophonia among individuals reporting ASMR relative to controls with respect to all subscales of the Misphonia Questionnaire includin10.7717/peerj.5351/supp-1Supplemental Information 1Output containing answers to individual questions and computed final scores. This data has been cleaned and anonymised.Click here for additional data file.10.7717/peerj.5351/supp-2Supplemental Information 2Results of supplemental analysis conducted on data contributed by individuals recruited outside of social media sites dedicated to ASMR who also reported to experience ASMRClick here for additional data file.10.7717/peerj.5351/supp-3Supplemental Information 3Click here for additional data file."} +{"text": "R groups attached to the core. The thermal stabilities of the different sulfenamides were examined by thermogravimetric analysis (TGA). Radicals generated by the homolytic cleavage of the S\u2013N or S\u2013N\u2013S bonds at an elevated temperature can effectively interact with the intermediate products of polymer thermolysis and provide excellent flame retardant properties. The choice of most suitable SN-structure varies depending on the polymer type. For polypropylene DIN 4102-1 B2 and UL94 VTM-2 classifications were achieved with only 0.5 to 1 wt % of sulfenamide, and, in some cases, no flaming dripping was observed. Also for LDPE thin films, sulfenamides offered the DIN 4102-1 B2 rating at low dosage. In the case of polystyrene, the very stringent UL94 V-0 classification was even achieved at a loading of 5 wt % of sulfenamide.A wide variety of molecules containing S\u2013N or S\u2013N\u2013S cores were synthesized, and their flame retardant properties in polypropylene (PP), low density polyethylene (LDPE) and polystyrene (PS) were investigated. In addition, polymers or oligomers bearing the sulfenamide functionality (SN) were also synthesized. It was shown that this radical generator family based on sulfenamides is very versatile in terms of structural modifications, and the thermal decomposition range can be easily adjusted by changing the In response to the urgent need to develop new high-performance and halogen-free fire resistant materials with low environmental impact , differeMost plastic materials require specific FRs for optimal fire safety; furthermore, FR materials must often fulfill very application-specific fire safety demands in order to pass various fire safety standards established for plastic components. Coating, construction, textiles, electric/electronic (E&E) and transportation industries have additionally various requirements on the additives used in their polymer materials; temperature stability, being solid or liquid, viscosity, polarity and miscibility are only a few properties which need to be matched between a suitable FR, the polymer, the production process and the product. Hence, the optimal flame retardant solution and the obtained level of flame retardancy depend on the polymer type and application. In this respect, flame retardants that can be compounded at temperatures \u2265230 \u00b0C, have high hydrolytic stability, and do not require high loadings (below 10% by weight) in order to achieve compliance with UL94 V-0 would be highly desired. In addition, they should have a reasonable price compared to the halogen-containing flame retardant systems.Along with phosphorus, nitrogen and silicon, sulfur is one of the known flame retardant elements, but compared to the others, sulfur containing compounds have been much less extensively studied as flame retardants. However, disulfide based radical generators alone or in combination with phosphorus compounds have shown good flame retardant effects both in polypropylene (PP) and polystyrene (PS) ,14. ReplR groups attached to S and N, very different kinds of chemical and physical properties can be obtained. Hydrophobicity, thermal properties and stability in different chemical environments can be tailored to fit the application-specific requirements. Both small molecules and polymers can be easily synthesized. The synthesis of first polysulfenamides using secondary diamines and dithiosuccinimid sulfenyl transfer agent was described in 2011 propionate].Synthesis and characterization of the SN compounds Ia\u2014VIb are described in a recently filed patent . All che1H and 13C NMR spectra were recorded on a Bruker Avance 600 . Thermogravimetric analyses (TGA) were performed using an SDT Q600 apparatus from TA Instruments under N2 atmosphere at a heating rate of 10 \u00b0C/min.\u00ae B 225 (0.3 wt %) were added. After one minute, flame retardant was added and the mixing was continued for five minutes. LDPE was processed similarly at 190 \u00b0C but without processing aids. Test films (200 \u03bcm and 1mm) were compression molded in a hot press at 210 \u00b0C for PP and at 135 \u00b0C for PE. Polystyrene formulation was compounded by twin screw extrusion with die temperature set at 200 \u00b0C, followed by injection molding at 200 \u00b0C into 1.6 mm thick strips (127 \u00d7 12.7 mm2) for UL 94 tests. All samples were conditioned three days prior to testing .Flame retardants were melt mixed in polypropylene 12 g/10 min (230 \u00b0C/2.16 kg)) and low density polyethylene ) in a Haake Rheocord melt blender (60 rpm). Polypropylene was melted first for 1 min at 210 \u00b0C, where after processing aids Ca-stearate and IrganoxThe flame retardant effect was investigated in accordance with a modified DIN 4102-1 B2. The flame application time was 15 s with a 40 mm flame height and edge ignition. Polystyrene samples were tested according to UL94 standard .In the course of this study, a series of radical precursors based on sulfenamide derivatives were prepared in order to explore their utility as novel flame retardants for polyolefins. The prepared sulfenamide compounds were divided into six groups based on their chemical structures and their thermal stabilities were examined by TGA studies.The general synthetic pathways for the preparation of sulfenamides have been reviewed in the literature ,31,32,332 or SO2Cl2 in dry aprotic solvents. As an example, synthesis of the compound IIc is described here , diphenyl carbonate and potassium carbonate were dispersed in mineral spirits (65 mL). The reaction mixture was refluxed at 180 \u00b0C for 8 h. The mixture was left to cool down to 50 \u00b0C and washed with warm water (3 \u00d7 60 mL). White precipitate appeared upon cooling down to RT, which was filtrated off and dried under reduced pressure at 90 \u00b0C to remove residues of water and phenol. Pure product was obtained in 51% yield, 5.5 g.1H NMR (CDCl3): 5.04 , 2.04 , 2.02 , 1.25 , 1.20 , 1.17 .13C NMR (CDCl3): 154.28, 72.70, 51.76, 44.05, 35.01, 28.81.2Cl2 (100 mL). The solution was cooled down to \u221218 \u00b0C and Cl2 gas was passed in over 20 min. The solution got a strong orange-red color. The cooling bath was allowed to warm up to \u22125 \u00b0C, and then the flask was removed from the bath and the solution was stirred gently for 30 min under argon flow. The complete conversion of the disulfide into phenylsulfenyl chloride was confirmed by 1H NMR without solvent removal.A three-necked reaction flask equipped with a reflux condenser fitted with an acid gas receiver (15% NaOH solution) was charged with phenyl disulfide and dry CH1H NMR (10% CDCl3): \u03b4 7.39\u20137.45 , 7.65\u20137.7 .2Cl2 was added dropwise. After complete addition, the reaction mixture was stirred at room temperature for 2 h. The organic phase was washed with water (3 \u00d7 10 mL), dried over Na2SO4 and evaporated. The product was recrystallized twice from diethyl ether and the title compound was obtained in 84% yield (0.7 g).Diamine A was dissolved in dry dichloromethane (5 mL) in an argon atmosphere, and triethylamine was added. Then, benzenesulfenyl chloride in CH1H NMR (CDCl3): 7.24 , 7.06 , 5.04 , 2.19, 2.16 , 1.80 , 1.36 , 1.28 .13C NMR (CDCl3): 154.2, 145.0, 128.0, 124.1, 121.7, 70.7, 61.2, 45.3, 32.2, 25.5.R-groups attached to S (R1) and N . Low basicity and increased number of substitutions of the nitrogen atom increase the stability. Thermal stability decreases, if R1 contains groups capable of anchimeric assistance in cleavage of the S\u2013N bond, whereas electron withdrawing substituents on R increase the thermal stability [Thermal stability of sulfenamides is affected by the tability . DelocalShort S\u2013N bond length correlates to the good thermal stability. If the distance between sulfur and nitrogen atoms is less than normal 1.73 \u00c5 for a single bond, the bond exhibits some \u03c0-character ,35,36. TThermal weight loss curves for sulfenamides Ic, IIc, IIIa, Vb, VIa and VIb are shown in All polypropylene, low density polyethylene and polystyrene formulations containing various sulfenamides as flame retardant additives successfully pass the DIN 4102-1 B2 classification. According to the DIN 4102-1 standard, a 20 mm flame is applied for 15 s, and the average burning time when the flame is removed is recorded. However, in many cases, the sample self-extinguished already before the flame was removed, and only melting was detected after that. Therefore, in The first group of synthesized sulfenamides based on 2,2,6,6-tetramethylpiperidinone, i.e., Ic\u2013Id, with different thioaryl groups exhibited all very similar and short burning times in thin polypropylene films of 200 \u03bcm .Additional fire tests were performed using Ic in thick polypropylene plaques of 1 mm in accordance with DIN 4102-1 standard and films of 300 \u03bcm based on a UL94 VTM test standard. The results revealed that even polypropylene plaques and films containing 0.5 wt % of Ic passed the criteria\u2019s set forth for DIN 4102-1 B2 see and UL94The prepared LDPE samples flame retarded with Ia and IIa passed the DIN 4102-1 B2 test. Both burning time and damage length were, however, somewhat longer than for the corresponding PP thin samples .The second group of sulfenamides was based on bis that are less volatile than group I sulfenamides. The samples self-extinguished even before flame removal . The rec\u00ae 5050 H and Sabo\u00aestab UV 94 are shown in The third group of sulfenamides consisted of polymeric sulfenamide structures. Tentative structures for the reaction products of UvinulSulfenamides belonging to group IV contain benzothiazole moieties, and they were commercially available. Traditionally, these types of sulfenamides are used as non-nitrosamine generating accelerators in rubber vulcanization. Compound IVb was thermally more stable than most sulfenamides used in the rubber industry, and was therefore selected to be the more potent candidate as a flame retardant for PP within this family of sulfenamides. Interestingly, this structure passed the DIN 4102 B2 flammability test, but it was not as effective as the 2,2,6,6-tetramethylpiperidine based sulfenamides of group I .One of the most impressive results was obtained when 5 wt % of sulfenimide IVa was used in 1.6 mm thick polystyrene sticks . Thus, tThe fact that IVa is such an effective FR in polystyrene was attributed to generation of different radicals depicted in In order to study amine moieties with a very high charge dissipation capacity, carbazole based sulfenamide derivatives of group V were synthesized. The thermal stability of compounds Va and Vb was expected to be high due to planarity of N. Both compounds exhibited excellent flame retardant performance at a loading of 1 wt % without any flame dripping. Compound Vb had slightly higher thermal stability and showed better flame retardancy than compound Va. Both compounds also passed the criteria for UL94 VTM-2.The sixth group of sulfenamides contained imide and imidazole moieties. In the imide and imidazole derivatives, the N atom is next to electron withdrawing carbonyl groups. In theory, this type of sulfenamides should have high thermal stabilities and excellent free-radical quenching capabilities. It was found that both VIa and VIb could effectively improve the flame retardancy of polypropylene films.We have shown that, when exposed to a small ignition source, various sulfenamide derivatives can help to self-extinquish films of PP, LDPE and PS already at a very low dosage ranging from 0.5\u20135 wt %. In general, at these very low loadings, various radical generators do not provide sufficient fire protection when the polymers are exposed to more harsh fire scenarios such as in cone calorimeter tests alone, but they can be used to enhance protection given by intumescent or char forming systems, as will be shown in our subsequent paper. However, fire often starts by a small ignition source. If the material around it self-extinguishes, it can effectively prevent fire from spreading. Studies related to their flame retardant mechanism are also under way."} +{"text": "Accurate risk assessment of an individuals\u2019 propensity to develop cardiovascular diseases (CVDs) is crucial for the prevention of these conditions. Numerous published risk prediction models used for CVD risk assessment are based on conventional risk factors and include only a limited number of biomarkers. The addition of novel biomarkers can boost the discriminative ability of risk prediction models for CVDs with different pathogenesis. The present study reports the development of risk prediction models for a range of heterogeneous CVDs, including coronary artery disease (CAD), stroke, deep vein thrombosis (DVT), and abdominal aortic aneurysm (AAA), as well as for Type 2 diabetes mellitus (DM2), a major CVD risk factor. In addition to conventional risk factors, the models incorporate various blood biomarkers and comorbidities to improve both individual and population stratification. An automatic variable selection approach was developed to generate the best set of explanatory variables for each model from the initial panel of risk factors. In total, up to 254,220 UK Biobank participants were included in the analyses. The derived prediction models utilizing Cox proportional hazards regression achieved consistent discrimination performance (C-index) for all diseases: CAD, 0.794 ; DM2, 0.909 ; stroke, 0.778 ; DVT, 0.743 ; and AAA, 0.893 . When validated on various subpopulations, they demonstrated higher discrimination in healthier and middle-age individuals. In general, calibration of a five-year risk of developing the CVDs and DM2 demonstrated incremental overestimation of disease-related conditions amongst the highest decile of risk probabilities. In summary, the risk prediction models described were validated with high discrimination and good calibration for several CVDs and DM2. These models incorporate multiple shared predictor variables and may be integrated into a single platform to enhance clinical stratification to impact health outcomes. Cardiovascular diseases (CVDs) include a range of chronic diseases that impair cardiac and vascular function, which continues to be the leading cause of death in the United States (US). It is projected that over 45% of the US population will suffer from one or more CVDs by 2035 . The heaAs with any chronic condition, appropriate prevention and selective treatment of CVDs are the most effective approaches to reduce their clinical and financial impact. Accurate risk assessment of an individual\u2019s propensity to develop CVDs is essential for personalized health care and primary prevention of these conditions. An increasing number of novel biomarkers have been linked to CVD risk \u201314, implIn this study, we sought to improve CVD risk stratification through the addition of multiple blood biomarkers in CVD risk prediction modeling. We report the development and validation of risk prediction models for a range of heterogeneous CVDs with different pathogenesis, including coronary artery disease (CAD), stroke, deep venous thrombosis (DVT), and abdominal aortic aneurysm (AAA), as well as for Type 2 diabetes mellitus (DM2), a prime CVD risk factor . The afoFig 1). Outcomes for coronary artery disease (CAD), Type 2 diabetes mellitus (DM2), stroke, deep venous thrombosis (DVT), and abdominal aortic aneurysm (AAA) were determined according to documentation using the following International Classification of Diseases (ICDs) for each of the diseases:Baseline data for 502,616 UK Biobank (UKBB) participants collected at assessment centers were used to derive the prediction models. Overall, 95% of the UKBB participants were self-described as white, with women comprising 54.4% of the participant population. The UKBB data was subsequently linked to hospital episode statistics (HES) data from hospitals in England, Scotland, and Wales codes for all CMD outcomes in the HES data. The following ICDs were used: I20\u2013I25 and T82 codes were used for CAD; E11-E14 codes for DM2; G46.3, G46.4, I63, I66, I67, and I693 codes for stroke; I82, O22.3, R09.89, and Z86.7 codes for DVT; and I71 and I79.0 codes for AAA.Self-reporting for CAD, DM2, and DVT.Self-reported medications for CAD and DM2 .Fig 1). However, participants with prior non-target CMD event(s) were not excluded. The above exclusions resulted in five CMD-specific datasets with samples sizes ranging from approximately 466,000 to 481,000. Incident cases were defined as CMD-positive cases per ICD-10 codes and had the date of the event recorded on the HES data after the baseline. Self-reported diagnoses and medications were only used to identify prevalent cases since this information is only available at baseline. Further exclusion of cases due to missing data produced a final five study populations used to develop the prediction models; these had sample sizes of 215,269 (CAD), 233,875 (DM2), 237,411 (stroke), 249,338 (DVT), and 254,220 (AAA) . The exit date was determined as the occurrence of either date of death, end of follow-up , or a CMD event.The age and date of a CMD event were determined based on primary or secondary ICD-10 codes in the HES data corresponding to the earliest hospitalization records. Individuals with more than one CMD diagnosis during a given admission were included in the study samples for corresponding CMD. The date of inclusion in the UKBB was defined as the participant\u2019s baseline and was used as the starting point for time-to-event calculations. Participants with a target CMD event before baseline were excluded from the study sample for the corresponding CMD modeling , systolic and diastolic blood pressure (SBP and DBP), physical activity, current and past smoking history, and family history. Physical activity was assessed as the metabolic equivalent of task (MET) and calculated in hours/week according to the \"Guidelines for Data Processing and Analysis of the International Physical Activity Questionnaire (IPAQ)\u201d . Family Additionally, 22 blood biomarkers, including three blood count tests and 19 biochemical markers, were considered as risk factors. We further considered novel risk factors: self-reported sleep apnea, congestive heart failure, arrhythmia, heart valve problem, irritable bowel syndrome, and hyperthyroidism. The arrhythmia category included atrial fibrillation, atrial flutter, Wolff-Parkinson-White (WPW) syndrome, irregular heartbeat, sick sinus syndrome, and supraventricular tachycardia. The heart valve deficiency category included mitral valve prolapse, mitral stenosis, mitral regurgitation/incompetence, aortic valve disease, aortic stenosis, and aortic regurgitation/incompetence.Fig 1). Training sets were used for model fitting and assessment and variable selection. Testing sets were solely used for assessing models\u2019 discrimination performance and calibration as well as for sensitivity analysis.Python 3.6.6 for Windows x64 was used for the preparation of datasets for each CMD according to the approaches described above. The datasets were further split into 80% training and 20% testing sets using the pseudorandom number generator algorithm with a constant initial seed value of 42 (The Recursive Feature Elimination (RFE) method (scikit-learn 0.20.0 Python library) was used to automatically construct the best set of predictor variables for each CMD model from the initially available panel of candidate risk factors. Multiple random forest binary classification models predicting the occurrence of a CMD event by the end of the follow-up period were constructed based on subsets of variables of decreasing size, and the models\u2019 performance was compared . ConsideThe RFE method was combined with a stratified two-fold cross-validation using the following procedure: 1) 40,000 samples were randomly selected from a training set and split into two equal sized subsets with preserved ratio of positive to negative CMD cases; 2) variables were recursively removed one-by-one, and a model using the remaining variables was fitted to one subset, and its accuracy was evaluated on another subset; 3) this process was run once on each subset, and average accuracy was calculated and used for ranking and removing the weakest variables and selecting the best subset of variables. The RFE variable selection was applied to each CMD separately. The gender variable was forced into all CMD-specific sets of explanatory variables.VIFi for each variable was calculated using the following formula:Ri2 is the coefficient of determination for each variable. Ri2 was calculated by regressing the variable against every other variable using ordinary least squares regression (statsmodels 0.9.0 Python library). Variables with the lowest VIF among all variables with VIF higher than 2 were removed one-by-one until all variables had VIF lower than 2. The VIF variable selection did not improve the calibration when applied to the rest of the CMD models.Additional variable selection based on a variance inflation factor (VIF) detecting correlation between variables was conducted for the DM2 model prior to RFECV to achieve better calibration. Linear Cox Proportional Hazard (PH) models were developed using lifelines 0.13.0 Python library. Continuous variables were scaled to a range between 0 and 1 to allow for a comparison of the magnitudes of regression coefficients. The discriminative ability of the risk prediction models was assessed by Harrell\u2019s concordance index (C-index) \u201326, whick-fold cross-validation was used to assess for overfitting leading to model optimism and to adjust estimates of discriminative ability for this optimism [optimism . A trainO1g is observed CMD events, E1g is expected CMD events, Ng is total observations, and \u03c0g is predicted probability for the gth risk decile group, and G is the number of groups. The testing set was divided into decile groups based on the predicted probability of CMD events for a time horizon of five years. Then, the number of observed CMD events and the sum of the predicted probabilities of CMD events (interpreted as the number of expected CMD events) were calculated in each decile group. The computed Hosmer-Lemeshow statistic was compared to a chi-squared distribution with eight (G-2) degrees of freedom to calculate the P-value. Calibration was visualized using a calibration plot using the predicted risk probabilities plotted against the observed risks for each decile group.Calibration of Cox PH models was evaluated by the Hosmer-Lemeshow goodness-of-fit test . The HosThe performance and sensitivity of prediction models were assessed in four subgroups of individuals with different age and CMD status. Multiple testing sets were created by applying age and disease filters to the general testing datasets. These subpopulations included (1) \u201chealthy\u201d participants without any of four non-target CMD at the baseline; (2) \u201cunhealthy\u201d participants with one or more pre-existing non-target CMD at the baseline; (3) participants with only one non-target CMD ; and (4) participants in the age categories <45, 45\u201355, 55\u201365, and 65\u201375 years.Table 1), as well as biochemical and clinical characteristics (S1 Table), showed no significant variation across different CMD study samples. Assessment of the participant\u2019s follow-up (median 6.1 years) reports, the incidence rates for CAD (3.32%), DM2 (2.65%), stroke (0.66%), DVT (2.44%), and AAA (0.17%) were observed in the corresponding study sample.The mean (SD) age at baseline was 56 (8.0) years across all CMD study samples, with women accounting for 54.0% to 55.6% of the participants in these samples. Gender-specific demographics, physiological and lifestyle characteristics, the number of CMD incident events (Table 2). The models for stroke and AAA, the diseases with lower numbers of incident events, demonstrated a lower reproducibility compared to the other models. Performance assessment in testing sets of the general population (Table 2) demonstrated that C-indexes for the CAD and stroke models were within the above 95% CIs. C-indexes for the DM2, DVT, and AAA models were outside of the CIs by 0.003, 0.001, and 0.005, respectively, consistent with the low degree of overoptimism estimated by the cross-validation method.The discriminative ability of all Cox PH CMD models estimated by five-fold cross-validation varied between the diseases with the highest and lowest C-indexes for DM2 and DVT, respectively. The optimism-corrected estimate of discrimination C-statistic was 0.794 for CAD, 0.909 for DM2, 0.778 for stroke, 0.743 for DVT, 0.893 for AAA. A low standard deviation of C-statistic values for the CAD, DM2, and DVT models implied their high reproducibility and good generalization to unknown data from the same population and a low degree of overoptimism (S2 Table). Among multiple biomarker predictors shared across different models, cystatin C and red blood cell distribution width were common risk factors for all four CVDs, but not for DM2. Comparison of the values of normalized regression coefficients (S2 Table) demonstrated that cystatin C was the most crucial risk factor for stroke, DVT, and AAA, and was superseded by glycated hemoglobin only in the CAD model. Glycated hemoglobin also was the most important risk factor for DM2 and was shared among stroke, DVT, and DM2; however, it was not a statistically significant variable for AAA. Overall, the CAD and stroke models shared the largest number of predictors among all diseases.The number of predictors in the best sets generated for different models varied from nine predictors for AAA to 40 for CAD (Table 2). This analysis demonstrated lower prediction in the \u201cunhealthy\u201d compared to the \u201chealthy population,\u201d as defined in the material and methods. The performance of the models was highest in middle age (45\u201365 years), but it significantly dropped in the subpopulations with pre-existing CMD.Broad range applicability and performance consistency for the developed risk prediction models for each disease was further determined by assessing the discriminative ability across subpopulations using sensitivity analysis of 33.8 (<0.0001), 77.8 (<0.0001), 12.8 (0.12), 45.3 (<0.0001), and 12.3 (0.14) for the CAD, DM2, stroke, DVT, and AAA models, respectively. Calibration plots demonstrated consistent overall calibration, but overestimation of CMD risk in the highest decile of risk probabilities for all except the DM2 model. DM2 risk was slightly overestimated in the lowest deciles and minimally underestimated in the highest risk decile. As expected, with a low number of events, the prediction model for AAA was poorly calibrated.To evaluate the calibration properties of the prediction models amongst the general population, the five-year absolute risk for each CMD event was calculated. Hosmer-Lemeshow test statistic produced Fig 3) to evaluate the discriminative ability of individual risk models. The mean risk probabilities for all CMDs exponentially decreased with increased survival time. A steeper exponential decay was observed for the DM2 and AAA models, which demonstrated the highest discriminative ability for these two diseases.The mean risk probability versus mean survival time for each decile was plotted , DVT 4040, AAA and DM2 Given the predominantly white UK Biobank population and the fact that both training and testing datasets were produced from the same population, the developed prediction models are unlikely to be generalizable to other populations, and their transportability requires further assessment in external validation studies. Low transportability is a common limitation of prediction models, including established CVD risk algorithms. It was reported that neither the Framingham (derived from a US cohort ) nor ASSComparison of risk factor associations in UK Biobank against representative, general population a recent study by Batty et al. demonstrConsidering computational limitations of non-linear survival models , binary S1 Table(XLSX)Click here for additional data file.S2 TableP-values, lower and upper 95% CI are presented.Regression coefficients (coef) and corresponding standard errors (se), (XLSX)Click here for additional data file."} +{"text": "Intestinal perforation is a rare adverse event of antineoplastic therapy. However, once it occurs, it is potentially fatal. This report describes a case of intestinal perforation caused by bevacizumab in a patient with ovarian cancer who concurrently developed neutropenic enterocolitis. A 66-year-old woman diagnosed with metastatic ovarian cancer received combination chemotherapy with carboplatin, gemcitabine, and bevacizumab. On day 14, she developed grade 4 pancytopenia and febrile neutropenia, which resulted in neutropenic enterocolitis and intestinal perforation. Emergency surgery was performed, and an intestinal perforation found in the ascending colon was closed. Postoperatively, she developed an intra-abdominal abscess requiring peritoneal drainage. She was discharged from the hospital on recovery. VEGF also plays a role in tumor growth and is the key target of certain antitumor drugs . Bevacizramework . The efframework \u20136. Gastrramework . SurgeryThis report describes our experience with a patient who developed intestinal perforation due to the combined effect of neutropenic enterocolitis and bevacizumab.2, day 1, every 3 weeks). She experienced grade 2 neutropenia during the first cycle; however, she recovered from this adverse event without the complication of infections. In addition, she achieved partial response in CT after two cycles of chemotherapy. Interval debulking surgery (IDS) was performed after seven cycles of this chemotherapy. Total hysterectomy, salpingo-oophorectomy, and infracolic omentectomy were performed for this patient. No major postoperative complications were observed in this case. On recovery, she received two cycles of adjuvant chemotherapy. Despite achieving complete response following treatment, she presented with recurrent peritoneal dissemination of the tumor, seven months after the last chemotherapy cycle. She was diagnosed with platinum-sensitive relapsed ovarian cancer and was prescribed combination chemotherapy with carboplatin , gemcitabine , and bevacizumab .A 66-year-old Japanese woman presented with symptoms of abdominal distension and anorexia and was diagnosed with ovarian cancer (clear cell carcinoma), stage IIIC. She received neoadjuvant combination chemotherapy with carboplatin and paclitaxel (175\u2009mg/mShe did not experience any adverse events for several days after administration of second-line chemotherapy. However, on day 14 of the first cycle, she presented to the hospital with fever and was subsequently diagnosed with febrile neutropenia owing to severe reductions in absolute neutrophil counts, which was evident from the laboratory data . She alsIn the present case, intestinal perforation was induced by bevacizumab in the presence of neutropenic enterocolitis. Intestinal perforation is a fatal condition, particularly in neutropenic patients; urgent surgical intervention is essential for its management. Prompt assessment and formulation of a management plan are key factors in improving patient outcomes. In the present case, treatment of the intestinal perforation was successful owing to early detection and treatment.1 antibody, has demonstrated clinical benefit in ovarian cancer and has been available in Japan since 2013. Although bevacizumab shows considerable benefits, several adverse events have been reported with its use. Gastrointestinal adverse events are common with bevacizumab, and nausea, anorexia, and constipation are frequent. However, gastrointestinal perforation is a rare event, occurring in 0.9% to 3% of these patients [Bevacizumab, a humanized anti-VEGF monoclonal IgGpatients \u201310; it lpatients . Bevacizpatients . A reporSeveral risk factors predispose patients to develop gastrointestinal perforation. Medical conditions associated with increased risk of perforation include prior bowel surgeries, bowel obstruction/ileus, diverticulitis, and rectovaginal nodularity . CertainIn the present case, neutropenic enterocolitis was probably responsible for increasing the risk of intestinal perforation associated with bevacizumab. Neutropenic enterocolitis occurs as a result of injury to gut mucosal integrity in severely neutropenic patients. It follows treatment with cytotoxic chemotherapy, including carboplatin and paclitaxel . It is hTwo factors, namely, the administration of bevacizumab and the occurrence of neutropenic enterocolitis, might have synergistically led to gastrointestinal perforation in the present case. The bowel wall might have been initially inflamed and damaged by bacterial invasion resulting from neutropenic enterocolitis; concurrent bowel ischemia induced by bevacizumab probably prevented healing of the intestinal mucosa, contributing to intestinal perforation. Since it is a rare complication, neutropenic enterocolitis has not been recognized as a risk factor for gastrointestinal perforation.The present patient also had thrombocytopenia of grade 4 on admission. Although severe pancytopenia is rare in patients receiving the gemcitabine/carboplatin plus bevacizumab regimen, it is a severe adverse event . Severe To the best of our knowledge, this is the first reported case of intestinal perforation resulting from neutropenic enterocolitis in a patient treated with gemcitabine/carboplatin plus bevacizumab for ovarian cancer. Neutropenic enterocolitis is very rare among patients treated with bevacizumab. However, it is necessary to recognize the considerable increase in the risk of bevacizumab-related gastrointestinal perforation in patients having neutropenic enterocolitis consequent to cytotoxic chemotherapy.In conclusion, bevacizumab in conjunction with neutropenic enterocolitis led to intestinal perforation in this patient with ovarian cancer. Patients undergoing treatment with bevacizumab should be carefully evaluated for concurrent neutropenia. In the event of intestinal perforation, it is essential to institute immediate and appropriate measures for management."} +{"text": "Drosophila melanogaster. To comprehensively determine how Hippo signalling interacts with other pathways in this regulation, we screened all known signalling pathway genes, and identified Hpo-dependent and Hpo-independent signalling requirements. Network analysis of known protein-protein interactions among screen results identified independent gene regulatory sub-networks regulating one or both of ovariole number and egg laying. These sub-networks predict involvement of previously uncharacterised genes with higher accuracy than the original candidate screen. This shows that network analysis combining functional genetic and large-scale interaction data can predict function of novel genes regulating development.Understanding the genetic regulation of organ structure is a fundamental problem in developmental biology. Here, we use egg-producing structures of insect ovaries, called ovarioles, to deduce systems-level gene regulatory relationships from quantitative functional genetic analysis. We previously showed that Hippo signalling, a conserved regulator of animal organ size, regulates ovariole number in The final shape and size of an organ is critical to organismal function and viability. Defects in human organ morphology cause a multitude of pathologies, including cancers, organ hypertrophies and atrophies e.g. . It is tDrosophila melanogaster female reproductive system is a useful paradigm to study quantitative anatomical traits. In these organs, the effects of multiple genes and the environment combine to produce a quantitative phenotype: a species-specific average number of egg-producing ovarian tubes called ovarioles. Fruit fly ovaries can contain as few as one and as many as 50 ovarioles per ovary, depending on the species and Engrailed and the large Maf factor Traffic Jam , and on E-cadherin dependent adhesion . Initialadhesion . Regulatadhesion .hippo; hpo) and Warts , which prevent the nuclear localisation of the transcriptional co-activator Yorkie . Yki and the transcription factor Scalloped together initiate the transcription of multiple target genes, including those that promote cell proliferation and survival. In the D. melanogaster larval ovary, loss of Hpo in the somatic cells causes an increase in nuclear Yki, leading to an increase in TFCs, TFs, ovariole number and egg laying in adults or the TRiP collections at the Bloomington Drosophila Stock centre (BDSC) .To systematically ascertain the function of signalling pathway genes and their interactions with Hippo signalling in the development of the ng genes . We idennogaster , and obthpo in the somatic cells of the larval ovary using traffic jam Gal4 driving hpo[RNAi] increased both ovariole number and egg laying of adult female flies over five days. To address batch variation (geneZ\u00a0=\u00a0number of standard deviations from the mean) for each candidate line relative to its batch controls. 190 genes had an egg laying |geneZ| below 1. Previous studies have shown that the egg laying of newly eclosed adult mated females correlates with ovariole number during the first five days for the 273 remaining candidate genes. For the third screen . We therefore selected the 67 genes with a |geneZ| above two for ovariole number Betweenness centrality measures the number of shortest paths amongst all the shortest paths between all pairs of proteins that require passing through a particular protein. (3) Closeness centrality measures the average shortest path that connects a given protein to all other proteins in the network. (4) Eigenvector centrality is a measure of the closeness of a given protein to other highly connected proteins within the network.The finding that these reproductive traits were regulated by the genes of all signalling pathways led us to consider the broader topology of putative gene regulatory networks in the analysis of our data. Previously characterised genes in the ovary are often pleiotropic and can regulate both ovariole number and egg laying . As withD. melanogaster proteins, from the combination of publicly available protein-protein interaction (PPI) studies in the DroID database . We then calculated the four centrality measures described above for all genes within the D. melanogaster PIN (geneZ| threshold) are reflected in an area under the curve (AUC) of more than 0.5. We found that the higher the centrality score, the greater the likelihood that a gene had |geneZ| values above our threshold for effects on ovariole number and egg laying r screen . Interesr screen .Drosophila PIN. We performed our modularity test using four commonly measured network metrics: (1) Largest Connected Component (LCC) (the number of proteins or nodes connected together by at least one interaction), (2) network density , (3) total number of edges, and (4) average shortest path (average of the minimum distances connecting any two proteins). We considered a sub-network to show modular features if they showed most of the following properties: higher LCC, higher network density, more edges, and shorter average shortest path length, when compared to a similarly sized, randomly sampled selection of genes from the PIN.We then asked whether the members of these four seed lists were more connected than would be expected by chance. In other words, we formally tested them for modularity as defined above. Meeting our criteria for modularity would suggest that the genes in these phenotypically separated seed lists might operate together as putative functional modules within the To determine whether these criteria would correctly identify signalling genes, which are known to function together as a module, we measured these four parameters in the original set of genes used in this study . We founBased on published molecular interactions, in addition to the four criteria described above, further evidence for putative functional modules of genes can also be obtained by applying algorithms that use either the shortest path method or the Shpo[RNAi] Egg Laying module , and much higher within group IV (Z score\u00a0>3) . This shWe then asked if these seven sub-groups were as connected to each other, as were the genes within each of the sub-groups, again using the edge density assessment as described above . This anUAS:RNAi lines for each connector, driven by tj:Gal4 to measure the effects of knocking down each of the connector genes , smoothened (smo) and costa (cos) . Howeverta (cos) , does not tissue . In anot cascade .hpo[RNAi] Ovariole Number sub-network is composed of genes that affect the Hippo signalling activity-dependent determination of ovariole number during development. Establishment of ovariole number occurs largely during the third instar stage of larval development in D. melanogaster (engrailed (En) and retinoblastoma-family protein (rbf), which we hypothesise could also be involved in the specification of ovariole number. bun and rbf have been implicated in the migration (The nogaster . During nogaster . TF specled (En) and the ac locus . A thirdac locus . Our hpoigration and endoigration of the ftwinstar, is essential to this process (hpo[RNAi] Ovariole Number sub-network further identifies Rho1 (Rho kinase (Rok) , was also identified by our hpo[RNAi] Ovariole Number sub-network. msn encodes a MAP kinase previously shown to regulate the polarisation of the actin cytoskeleton during oogenesis as necescalation . Given tskeleton , we propogenesis , but hasdiscs large 1 (dlg1) and prickle (pk) in the ovariole sub-network. During the maturation of the TFs during larval development, the TFCs undergo significant cell shape changes, coincident with localised expression of beta-catenin and actin to the lateral edges of the TFCs .This study did not generate new unique reagents. This study generated new python3 code available on GitHub: extavour@oeb.harvard.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Cassandra G. Extavour (Drosophila melanogaster were obtained from publicly accessible stock centers and maintained as described in \u2018Fly Stocks\u2019 below. Genotypes and provenance are provided in the Key Resource Table. Candidate genes were randomly assigned to batches for screening (see the Wild type and mutant lines of Drosophila food (Drosophila Stock Centre (BDSC) and from the Vienna Drosophila Resource Centre (VDRC). See Key Resources Table for complete list of stocks used in this study. Oregon R was used as a wild-type strain. The genotype of the traffic jam:Gal4 line used in the screen was y w; P{w[+mW.hs]=GawB}NP1624 . The hippo RNAi line used in the screen was y[1] v[1]; P{y[+t7.7]v[+t1.8]=TRiP. HMS00006}attP2 .Flies were reared at 25\u00b0C at 60% humidity with standard ila food containiila food . RNAi liScreen design\u2019 below) with two males in a vial containing standard food and yeast granules (day one) and then transferring them into a fresh food vial without yeast granules for a 24 hr period. Eggs from vials were then counted by visual inspection of the surface of the food in the vial. Males and females were transferred to fresh food vials without yeast granules, every day thereafter until day six. All egg -laying measurements reported and analysed in the paper are the sum of the eggs laid by three adult female flies over the five days of this assay (days two through six without yeast granules). Data from any vial in which either a female or male died, during the course of the experiment, were not included in the analysis.Adult egg laying was quantified by crossing three virgin females of the desired genotype . Ovarioles were separated from each other with No. 5 forceps (Fine Science Tools) and counted by counting the number of germaria under a ZEISS Stemi 305 compact stereo microscope with a NIGHTSEA stereo microscope UV Fluorescence adaptor.hpo[RNAi] Egg Laying), 463 candidate genes (hpo[RNAi] background on the number of eggs laid in the first five days of mating (see \u2018Egg and ovariole number counts\u2019 above) by adult females. These females were the F1 offspring of UAS:candidate gene RNAi males crossed to P{w[+mW.hs]=GawB}NP1624; P{y[+t7.7] v[+t1.8]=TRiP.HMS00006}attP2 virgin adult females (hpo[RNAi] Egg Laying). All genes that yielded an egg laying count with a |geneZ|\u00a0>\u00a01 (see \u2018Gene selection based on Z score and batch standardisation\u2019 below) were selected to undergo two secondary screenings virgin adult females were scored for ovariole number (see \u2018Egg and ovariole number counts\u2019 above). (hpo[RNAi] Ovariole Number).In the primary screen virgin adult females. Because the control group in each batch had slightly different distributions of egg laying and ovariole number values (geneZ) to its batch control group was used as a discriminant. This approach standardises for batch effects and allows the comparison of all genotypes within and across the primary and secondary screens with a single metric (geneZ).Candidate genes were screened in batches with an average size of 50 genes. For each batch, control flies were the female F1 offspring of Oregon R males crossed to r values , it was b and \u03c3b respectively. Then, using the number of eggs laid by adult females of a candidate gene RNAi (genex) of the same batch, the Z score for the egg laying count of that gene (geneZ) was calculated as Firstly, the mean and standard deviation of the eggs laid by the control genotype for a batch were calculated as \u03bcgeneZ|\u00a0>\u00a02 was applied for ovariole number phenotype. Egg laying counts were derived from measurements of three females in a single vial per gene. We therefore chose to be more conservative in our Z score comparisons for the egg laying phenotype, than for ovariole number phenotype, and applied a stringent threshold of |geneZ|\u00a0>\u00a05 to select genes of interest. All genes with |geneZ| values above these thresholds are referred to throughout the study as \u2018positive candidates\u2019. Ovariole numbers were derived from counts of the number of ovarioles per ovary for 20 ovaries per candidate gene, and a threshold of |To study the enrichment of a particular signalling pathway in a group of candidate genes that had similar phenotypic effects revealed by the screen, custom scripts were generated to implement two different methods .D. melanogaster signalling genes 10,000 times, and counted the number of genes (M) that were members of the signalling pathway S. Positive candidates in each of the three screens were sorted by their presence in signalling pathways and counted. The Z score was then calculated by comparing the experimentally observed number of positive candidates in each signalling pathway against the randomly sampled null distribution.The first method is a numerical method that uses random sampling to calculate the null distribution of the number of members (M) of a signalling pathway (S) that would be expected at random in a set of genes of size (N). The script randomly sampled N genes from among the 463 tested D. melanogaster signalling genes, and the known attribution to a pathway S of each gene.The second method used the hypergeometric p-value to calculate the probability of M members of a signalling pathway being in a group of N genes, given a starting population of 463 tested Drosophila melanogaster. However, there exist many smaller networks from different screens, as well as literature extractions. We therefore combined data from these sources and then created a PIN for use in the present study, as follows:There is no standard complete Protein-Protein Interaction network (PIN) available for http://www.droidb.org. The version DroID_v2018_08 was used. All available datasets were first downloaded from that database using this link: http://www.droidb.org/Downloads.jsp. The description of all of these datasets can be found here: http://www.droidb.org/DBdescription.jsp.Several screens assessing protein-protein interactions have been centralised in a database called DroID: We used the datasets from all screens that assessed direct protein-protein interactions and did not use the interolog database (predicted protein interaction based on mouse human and yeast PPIs). These direct assessment screens were seven in total, as follows:Finley Yeast Two-Hybrid Data Curagen Yeast Two-Hybrid Data Hybrigenics Yeast Two-Hybrid Data Perrimon co-AP complex DPiM co-AP complex PPI from other databases PPI curated by FlyBase We did not consider self-loop edges from proteins predicted to interact with themselves (homotypic or self-interactions). An important element to note is that the PPIs curated by FlyBase is a literature-based PPIs. FlyBase protein-protein interactions are experimentally derived physical interactions curated from the literature by FlyBase and does not include FlyBase-curated genetic interactions.We concatenated the seven datasets listed above into a single unique database. A custom python script was created that downloads and reads each of the above seven unique PPI tables, and generates a single PIN (see 01_PIN_builder.ipynb). From this concatenation, a single-edge undirected network was created and saved. This network is hereafter referred to as the PIN (see 01_PIN_builder.ipynb). The PIN contains 10,632 proteins (nodes) and 85,019 interactions (edges), giving a network density of 0.0015.The centrality of a node is often used as a measure of a node\u2019s importance in a network. Within a PIN, the centrality of a gene reflects the number of interactions in which the gene directly or indirectly participates. Four different centrality metrics were computed for all genes in the PIN using the networkx python library:Betweenness reflects the number of shortest paths passing through a gene.Eigenvector is a measure of the influence of a gene in the network.Closeness measures the sum of shortest distance of a gene to all the other genes.Degree centrality corresponds to the normalised number of edges of a gene in the network.While there exist more centrality measures, these four are commonly used to assess biological networks. These computed centrality parameters of the genes measured in the screen were computed with 03_ROC_curve_analysis_of_network_metrics.ipynb, and are reported in the geneZ| being above or below the appropriate |Z score| threshold (>5 for egg laying and\u00a0>2 for ovariole number). The Area Under the Curve (AUC) measures the extent of correlation between centrality and effect of a gene on measured phenotype. AUC above or below 0.5 indicates a positive or negative correlation respectively, while an AUC of 0.5 indicates no correlation of the parameters. The scikit-learn python package was used to calculate the AUC of each ROC curve plotted .To check whether the centrality of a gene in the network could predict the phenotypic effect produced by RNAi against that gene, ROC curves were plotted for the four aforementioned centrality measures of each gene in each screen. A ROC analysis is used to measure the correlation between a continuous variable and a binary outcome (above or below Z score threshold). Therefore, for each screen, measured genes were rank ordered from high centrality to low centrality, and plotted against the binary outcome of |We assessed the modularity of the networks by comparing the network metrics of each sub-network to a degree-controlled randomly sampled network. To generate this degree controlled random network, we applied a previously developed method . In shorgeneZ| value was above the threshold and created three seed lists, respectively (hpo[RNAi] egg laying and hpo[RNAi] ovariole \u2018seed\u2019 list). A fourth list consisting of the intersection of the aforementioned seed lists was also collated and called the core \u2018seed\u2019 list (hpo[RNAi] Egg Laying seed list and CG6104 in the hpo[RNAi] Ovariole number seed list were absent from the PIN) and were therefore eliminated from further network analysis. The removal of these two genes accounts for the variation in the number of positive candidates in Network modules were assessed using the previously published Seed Connector Algorithm (SCA) , implemeed\u2019 list . Genes wThe modularity of the sub-networks was then assessed using four network metrics namely Largest Connected Component (LCC), number of edges, network density and average shortest path in the LCC. Each metric for each module was assessed using distance of the network metric to a null distribution. Initially, the null distribution was calculated by taking 1000 samples of 463 genes randomly selected from the PIN and calculating the above metrics. We found that the 463 genes selected in the signalling screen were already more connected than the null distribution of sets of 463 genes randomly selected from the PIN . TherefogeneZ| value was above the threshold in more than one and possibly all three of the screening paradigms. For example, if a gene was found in the hpo[RNAi] Ovariole Number and Egg Laying sub-networks it is then assigned to the dual positive group hpo[RNAi] Ovariole Number/Egg Laying in To build the meta network, the genes from all four sub-networks were concatenated into one network. This network was then visually sorted in an approach akin to projecting the network onto a Venn Diagram. The meta network was sorted by which of the three screens the gene was positive in. The intersections were genes whose |The number of samples across the different screens were as follows:Hpo[RNAi] Egg Laying and Egg Laying screensControls: five vials of three females and two malesSample: one vial of three females and two malesHpo[RNAi] Ovariole number screenControls: 20 flies, two ovaries per fly considered as independent measurementsSample: 10 flies, two ovaries per fly considered as independent measurementsgeneZ) to the control distribution. For each batch, the measurements for controls were pooled into a distribution, and the mean and standard deviation was computed. Then each sample was compared to its respective batch and its Z score computed (see \u2018Gene selection based on Z score and batch standardisation\u2019 for formula).Despite best efforts to maintain the exact same condition between each experiment, some variation was measured between the batches. Control flies showed variations in both measured phenotypes, ovariole number and egg laying . In ordehttps://www.scipy.org/) and scikit-learn (https://scikit-learn.org/) python package. Statistical tests and p-values are reported in the figure legends. All statistical tests can be found in the Ipython notebooks mentioned below.All statistical analyses were performed using the scipy stats module (https://cytoscape.org/) can be reproduced by running the aforementioned python3 code. The raw data, calculations made with these data, and code used for calculations and analyses (Ipython notebooks) are available as supplementary information. For ease of access, legibility and reproducibility, the code and datasets have been deposited in a GitHub repository available at https://github.com/extavourlab/hpo_ova_eggL_screen.This study generated a series of python3 Ipython notebook files that perform the entire analysis presented in this study. All the results presented in this paper, including the figures with the exception of the network visualisations, which were created using Cytoscape3 ; Luke Lambourne (Reviewer #3).Thank you for submitting your article \"Topology-driven protein-protein interaction network analysis detects genetic modules regulating reproductive capacity\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to proceed without manuscripts, like yours, that they judge can stand as This work aims to predict protein-protein interaction in a developmental context with a combination of approaches drawn from developmental genetics and system biology. This work represents an elegant study that combines computational biology and molecular developmental biology to explain how organ systems are formed.Although it is our usual practice to combine the reviews into a single consensus, in this case I think the independent reviews offer distinct takes on the manuscript and suggestions for revisions and stand better on their own, although to expedite your revisions, I will summarize the main issues to address here:Analysis:Several issues were raised by reviewer 3 and during discussion:1) Additional analysis is needed to support claim of non-random modulesIn particular, the results of Figure 4 show that the positive pairs in the screen on average, tend to have higher degree in the network, which could be responsible for the higher connectivity of the seed genes, rather than them forming modules. This should be addressed with some form of control analysis.2) Module separationAs a general rule, it is not surprising that the connectivity is higher within the output of the SCA algorithm, which returns a subnetwork of connected nodes, to the connectivity between two different SCA produced sub-networks. This is expected to happen with random lists of seed genes, and does not prove that functionally separate sub-networks have been identified. The claim is confusing given that there are shared subnetworks which seems to contradict the idea of separate modules. Reviewer 3 offers several suggestions for how to make this analysis more convincing.Writing and presentation:eLife. However there is a sense that the manuscripts lacks a clear take home message. This is not required for publication per se, but we all feel the manuscript could be made stronger in revisions by attempting to distill a take home message for readers.1) In addition to suggestions about presentation in the reviews below, during discussion all three reviewers highlighted the value of the overall approach and felt that the manuscript merits publication in 2) The experimental validation results are not presented clearly. That section could benefit from more clarity in what specific aspects of the predictions are being tested and how the specific tests address them.Reviewer #1:Drosophila ovary development and the rate of egg production. An earlier study from this lab identified a role for hippo signaling in these processes. Here, they performed an RNAi-based screen through 463 candidate genes involved in signal transduction to identify genetic modifiers of the hippo[RNAi] phenotype as well as genes that regulate the rate of egg laying through a potentially hippo-independent mechanism. The screens were well-designed and produced quantitative data that the authors could use to rank-order genes according to the strength and direction of the phenotype. Their strategy for assessing the rate of egg laying is in line with standard approaches in the field and their method of counting ovariole number is entirely appropriate. These screens identified a long list of genes, and they took a systems biology approach to assess the individual and combinatorial contributions of these genes to ovary development and function. These types of analyses are outside my area of expertise so I could not assess whether the appropriate computational methods were chosen and applied correctly. However, the writing of this section was clear to a non-expert and, taking their claims at face value, the data seem to support their interpretations.In this study, the authors investigate the signaling pathways that regulate In the final section of the manuscript they describe how their network analysis identified additional genes not uncovered by their screens and show that RNAi knockdown of these additional genes do, indeed, cause phenotypes in egg laying rates and ovariole number. This is a nice validation of the approach. As a minor point, the data presented in Figure 7 only summarize how many genes in the list showed a phenotype in each category but did not indicate what the genes are, which ones are associated with which phenotype, or what the z-scores were in each case . If it has not been included already, the authors should provide this for completeness and to allow comparison to the other data in the study.Overall, I think this study will be a useful though perhaps somewhat limited contribution to the field. The results of the screens provide a large amount of data for the field to build on and I appreciate their holistic approach to describing the interplay between genes and pathways in the regulation of processes as complex as development and oogenesis. At the same time, I struggled a bit to extract the most important take-away messages that change how I think about these processes. For example, they state in the Discussion that the core module consists of housekeeping genes and hedgehog pathway genes, but this could have largely been predicted from previous publications on this topic. Likewise, terminal filament formation is known to involve cell migration and, consistent with this, they find that several genes involved in cytoskeletal dynamics are important for proper specification of ovariole number. Though some of these genes have not been studied in this context, their involvement here is not too surprising. Nonetheless, it may be that the main innovation of this study is in the systems biology approach they have used to analyze the data, so I look forward to the online discussion with the other reviewers who have more expertise in this area.Reviewer #2:A general assessment of the workThis manuscript aims to predict protein-protein interaction in a developmental context. By using tissue-specific RNAi screening and systems biology approaches, the authors found known and unknown genes involved in ovarian development and function. A plethora of unknown genes was functionally tested, giving support to the topology-driven network analysis conducted, and the developmental regulatory modules found. This work represents an elegant study that combines computational biology and molecular developmental biology to explain how organ systems are formed.Numbered summary of any substantive concernsThis article is generally well-structured, the data analysis is performed in a proper fashion, and the results are nicely presented, giving support to the major conclusions. I have no major concerns with regard to this manuscript, only some minor edits.1) I am wondering if authors can give more detail regarding the paragraph starting \"To choose candidates.\u2026\" I might be wrong, but have the feeling that this approach might produce bias in the results obtained by the authors.gene scores of positive candidate genes sorted by centrality metrics. However, I wonder why the authors did not calculate p-values (significance level) between 1st and 5th quantile comparisons of Zgene scores shown in this figure? Significance levels in these comparisons might give strong support to the weak prediction of a gene that would affect a specific phenotype.2) In Figure 4\u2014figure supplement 2, the authors show a comparison of Z3) In Figure 6, for the sake of clarity, it is necessary to show the sloppy paired 1 gene in the meta-network analysis . As is now shown in this figure, readers cannot see easily the importance of this gene within sub-network VII, which I think is one point that the authors want to highlight.4) In step 3 in the protein-protein interaction network (PPI) building section (\u201cMethod Details\u201d subsection), authors indicate that a custom python script was created to download and reads each of the PPI tables from DroID database, but I could not find this python script in the Github repository in the paper. Please, can authors upload this script in the repository?Reviewer #3:Drosophila with protein-protein interaction networks. Highly interesting, interdisciplinary work lying at the intersection of developmental and network biology. They identify genes regulating ovariole number and egg laying and analyze those genes in the context of PPI networks, predicting and experimentally testing additional novel reproductive regulatory genes with some success. However there are serious issues with the analysis supporting key claims in the manuscript related to network topology.The authors present a novel study integrating the results of performing screens for regulators of reproduction in 1) The claim of non-random modules is not supported by the analysis.\u2013 The claim in the section title \"Genes regulating egg laying and ovariole number regulation form non-random interaction modules\" (L260-L261) is supported by Figure 5\u2014figure supplement 4. In that figure both the seed genes and results from the SCA algorithm are compared to the subnetworks formed by randomly selected genes from the initial screen. This is a sensible comparison for the seed genes but not for the output of the SCA algorithm, since that algorithm builds a LCC and so will obviously have a much larger LCC and be much more connected than randomly selected genes from the network.\u2013 The results of Figure 4 show that the positive pairs in the screen on average, tend to have higher degree in the network, which could be responsible for the higher connectivity of the seed genes, rather than them forming modules. To account for this, the seed genes could be compared to a null distribution of degree-controlled randomized networks.\u2013 The text reads \"decreased average shorted path\" (L331) but it is increased in 7 of 8 cases in the figure.2) The claim of separate modules is not supported by the analysis.\u2013 Similarly to point 1, it is not surprising that the connectivity is higher within the output of the SCA algorithm, which returns a subnetwork of connected nodes, to the connectivity between two different SCA produced sub-networks, that will happen with random lists of seed genes, it does not prove that functionally separate sub-networks have been identified.\u2013 There are sizable shared subnetworks which seems to contradict the idea of separate modules.2 \u2013 x) / 2 where x is the number of nodes in the group (assuming self-interactions are not considered), the edge density calculations between the different groups are correct.\u2013 The heat-map Figure 6B, shows very low edge density within the groups , in contradiction to what is written in the text. This appears to be due to a miscalculation in the file 08_MetaModule_Analysis.ipynb in the Github repository: within the group the number of edges should be divided by .3) The results of experimentally testing the predictions are presented in an unclear way, making it difficult for the reader to assess their accuracy.The results in Figure 7A/B/C appear to show that the predictions from the modules are just as good for the phenotypes not associated with the module as for those associated with the module, e.g. for the EL phenotype there is one correct prediction from both the Core and EL modules and 9 correct predictions from the hpo[RNAi] ON/EL modules, with a similar number of predictions. So it would seem that these results suggest that, although there is positive predictive value overall relative to the initial signaling genes list, that the separate modules predicting their associated phenotype separately from the others is not supported by the data. This is not discussed in the text.\u2013 From what I could understand, the fractions for the initial signaling gene lists for the EL and hpo[RNAi] ON phenotypes are not exactly consistent with the compared fractions of the predictions, due to the filtering applied on the hpo[RNAi] of |Z| > 1, for the initial screens. Perhaps the fractions for the 2 affected bars in Figure 7C could also be calculated with this threshold applied to remove genes?\u2013 There are no p-values calculated, comparing the predictions to the original screens and no error bars shown in Figure 7.\u2013 The title of Figure 7C could be changed to make it clearer that this contains predictions from all modules.\u2013 There is no \u201cAll screens\u201d column in either Figure 7C or Figure 7D for comparison. 1) Although it is our usual practice to combine the reviews into a single consensus, in this case I think the independent reviews offer distinct takes on the manuscript and suggestions for revisions and stand better on their own, although to expedite your revisions, I will summarize the main issues to address here:2) Analysis:Several issues were raised by reviewer 3 and during discussion:(2.1) Additional analysis is needed to support claim of non-random modulesIn particular, the results of Figure 4 show that the positive pairs in the screen on average, tend to have higher degree in the network, which could be responsible for the higher connectivity of the seed genes, rather than them forming modules. This should be addressed with some form of control analysis.We understand the concerns of the reviewer and we have modified our analysis to better support the claim of putative non-random modules. Firstly, we performed a new analysis, comparing the network metrics of the seed genes, to two different control groups: (a) a random selection of the same number of genes, chosen from among the signalling candidates, and (b) a degree-controlled random network of the same number of genes . Our results indicate that in most cases, the seed gene lists display higher network metrics than both null distributions. We therefore interpret this as evidence that the seed genes may constitute a non-random putative interaction module, independent of application of the SCA.On application of the SCA, we find that there is reduction of the difference in network metrics when compared to the two null distributions described above, of similarly sized seed lists selected from either (a) the signalling genes or (b) a degree-controlled seed list . We interpret this result to mean that though the SCA is able to predict genes that may function in the network, its application does not increase the modularity of the sub-network in this setting. We believe that this may also explain why genes predicted by the SCA are more likely to affect any of the three phenotypes measured, rather than the specific subnetwork in which they were predicted. We present and discuss these new control analyses in the revised manuscript paragraph six subsection \u201cGenes regulating egg laying and ovariole number regulation form non-random gene interaction networks\u201d and subsection \u201cBuilding degree-controlled randomized networks\u201d, and in the new figure supplements Figure 4\u2014figure supplement 3 and Figure 5\u2014figure supplement 5. We also qualify our description of the possible relationship between genes in the sub-networks under study, by calling them \u201cputative modules\u201d only when we describe them as predicted by the SCA, and subsequently as \u201csub-networks\u201d throughout the revised manuscript. This comment is addressed in more detail in response to reviewer 3\u2019s comments 2.1 and 2.2.(2.2) Module separationAs a general rule, it is not surprising that the connectivity is higher within the output of the SCA algorithm, which returns a subnetwork of connected nodes, to the connectivity between two different SCA produced sub-networks. This is expected to happen with random lists of seed genes, and does not prove that functionally separate sub-networks have been identified. The claim is confusing given that there are shared subnetworks which seems to contradict the idea of separate modules. Reviewer 3 offers several suggestions for how to make this analysis more convincing.We understand the reviewers' point, and have addressed this in two major ways. First, we performed the suggested control analyses as described in response to comment 1 above, and in light of those results, are more conservative in our discussion and interpretation of the \u201cconnectedness\u201d of the genes in phenotypically linked sub-networks. Second, we have revised the text to better convey what we meant in the original manuscript by \u201cseparate modules,\u201d and now explain that one interpretation of the data is that genes in such sub-networks may potentially function rather independently of other sub-networks, in regulating a given phenotype, or pairs of phenotypes We have more detailed explanations of this point in response to reviewer 3's comment 3.1, 3.2 and 3.6.(3) Writing and presentation:(3.1) In addition to suggestions about presentation in the reviews below, during discussion all three reviewers highlighted the value of the overall approach and felt that the manuscript merits publication in eLife. However there is a sense that the manuscripts lacks a clear take home message. This is not required for publication per se, but we all feel the manuscript could be made stronger in revisions by attempting to distill a take home message for readers.We take this point, and in response have modified the Discussion section to better present what we believe are the most important take home messages of the paper. We have addressed this in further detail in response to reviewer 1\u2019s comment 4.(3.2) The experimental validation results are not presented clearly. That section could benefit from more clarity in what specific aspects of the predictions are being tested and how the specific tests address them.To address this concern, we have made significant modifications to the presentation of experimental validation results. We have modified original Figure 7C as per the advice of the reviewer, and merged it with original Figure 7D to create a new Figure 7C, which we believe provides a better understanding of our results (discussed in detail in response to reviewer 3\u2019s comment 4.2 ). We have also modified the description of Figure 7 in the revised results and Discussion .Reviewer #1:DrosophilaDrosophila ovary development and the rate of egg production. An earlier study from this lab identified a role for hippo signaling in these processes. Here, they performed an RNAi-based screen through 463 candidate genes involved in signal transduction to identify genetic modifiers of the hippo[RNAi] phenotype as well as genes that regulate the rate of egg laying through a potentially hippo-independent mechanism. The screens were well-designed and produced quantitative data that the authors could use to rank-order genes according to the strength and direction of the phenotype. Their strategy for assessing the rate of egg laying is in line with standard approaches in the field and their method of counting ovariole number is entirely appropriate. These screens identified a long list of genes, and they took a systems biology approach to assess the individual and combinatorial contributions of these genes to ovary development and function. These types of analyses are outside my area of expertise so I could not assess whether the appropriate computational methods were chosen and applied correctly. However, the writing of this section was clear to a non-expert and, taking their claims at face value, the data seem to support their interpretations.(1) In this study, the authors investigate the signaling pathways that regulate (2) In the final section of the manuscript they describe how their network analysis identified additional genes not uncovered by their screens and show that RNAi knockdown of these additional genes do, indeed, cause phenotypes in egg laying rates and ovariole number. This is a nice validation of the approach. As a minor point, the data presented in Figure 7 only summarize how many genes in the list showed a phenotype in each category but did not indicate what the genes are, which ones are associated with which phenotype, or what the z-scores were in each case . If it has not been included already, the authors should provide this for completeness and to allow comparison to the other data in the study.We appreciate the comments of the reviewer, which made us realize that we should emphasize more clearly where to find this information within the manuscript. The novel genes (i.e. connector genes) tabulated in original Figures 7, Figure 7\u2014figure supplement 1 and Figure 7\u2014figure supplement 2, are indicated as triangles with the gene names shown beside them, in each of the four putative modules generated by the SCA, shown in original Figure 5B and Figure 5\u2014figure supplement 1-3. The genes are also listed in Table 4 and can be identified under the column header [ModuleName]_Connector. To highlight this for the reader, we have added new text to the revised legend of Figure 7 as follows: \u201cThe connector genes are listed in Table 4, and can be identified under the column header [ModuleName]_Connector. The raw data for egg laying and ovariole number for each of the connector genes can be found within Table 4.\u201d.(3) Overall, I think this study will be a useful though perhaps somewhat limited contribution to the field. The results of the screens provide a large amount of data for the field to build on and I appreciate their holistic approach to describing the interplay between genes and pathways in the regulation of processes as complex as development and oogenesis.We thank the reviewer for this comment. The aim of the project was to indeed identify more genes that affect the development of the ovaries, both to advance the field of ovary development and also to provide an example of how such large datasets may be better used to enhance our understanding of complex developmental processes.(4) At the same time, I struggled a bit to extract the most important take-away messages that change how I think about these processes. For example, they state in the discussion that the core module consists of housekeeping genes and hedgehog pathway genes, but this could have largely been predicted from previous publications on this topic. Likewise, terminal filament formation is known to involve cell migration and, consistent with this, they find that several genes involved in cytoskeletal dynamics are important for proper specification of ovariole number. Though some of these genes have not been studied in this context, their involvement here is not too surprising. Nonetheless, it may be that the main innovation of this study is in the systems biology approach they have used to analyze the data, so I look forward to the online discussion with the other reviewers who have more expertise in this area.Drosophila melanogaster , and potentially in other species of insects as well. Understanding the gene regulatory networks that regulate egg laying and ovariole development could provide a framework to understand the key regulatory steps during this process that may be modified over evolutionary time, to yield the wide diversity of ovariole numbers and fecundities displayed by extant insects. We suggest that, given our success in applying a network approach to the results of a traditional forward genetic screen, the field of developmental genetics should find it fruitful to apply network analyses to the interpretation of large scale transcriptomic and proteomic data.\u201dWe appreciate this point, and in response have added the following text to the beginning of the revised Discussion to help clarify the take home messages of the work, as follows: \u201cIn this study, we have identified many novel genes that regulate either or both of egg laying and ovariole number. Though the development of the insect ovary has been studied for over 100 years, our understanding of the genetic mechanisms that regulate the development of the ovary is sparse. The female reproductive system and its ability to produce eggs are one of the key determinants for the survival of a species in an ecological niche. The genes we have uncovered here are possible targets for the regulation of the construction and function of the reproductive system in Reviewer #2:[\u2026](2) Numbered summary of any substantive concernsThis article is generally well-structured, the data analysis is performed in a proper fashion, and the results are nicely presented, giving support to the major conclusions. I have no major concerns with regard to this manuscript, only some minor edits.(2.1) I am wondering if authors can give more detail regarding the paragraph starting \"To choose candidates.\u2026\" I might be wrong, but have the feeling that this approach might produce bias in the results obtained by the authors.We acknowledge the concerns of the reviewer in this section, where we state our reasons for the setting of thresholds of phenotypic significance in the screens. We considered running our egg laying screen with multiple vials containing three flies each, but as we endeavoured to test many genes for multiple phenotypes with as high throughput but accurate a method as possible, we settled on using only a single vial of three flies to measure egg laying. Aiming to mitigate this bias, we did, however, set our thresholds for positive candidates in the egg laying at a higher value of |5| when compared to an ovariole number threshold of |2| to account for the difference in statistical power. We also address this concern in the Materials and methods section.gene scores of positive candidate genes sorted by centrality metrics. However, I wonder why the authors did not calculate p-values (significance level) between 1st and 5th quantile comparisons of Zgene scores shown in this figure? Significance levels in these comparisons might give strong support to the weak prediction of a gene that would affect a specific phenotype.(2.2) In Figure 4\u2014figure supplement 2, the authors show a comparison of Zgene scores between the 1st and 5th quintile. We have modified Figure 4\u2014figure supplement 2 by adding asterisks to indicate significant differences reflected by these p-values, which are shown under each comparison. Significance thresholds were set at 0.05 and are indicated in the revised figure legend, which now contains new text as follows: \u201csignificant differences are indicated by asterisks. p-values are displayed below every bar plot.\u201d We also revised the text in subsection \u201cCentrality of genes in the ovarian protein-protein interaction networks can predict the likelihood of loss of function phenotypic effects\u201d that describes the relationship between the centrality of genes and the strength of phenotypic effect to include the relevant pvalue < 0.05 in Figure 4\u2014figure supplement 2\u201dWe have taken this suggestion and calculated the significance for the differences of Z(2.3) In Figure 6, for the sake of clarity, it is necessary to show the sloppy paired 1 gene in the meta-network analysis . As is now shown in this figure, readers cannot see easily the importance of this gene within sub-network VII, which I think is one point that the authors want to highlight.This comment by the reviewer made us realize that we needed to clarify that our intention with the reference to slp1 in the legend of Figure 6A was simply to explain that it is a gene that is present both as a seed gene in the Egg Laying sub-network as well as a novel predicted gene (connector) in the hpo[RNAi] Egg Laying sub-network. For improved clarity, we have removed all mention of slp1 from the revised legend of Figure 6A and have instead explained its presence in two sub-networks within the revised \u201cMeta network\u201d section of the Materials and methods.(2.4) In step 3 in the protein-protein interaction network (PPI) building section (\u201cMethod Details\u201d subsection), authors indicate that a custom python script was created to download and reads each of the PPI tables from DroID database, but I could not find this python script in the Github repository in the paper. Please, can authors upload this script in the repository?We thank the reviewer for pointing this out, and have both confirmed that the script is in the repository and also added a reference to this script in subsection \u201cProtein-Protein Interaction Network (PIN) building\u201d.Reviewer #3:Drosophila with protein-protein interaction networks. Highly interesting, interdisciplinary work lying at the intersection of developmental and network biology. They identify genes regulating ovariole number and egg laying and analyze those genes in the context of PPI networks, predicting and experimentally testing additional novel reproductive regulatory genes with some success. However there are serious issues with the analysis supporting key claims in the manuscript related to network topology.(1) The authors present a novel study integrating the results of performing screens for regulators of reproduction in We thank the reviewer for their very careful and thorough analysis of our work. We are also grateful for their appreciation of the key aspects and novelty of our work. We understand their reservations on some aspects of the manuscript and have attempted to address them with more analyses or through clearer explanations.(2) The claim of non-random modules is not supported by the analysis.We have both modified our analyses and performed new ones to more carefully assess this claim, and to more conservatively interpret the results of these assessments . We have a more detailed explanation below in our response to comment 2.1.(2.1) The claim in the section title \"Genes regulating egg laying and ovariole number regulation form non-random interaction modules\" (L260-L261) is supported by Figure 5\u2014figure supplement 4. In that figure both the seed genes and results from the SCA algorithm are compared to the subnetworks formed by randomly selected genes from the initial screen. This is a sensible comparison for the seed genes but not for the output of the SCA algorithm, since that algorithm builds a LCC and so will obviously have a much larger LCC and be much more connected than randomly selected genes from the network.We understand the concerns of the reviewer, and in response we have modified our analysis to better address this interpretation. Firstly, we compared the network metrics of the seed genes both to a similarly sized random selection of genes from the signalling candidates, and to a degree-controlled random network . Our results, shown in new Figure 4\u2014figure supplement 3, indicate that the seed gene lists display higher network metrics than both null distributions in most cases. We therefore conclude that, independent of application of the SCA, the groups of seed genes display many features of modularity by this assessment, and this analysis thus suggests that they may function as putative modules. We report this in subsection \u201cGenes regulating egg laying and ovariole number regulation form non-random gene interaction networks\u201d of the revised manuscript.Secondly, to directly address this important point by the reviewer, we assessed these network metrics in the putative modules generated by application of the SCA. We found that there was an overall reduction of network metrics when compared to null distributions of similarly sized seed lists selected from either the signalling genes or a degree-controlled seed list . We therefore conclude that though the SCA may be able to correctly predict genes that may function in the network , it does not increase the \u201cmodularity\u201d of the sub-networks. We therefore conservatively refer to these connected groups of genes as \u201csub-networks\u201d throughout the remainder of the manuscript. These analyses and interpretations are explained and presented in paragraph six of subsection \u201cGenes regulating egg laying and ovariole number regulation form non-264 random gene interaction networks\u201d.(2.2) The results of Figure 4 show that the positive pairs in the screen on average, tend to have higher degree in the network, which could be responsible for the higher connectivity of the seed genes, rather than them forming modules. To account for this, the seed genes could be compared to a null distribution of degree-controlled randomized networks.We understand the concern of the reviewer, and have implemented this suggestion as described in response to this reviewer\u2019s comment 2.1 above, using the method of Guney and colleagues .The revised Materials and methods section now contains the following text explaining the methodology for obtaining the degree-controlled randomized network, and mentioning the sections in the python code where this is applied, as follows. \u201cBuilding degree-controlled randomized networks. We assessed the modularity of the networks by comparing the network metrics of each sub-network to a degree-controlled randomly sampled network. To generate this degree controlled random network, we applied a previously developed method . In short, nodes in the PPI are binned by degree with the minimum size of each bin being set at 100 nodes. Bins are constructed iteratively from the lowest degree to the highest degree in the network. To sample a set of nodes, the sub-network degree distribution is computed, using the bin cut-off, from the PPI. Then, nodes are randomly selected from each bin to match this degree distribution (see 05.2_Degree_Controlled_Testing.ipynb for code implementation).\u201d(2.3) The text reads \"decreased average shorted path\" (L331) but it is increased in 7 of 8 cases in the figure.Our new analyses with more appropriate control groups for the SCA-generated putative modules do indeed often display relatively increased paths. This new result is described in paragraph six of subsection \u201cGenes regulating egg laying and ovariole number regulation form non-random gene interaction networks\u201d, and shown in new Figure 5\u2014figure supplement 5.(3) The claim of separate modules is not supported by the analysis.We believe that this comment refers to our interpretation of the data shown in original Figure 6, which is discussed in subsection \u201cLow edge densities between sub-networks suggest genetically separable mechanisms of ovariole number and egg laying\u201d. This comment from the reviewer helped us realize that, in addition to performing the additional control analyses suggested in this reviewer\u2019s comment 2.1, which encouraged us to be more conservative with our description of these gene groups and refer to them simply as \u201csub-networks\u201d in the revised manuscript, we should improve the clarity of our interpretation of the groups of genes we present in Figure 6. In the \u201cmeta network\u201d shown in this figure, we first separated genes into the seven different groups based on the phenotype(s) affected in their loss of function condition as assessed in our initial screens . Of these seven groups, three groups included genes that affected only one of the three measured phenotypes , three groups included genes that affect two of the three phenotypes , and the seventh group included genes that affected all three phenotypes . The differences in protein-protein interactions between these seven different phenotypically binned groups suggested the possibility that each phenotype could be largely regulated by genes that do not interact very much with genes regulating another phenotype. We have added new text to this section in the revised manuscript with the aim of improving the explanation of our thought process for suggesting this interpretation. We further address related points in our responses to this reviewer\u2019s comments 3.1 and 3.2 below.(3.1) Similarly to point 1, it is not surprising that the connectivity is higher within the output of the SCA algorithm, which returns a subnetwork of connected nodes, to the connectivity between two different SCA produced sub-networks, that will happen with random lists of seed genes, it does not prove that functionally separate sub-networks have been identified.We take this point by the reviewer, and in response to this and other reviewer comments, are more conservative in our interpretation of the functions of these gene groups in the revised manuscript. The seed lists for the SCA included any gene that affected a phenotype, unlike the seven groups of the meta network, which included genes that affected only one, two, or all three phenotypes. We have more detailed explanations in our responses to this reviewer\u2019s comments 3.2, 3.4 and 3.6.(3.2) There are sizable shared subnetworks which seems to contradict the idea of separate modules.We believe that we have addressed this comment by the modifications described in our response to comment 3 above. We hope that our revised section on low edge densities clarifies that we intended to communicate an interpretation of separable putative genetic interaction groups affecting one or more phenotypes, rather than independent functional modules, combined with our more conservative interpretations of these groups as simply \u201csub-networks,\u201d has clarified our logic and intent here.2 \u2013 x) / 2 where x is the number of nodes in the group (assuming self-interactions are not considered), the edge density calculations between the different groups are correct.(3.3) The heat-map Figure 6B, shows very low edge density within the groups , in contradiction to what is written in the text. This appears to be due to a miscalculation in the file 08_MetaModule_Analysis.ipynb in the Github repository: within the group the number of edges should be divided by and updated the figure with the new densities. We are here attaching the new version of the figure for simplicity. This new, corrected result does indicate higher edge densities within groups, than between groups .(3.4) An alternative approach to this analysis could be to look at the connectivity of only the seed genes and not the connector genes in the different groups and comparing to a random assignment of group.We thank the reviewer for this suggestion. We have implemented it by adding the results of a new analysis, shown in new Figure 6\u2014figure supplement 1 and described in the revised Results section in subsection \u201cLow edge densities between sub-networks suggest genetically separable mechanisms of ovariole number and egg laying\u201d. In this analysis, we ask if the seven groups of genes that we bin by phenotype in Figure 6A \u2013 considering either the seed genes alone, or the seed genes plus the connector genes predicted by the SCA \u2013 show connectivity different from that expected from a random group of genes of the same size. We found that the connectivity of the seed genes alone was not greater than expected by chance, but that including connector genes in the group changed their connectivity to be either higher or lower than that of a control group of randomized genes. This analysis helped clarify the internal structure of the meta network.(3.5) Identifying modules of genes which affect one phenotype and not another is presumably made more difficult by the decision to filter candidate genes for two of the phenotypic screens based on the results of the first screen.Drosophila remains unaltered through to and during adulthood, even if oogenesis within those ovarioles suffers congenital or age-related defects . Because previous work suggested that ovariole number in Drosophila could have at least some predictive relation to egg laying , we reasoned that scoring the latter phenotype in a primary screen could be an effective way to uncover ovariole number regulators . While our results showed that this was true in many cases, it was also clear that these two traits can vary independently , highlighting the fact that ovariole number is not the only determinant of egg laying. Egg-laying dynamics, even during the limited five day assay used in our study, are likely influenced not just by a single anatomical parameter such as ovariole number, but rather by many biological, biomechanical, hormonal and behavioural processes.\u201d. We have left this text unchanged in the revised manuscript.We agree with the reviewer that this screening strategy is likely to lead to us missing some genes that affect the second phenotype, if they did not pass our threshold for analysis based on the first screen. For this reason, we had included this explanatory text in the original manuscript \u201c Once established during larval life, ovariole number in (3.6) The prediction results do not appear to be consistent with separate modules (see point 3 below).Thanks to this comment and others by this reviewer, we realised through our new analyses that the SCA results indeed do not support a prediction of functionally separate modules. We have therefore modified the text of the manuscript throughout the revised manuscript to reflect our new, more conservative interpretation of our results, as described in our response to this reviewer\u2019s comment 2.1.(4) The results of experimentally testing the predictions are presented in an unclear way, making it difficult for the reader to assess their accuracy.To address this concern of the reviewer we have made significant modifications to the presentation of experimental validation results. We have modified Figure 7C as per the advice of the reviewer, and merged it with Figure 7D for better comparison (discussed in further detail in our response to this reviewer\u2019s comment 4.2). We have also modified the description of the new Figure 7 in the Results and the Discussion on the consequence of this result .(4.1) The results in Figure 7A/B/C appear to show that the predictions from the modules are just as good for the phenotypes not associated with the module as for those associated with the module, e.g. for the EL phenotype there is one correct prediction from both the Core and EL modules and 9 correct predictions from the hpo[RNAi] ON/EL modules, with a similar number of predictions. So it would seem that these results suggest that, although there is positive predictive value overall relative to the initial signaling genes list, that the separate modules predicting their associated phenotype separately from the others is not supported by the data. This is not discussed in the text.We have incorporated this important observation by the reviewer into a revised section of the Discussion, and have differentiated between prediction rates within the module, and prediction rates for genes involved in any of the phenotypic classes.(4.2) From what I could understand, the fractions for the initial signaling gene lists for the EL and hpo[RNAi] ON phenotypes are not exactly consistent with the compared fractions of the predictions, due to the filtering applied on the hpo[RNAi] of |Z| > 1, for the initial screens. Perhaps the fractions for the 2 affected bars in Figure 7C could also be calculated with this threshold applied to remove genes?We understand the comment of the reviewer as follows in the original Figure 7C, we compare the number of connectors from any of the three modules that are above the |Z| gene threshold of the screen. The |Z| gene threshold for the connectors in the Egg Laying and hpo[RNAi]EL screens are |5| while the |Z| gene threshold for the connectors in hpo[RNAi]ON Screen is |2|. Given that we are comparing the results of the connectors with the original signalling candidate screen results, the reviewer suggests that we utilise the same thresholds that we used in the original screen. We agree that this is logical, and accordingly, modified the thresholds for the connectors in what has become revised Figure 7C. The genes from the connector list were first subject to a threshold of |1| and the genes that were above this threshold were then counted depending on whether they were above the |Z| gene threshold |5| in Egg Laying and hpo[RNAi]Egg Laying screens and |2| in the hpo[RNAi]Ovariole Number screen. Application of this new method does not change the number of genes in the hpo[RNAi]Egg Laying screen, but does reduce the number of genes in the Egg Laying and hpo[RNAi]Ovariole Number screen. The results of these calculations have been added to the manuscript as new Figure 7\u2014figure supplement 2.(4.3) There are no p-values calculated, comparing the predictions to the original screens and no error bars shown in Figure 7.This comment by the reviewer made us realize that reorganizing the figure could help with its interpretation and avoid the confusion inadvertently caused by the original version. Figures 7A and 7B, which remain unchanged from the original version in this revised manuscript, show the percentage of connector genes within each phenotypic bin, and therefore do not have error bars or p-values. In modified Figure 7C, the prediction rate comparisons between the connector candidates and the signalling candidates now indicate calculated p-values with asterisks. We have modified and merged former Figures 7C and 7D into a single new Figure 7C, shown above in our response to this reviewer\u2019s comment 4.2.(4.4) The title of Figure 7C could be changed to make it clearer that this contains predictions from all modules.gene| above the respective threshold in any of the three phenotypic screens . Positive connector and signaling candidates that were above the |Zgene| threshold in all three phenotypic screens are indicated in an \u201cAll phenotypes\u201d column. Statistical significance was computed using the binomial test, comparing the probability of a positive candidate amongst the connectors to the probability of a positive candidate amongst the signalling candidates .\u201dWe have taken this suggestion by modifying the title of Figure 7C to read \u201cPositive prediction rates of all unique connectors compared to signalling candidates\u201d. We have also modified the legend of Figure 7C to improve the clarity of this point. The modified legend now reads as follows: \u201cProportion of all unique connector genes (dark grey bars) predicted by all four sub-networks compared to the proportion of signaling candidate genes (light grey bars) with |Z(4.5) There is no \u201cAll screens\u201d column in either Figure 7C or Figure 7D for comparison.To address this comment, we have added an \u201cAll screens\u201d column to both revised Figure 7C and 7D, which are discussed in detail in our response to this reviewer\u2019s comment 4.2."} +{"text": "Brassica napus to 11.3 Mbp in Silene conica. To date, in comparison with the number of plant species, only a few plant mitogenomes have been sequenced and released, particularly for conifers (the Pinaceae family). Conifers cover an ancient group of land plants that includes about 600 species, and which are of great ecological and economical value. Among them, Siberian larch (Larix sibirica Ledeb.) represents one of the keystone species in Siberian boreal forests. Yet, despite its importance for evolutionary and population studies, the mitogenome of Siberian larch has not yet been assembled and studied.Plant mitochondrial genomes (mitogenomes) can be structurally complex while their size can vary from ~\u2009222 Kbp in Two sources of DNA sequences were used to search for mitochondrial DNA (mtDNA) sequences: mtDNA enriched samples and nucleotide reads generated in the de novo whole genome sequencing project, respectively. The assembly of the Siberian larch mitogenome contained nine contigs, with the shortest and the largest contigs being 24,767\u2009bp and 4,008,762\u2009bp, respectively. The total size of the genome was estimated at 11.7 Mbp. In total, 40 protein-coding, 34 tRNA, and 3 rRNA genes and numerous repetitive elements (REs) were annotated in this mitogenome. In total, 864 C-to-U RNA editing sites were found for 38 out of 40 protein-coding genes. The immense size of this genome, currently the largest reported, can be partly explained by variable numbers of mobile genetic elements, and introns, but unlikely by plasmid-related sequences. We found few plasmid-like insertions representing only 0.11% of the entire Siberian larch mitogenome.Silene conica (11.3 Mbp). Similar to other species, the Siberian larch mitogenome contains relatively few genes, and despite its huge size, the repeated and low complexity regions cover only 14.46% of the mitogenome sequence.Our study showed that the size of the Siberian larch mitogenome is much larger than in other so far studied Gymnosperms, and in the same range as for the annual flowering plant To date, the complete assembly and accurate annotation of plant mitogenomes remain challenging due to their complex and often perplexing structure.Since the first plant mitogenome of the common liverwort has been sequenced , not as Brassica napus ), sugar pine \u2013 Pinus lambertiana (3.9 Mbp) [Pinus sylvestris (986 Kbp [https://www.ncbi.nlm.nih.gov/assembly/GCA_900143225.1/]), and Siberian larch \u2013 Larix sibirica (11.7 Mbp) presented here.The number of evolutionary and systematic studies based on comparative analysis of complete plant mitogenome sequences has significantly increased in the last decade, notably due to the advent of better sequencing methods. However, there are still very little published comparative mitogenome studies in gymnosperms, one of the oldest among the major plant clades comprising 14 families with more than 1000 species. Among all plant mitochondria genomes available, few gymnosperm mitogenomes have been fully assembled and properly annotated: 415 Kbp) , a ginkg347\u2009Kbp) , a gneto979\u2009Kbp) , the Jap5.9 Mbp) , Norway (4.3 Mbp or 4.9 M(4.3 Mbp ), loblol1.25 Mbp or 1.19 In this study, we report on the sequencing, assembly, and annotation of the mitogenome of Siberian larch, a key species for the Siberian boreal forest and ecosystem. Furthermore, several methods were employed to verify and evaluate the correctness of the assemblies.Based on ~\u200919.7 million of paired-end (PE) nucleotide reads produced by Illumina HiSeq 2000 using mitochondrial DNA (mtDNA) enriched samples and\u2009~\u2009625.5 million of mate-pair (MP) nucleotide reads produced by Illumina HiSeq 2000 for the whole nuclear genome assembly a prelimTo improve the assembly of the Siberian larch mitogenome, we used the same PE reads as in the preliminary CLC assembly generated by Illumina HiSeq 2000 and long reads generated by Oxford Nanopore MinION and MaSuRCa v.3.2.8 pipeline. MP reads were used only for the preliminary CLC assembly, but not for the MaSuRCa assembly. After mapping the MaSuRCa assembly to plant mitochondrial database, we finally assembled nine mitochondrial contigs resulting in a total length of 11.7 Mbp. The largest contig was 4,008,762\u2009bp long.C. elegans genome.To further evaluate the accuracy of the final assembly, we ran REAPR v1.0.18 , which rLINEs), and 9 short interspersed nuclear elements (SINEs). In total, 7691 REs were identified using a combined repeat library, which represent 11.14% of the 11.7 Mbp mitogenome assembly. LTRs, LINEs, simple repeats, and class II DNA transposons were the most abundant among classified repeats in the assembled contigs, respectively. RepeatModeler found 122 mobile elements, among which TEclass identified 27 DNA transposons, 38 long terminal repeats (LTRs), 12 long interspersed nuclear elements repeat families Karst (NCBI GenBank accession number AJ225562) was found in contig 1 in the 608,296\u2013608,337 position, yet with 100% identity were searched by comparing the mtDNA against the chloroplast DNA (cpDNA) of Siberian larch . Twenty The transcriptome data used to verify RNA editing sites are provided in Table\u00a0nad1 and nad4. Maximum number of the editing sites (80) was observed for the nad5 gene. Editing the RNA helped us identify start codons in atp6, rps3, rps10, rps19, and mttB genes and stop codons in nad4L, sdh3, and atp9 genes.Among 438 RNA editing sites predicted by the PREP-Mt program almost 90% were also verified by aligning RNA-seq reads to the annotated mitochondrial genes and then by calling variants using the CLC Genomic Workbench. Many additional sites were found only by variant-calling. They were likely specific for Siberian larch and included in the final annotation. In total, 864 C-to-U RNA editing sites were found for 38 among 40 protein-coding genes. The RNA editing was not found only for two genes, Cycas, Ginkgo, and Welwitschia mitogenomes were carried out specifically using enriched mtDNA. Our study benefits from both approaches by simply having more mitochondrial reads, but mtDNA enrichment was not as critical as having long reads.The whole genome sequencing data were used in the previous studies of conifer mitogenomes, while sequencing and assembly of Almost all studies that used whole genome sequencing data followed similar strategies to assemble and verify putative mitochondrial sequences. They separated mitochondrial sequences from nuclear and chloroplast by their length, depth of coverage, and GC content , 36, 38,Gene content reported for available annotated mitogenomes varies noticeably as well as the amount of repeats, showing no correlation between genome size, number of genes and repeats Table\u00a0.Table 5Cycas taitungensis and 10 other plants revealed that first DNA transfer from cpDNA to mtDNA occurred at least as far back as to the common ancestor of extant gymnosperms and angiosperms, about 300 mya [trnL, trnT, rps4, trnS, ycf3, psaA, and psaB . Analysis of the first gymnosperm mitochondrial genome of 300 mya . Later, 300 mya . MitogenThe reasons for extremely large size of mitogenomes in plants are still not fully understood, but at least can be partly explained by a variable number of mobile genetic elements, introns and plasmid-related sequences and coulWelwitschia to 1102 in Taxus, 1179 in Pinus, 1206 in Cycas, and 1306 in Ginkgo mtDNA needed for long read sequencing. Moreover, the obtained nucleotide sequences contained a significant contamination from nuclear and chloroplast genomes. It appears that the protocol we used to isolate mtDNA from conifer needles would require further adjustments. Such improvements would likely focus on the damaging effect of larch phenolic compounds on mitochondrial membranes during the homogenization step, which would thus increase the amount of intact organelles. In turn, this should result in a larger yield of intact mtDNA molecules. To achieve this, it seems necessary to (i) shorten the mitochondrial isolation procedure as much as possible and (ii) find a better buffer composition for both extraction and washing media. Note that the use of fresh material instead of cryo-preserved one would also likely contribute to a better isolation procedure.However, it is very important to notice that even with the best possible protocol for mtDNA enrichment and intact mtDNA molecules, it would still be very hard (if not currently impossible) to verify whether a particular large plant mitogenome assembly represents a single circular molecule or not. All subgenomic circles could theoretically be detected, if (and only if) extremely long reads comparable with the sizes of these subgenomic circles would be generated, which is still not feasible considering the huge length of conifer mitogenome. For unambiguous alternative assemblies reads should be as long as several Mbp. However, even though mitogenome would be assembled as a single circular structure, this would not guarantee its circular nature. Additionally, there could be alternative physical structures, which can all produce circular genome maps - such as head-to-tail concatamers and circularly permuted linear molecules to 11.7 Mbp in Siberian larch (this study). We succeeded in producing a high-quality assembly of the Siberian larch mitogenome using both short and long nucleotide sequencing reads generated by the Illumina HiSeq 2000 and MinION. The final assembly of the Siberian larch mitogenome consists of 9 contigs with a total length of 11,662,539\u2009bp . The longest contig is 4,008,762\u2009bp, the shortest - 24,767\u2009bp. Finally, 40 protein-coding, 34 tRNA, and 3 rRNA genes were annotated. This mitogenome is currently the largest one among publicly known. The assembled genome is of sufficient quality for further detailed studies and comparative analyses with other plant mitogenomes.Mitogenomes of conifer species are still poorly studied - only five genomes have been published to date. The sizes of published mitogenomes vary widely: from 986 Kbp in Scots pine BSA, 0.3% (w/v) PVP-40, 0.3% (w/v) PVPP, 3\u2009mM DTT, 3\u2009mM cysteine, pH\u20097.4. Homogenization was carried out in a blender at 4\u2009\u00b0C in series of 9 times for 3\u2009s each and 3 times for 10\u2009s each, followed by the grinding in a cooled mortar using pestle for 50\u201360\u2009s. The homogenate was filtered through 4 layers of gauze and 2 layers of 50\u2009\u03bcm nylon gauze. Then, 50\u2009ml of EM were added to the homogenate while filtering . The homogenate was centrifuged at 2500\u2009g in F0685 Beckman Rotor for 5\u2009min. The supernatant was centrifuged at 5000\u2009g for 10\u2009min. The pellet was discarded. The supernatant was centrifuged again at 13,000\u2009g for 15\u2009min. The pellet was resuspended in 100\u2009ml of the washing medium (WM): 0.3\u2009M mannitol, 30\u2009mM MOPS, 0.2% (w/v) BSA, pH\u20097.4. The resulting suspension was centrifuged at 13,000\u2009g for 15\u2009min. The pellet was resuspended in 25\u2009ml of the WM. The suspension was then centrifuged at 13,000\u2009g for 15\u2009min. The pellet was resuspended in 1\u2009ml of the WM. The resulting suspension was centrifuged at 11,000\u2009g for 10\u2009min (HL 081\u2009F45\u201324-11 Rotor) using the 5415R centrifuge . The final mitochondrial pellet was resuspended in the WM without BSA in a 1: 1 weight/volume ratio.To enrich total DNA with mtDNA the following mitochondria isolation protocol was used. The Siberian larch needles were stored either at 4\u2009\u00b0C for 4\u201313\u2009days or at \u2212\u200980\u2009\u00b0C for 10\u201312\u2009months after they were collected from a reference Siberian larch tree . For lon2 were added to 200\u2009\u03bcl of the final mitochondrial suspension. The mixture was incubated for 20\u2009min at 25\u2009\u00b0C. Then, 1\u2009ml of 0.3\u2009M mannitol, 30\u2009mM MOPS, 10\u2009mM EDTA, 10\u2009mM EGTA, pH\u20097.4 was added. The resulting mixture was centrifuged at 11,000\u2009g for 5\u2009min. The pellet was resuspended in 1\u2009ml of 0.3\u2009M mannitol, 30\u2009mM MOPS, pH\u20097.4 and centrifuged again at 11,000\u2009g for 5\u2009min. Then, DNA was isolated from the resulting pellet using DNeasy Plant Mini Kit from QIAGEN according to the manufacturer\u2019s protocol.To isolate mtDNA, 3\u2009\u03bcl of DNase from Sigma-Aldrich (1\u2009mg/1\u2009ml) and 3\u2009\u03bcl of 1\u2009M MgClThe isolation of high-quality HMW DNA for further sequencing on MinION was carried out using the modified CTAB-method . Fresh nAll sequencing was performed in the Laboratory of Forest Genomics . The PE library with the mean insertion size of 700\u2009bp was prepared from the enriched mtDNA using the Illumina TruSeq DNA LT Sample Prep Kit according to the Illumina TruSeq DNA Sample Preparation Guide . Three MP libraries were prepared from the total non-enriched DNA using Nextera Mate Pair Library Prep Kit . These PE and 3 MP libraries were sequenced with 2\u2009\u00d7\u2009100\u2009cycles on the HiSeq 2000 platform using the Illumina TruSeq SBS Kit v3 . More detailed data on Illumina libraries and obtained reads are provided in Table\u00a0The total non-enriched DNA library was sequenced on the MinION with use of R9 FlowCells (FLO-MIN106).http://www.bioinformatics.babraham.ac.uk/projects/fastqc). The de novo transcriptome assembly was generated using Trinity v. 2.8.4 (https://github.com/trinityrnaseq/trinityrnaseq/wiki).RNA was isolated from autumn buds of a reference Siberian larch tree using thThe quality of Illumina reads was assessed using FastQC v. 0.11.5 . Adapterhttps://github.com/bcgsc/abyss/tree/master/Sealer). The resulting assembly of Illumina reads consisted of 1,216,421 contigs with a total length of 516,8 Mbp, N50\u2009=\u2009436\u2009bp and the longest contig 1,449,395\u2009bp.The obtained PE and MP sequence reads were assembled de novo into contigs using the CLC Assembly Cell v. 5.0.0 . ScaffolThe basecalling and the quality evaluation for MinION reads were performed using Albacore . After tThe hybrid assembly using long MinION reads and short PE Illumina reads was carried out using MaSuRCA v. 3.2.8 . This hyTo mine mitochondrial contigs from this assembly, the BLAST search against all mitochondrial plant sequences available in the NCBI GenBank was used. After mapping this assembly to plant mitochondrial database, we finally assembled 9 mitochondrial contigs with total length of 11.7 Mbp. The largest contig was 4,008,762\u2009bp long.To further evaluate the accuracy of the hybrid assembly we ran REAPR v1.0.18 . It usesMitogenome of Siberian larch was checked for homology with other plant mitogenomes existing in the NCBI GenBank database using the NCBI BLAST algorithm. Mitofy was alsoRNA editing sites were predicted using PREP-Mt with a cTo search for MTPTs we used CLC Genomics Workbench whole genome alignment tool and Siberian larch chloroplast genome sequence .RepeatModeler v.1.0.11 was usedPicea abies (L.) Karst and deposited at the NCBI GenBank under accession number AJ225562 and 2) 891 broad host range plasmids (NCBI GenBank taxid:36549). The blast was performed with different stringency searching for highly similar sequences (megablast), more dissimilar sequences (discontiguous megablast), and somewhat similar sequences (blastn).To test whether the Siberian larch mitogenome contains plasmid-like sequences all nine contigs assembled in this study were blasted against 1) a linear plasmid sequence identified in mitochondria of Additional file 1. The Siberian larch mitogenome assembly evaluation using REAPR v1.0.18.Additional file 2. Plasmids matching inserts in the nine Siberian larch mitogenome contigs and their NCBI GenBank accession numbers."} +{"text": "Background: Chikungunya fever is caused by the chikungunya virus. Numerous factors affect the risk of chikungunya transmission. This study explored the epidemiological characteristics, differences, and trends in domestic and imported cases of chikungunya fever in Taiwan in terms of patient sex, age, month of confirmation, and area of residence from 2007 to 2019. Methods: Public annual chikungunya data from Taiwan\u2019s Centers for Disease Control (CDC) were analyzed. In total, 21 confirmed domestic and 198 imported cases of chikungunya were reported. Of the domestic cases, one was sporadic and reported in July 2019, and 20 were attributed to a cluster event during August and September 2019. Results: In a comparison between domestic and imported cases reported from July to October 2019, differences in sex were nonsignificant (p = 0.555), whereas significant differences were observed for age (p < 0.001), month of confirmation (p = 0.005), and place of residence (p = 0.001). An age of 69\u201369 years (odds ratio (OR) = 6.66, 95% confidence interval (95%CI) = 2.15\u201320.65), month of confirmation of September and place of residence of New Taipei City were identified as potential risk factors. Additionally, domestic cases in August and September 2019 increased in proportion to the increase in imported cases during July and August 2019. Increased domestic patients may have been caused by the domestic mosquitoes that transmitted the virus by biting the imported patients to Taiwan. This is the first report comparing domestic and imported cases of chikungunya from surveillance data from the Taiwan CDC from 2007 to 2019. Conclusion: This study highlights the importance of longitudinal and geographically extended studies to understand the implications of zoonotic disease transmission on Taiwan\u2019s population. Critical data were identified to inform future surveillance and research efforts in Taiwan. Frequent international interactions , global climate change, and changes in living environments have resulted in the rapid spread of various emerging and recurrent infectious diseases worldwide\u2014particularly vector-borne diseases\u2014and their increased frequency. Chikungunya is an acute infectious disease transmitted by vector mosquitoes [Alphavirus of the Togaviridae family [The chikungunya virus is a positive-sense single-stranded RNA virus classified into the genus e family . It has e family . Since 2e family .Aedes aegypti and Aedes albopictus [Chikungunya epidemics have been mainly distributed in tropical and subtropical regions of Africa, Asia, and the Americas . Chikungbopictus . IndividThe clinical symptoms of chikungunya are fever, joint pain or arthritis, headache, nausea, fatigue, and muscle pain, and approximately half of patients develop a rash that lasts approximately 3\u20137 days . Most paBecause Taiwan is an island, most infectious diseases (including vector-borne disease) spread to Taiwan via traffic vehicles entering its seaports and airports. Since chikungunya was declared a category-2 infectious disease in the Communicable Disease Control Act in October 2007 and the beginning of the chikungunya network surveillance report in December 2007, Taiwan has had an average of 16 confirmed imported and domestic cases per year . AdditioThis study did not require ethical approval because it involved information freely available in the public domain. Analysis was performed on open-source data sets in which data are properly anonymized.This study employed the TNIDSS public network database , which iA cluster infection is an aggregation of cases grouped in terms of location and time that are suspected to be greater than the number expected, even though the expected number may not be known.In Taiwan, chikungunya is a notifiable communicable disease, which is a disease classification determined by the central government in the Communicable Disease Act. For such diseases, doctors or medical institutions must report confirmed cases to the competent health authority and enact measures such as treatment or event isolation pursuant to the act. Notifiable communicable diseases are typically characterized by rapid spread, severe symptoms, or high mortality rates. Therefore, when doctors encounter patients suspected to have chikungunya, they must report these patients to the competent health authority within 24 h. Suspected patients with chikungunya exhibit associated clinical conditions as well as one of two epidemiological conditions . Patients then receive a test at the hospital; test results are deemed positive if they exhibit one of the following conditions: (1) the separated clinical specimen (blood) contains the chikungunya virus, indicated by nucleic acid testing yielding a positive result; (2) acute-phase serum (or serum collected the first time) tests positive for one of two specific antibodies for the chikungunya virus; and (3) the paired serum (serum in the recovery and acute phases) exhibits seroconversion for one of two specific antibodies (IgM or IgG) or an increase in antibody level by \u22654 times. The positive results of the PCR viral tests could also represent non-live virus. In this study, in addition to PCR positive results, other epidemiological information or clinical characteristics could be considered in the definition of positive patients. According to the Taiwan CDC, a confirmed case of chikungunya must meet one of the above criteria.p < 0.05 were considered statistically significant.According to the research framework, this was a retrospective historical study on all imported and domestic cases of chikungunya since 2007. On the basis of the TNIDSS, the numbers of imported and domestic confirmed cases of chikungunya from January 2007 to October 2019 were determined, and the distribution, differences, and results of epidemiological characteristics were consolidated. Descriptive data are expressed as means and summary statistics. Categorical variables were compared using chi-square tests, which were performed using SPSS . Chi-square tests were two-sided with an \u03b1 level of 0.05. Values for which During the study period (January 2007\u2013October 2019), 219 confirmed cases were reported , of whicp = 0.555), whereas a significant difference in age (p < 0.001), month of confirmation (p = 0.005), and place of residence travel notice for Myanmar and level-1 (watch) travel notices for Thailand, India, and the Maldives on 22 October, 2019 [Research outside Taiwan has demonstrated that hot environments are associated with rapid disease transmission by vector mosquitoes . For exaAedes aegypti and Aedes albopictus) that bite natural hosts. The chikungunya virus is maintained in nature in both urban and sylvatic cycles involving mosquito vectors and human or vertebrate animal hosts [Aedes albopictus, and they had a level-6 container index, indicating an extremely high risk of vector mosquito transmission. Laboratory results indicated that the DNA sequence of the virus in an imported case from Myanmar had high affinity to that of the domestic cases. Therefore, this study inferred that the genotype of the virus strain that infected the chikungunya cluster cases in 2019 originated in Myanmar. A study also indicated that two genotypes of chikungunya virus strains were imported to Taiwan between 2006 and 2014, namely ECSA and Asian [Zoonoses refer to infectious diseases that are transmissible between animals and humans. Such transmission occurs either directly or indirectly through vectors between humans and animals to transport pathogens from one organism to another. Types of pathogens include viruses, bacteria, mold, parasites, and protozoa. Their modes of transmission are direct contact with or inhalation of pathogens, consumption of food or water containing pathogens, and vector bites ,26. Chikal hosts . A studyal hosts . Additioal hosts . Populatal hosts and dengal hosts , which aal hosts , the 20 nd Asian . The ECSBecause the government regards the epidemic as a war, this study referred to the Taiwan CDC\u2019s promotion of epidemic prevention and reveIn addition to the aforementioned preventive measures to control infectious disease, vaccine development is an epidemic prevention strategy to induce the human body\u2019s immunity to external pathogens and achieve community immunity. Although the chikungunya virus is not associated with a high mortality rate, its severe and chronic symptoms have motivated experts to rapidly develop a vaccine. Vaccine development for the chikungunya virus began in 1960, when large-scale spread occurred in Thailand. Regarding progress until 2018, Reisinger and colleagues assessed the immunogenicity, tolerability, and safety of a live attenuated measles-vectored vaccine that expresses the chikungunya virus structural protein in a double-blind, randomized, placebo-controlled, and active-controlled phase-2 trial. The results revealed that the vaccine has a strong safety and immunogenicity profile, and plans for a phase 3 study are in preparation ProblemsApart from chikungunya, the most severe and deadly vector-borne disease that has spread widely and caused cluster infections in Taiwan is dengue fever, which has particularly affected Southern Taiwan. Both dengue fever and chikungunya are transmitted by the same types of vector mosquitoes. Dengue fever has been detected widely throughout Southern Taiwan and sporadically in the north . Warmer This study has two limitations. First, the Taiwan CDC\u2019s TNIDSS (infectious disease statistics) includes only basic epidemiological data of patients with chikungunya and provides no clinical data. Therefore, this study could not compare clinical data between patients in terms of their differences or trends. Second, data provided on TNIDSS contain no information about the genotypes or strains of the chikungunya virus. Accordingly, this study could not analyze (1) the type of chikungunya virus strain that spread to Taiwan or (2) the affinity between virus strains in Taiwan and other countries. This study has the advantage of using diverse data provided by Taiwan\u2019s public sector on its online platform . This open platform has stored all historical data, which researchers can use to conduct statistical analyses or create academic value. Such data are worth exploring to expand the monitoring of infectious diseases and their characteristics, thereby continually increasing the capacity of scientific research.This study was the first in Taiwan to analyze the epidemiological characteristics and trends of imported and domestic cases of chikungunya from 2007 to 2019. On the basis of data from the Taiwan CDC, this study indicated that one domestic sporadic infection and 20 cases of cluster infection occurred. Moreover, domestic cluster infections of chikungunya in Taiwan exhibited significant differences in age (patients aged 60\u201369 accounted for the largest proportion), month of confirmation (domestic confirmed cases increased in the month after imported cases increased), and place of residence (New Taipei City accounted for the largest proportion). This information will be useful for policy makers and clinical experts in directing prevention and control measures regarding the chikungunya virus, which causes illness among the Taiwanese population. This study highlights the importance of longitudinal and geographically extended studies to understand the implications of zoonotic disease transmission in the Taiwanese population. Critical data were identified to inform future surveillance and research efforts in Taiwan."} +{"text": "Adequate bowel preparation is essential for a high-quality colonoscopy. Many randomized controlled trials (RCTs) have investigated bowel preparation protocols, including split-dose and low-volume regimens. However, RCTs are conducted in an ideal, controlled setting, and translation of trial results to clinical practice is challenging. In this study, we compared the quality of bowel preparations of real-world patients from clinical practice with those enrolled in several prospective trials.Bowel preparation quality, defined by the Ottawa Bowel Preparation Scale (OBPS), from four RCTs and one prospective trial were compared with two observational diary studies. Bowel preparations were polyethylene glycol preparation (PEG) or sodium picosulfate plus magnesium citrate (P/MC) taken via traditional or split-dose timing regimen. Age, sex, average number of bowel movements per day, comorbidities, colonoscopy indication and colonoscopy completion rates were also collected.Patients enrolled in prospective trials had a better OBPS by one point when compared with those in observational clinical practice studies (P<0.049), after controlling for age, sex, colonoscopy indication and type of bowel preparation used. We also found that each 10-year increase in age was associated with a 0.2 point increase in OBPS (P=0.008), and men were associated with a 0.5 increase in OBPS when compared with women P=0.014).Patients from clinical practice have higher OBPS than prospective trial patients. Increased age and male sex were also associated with increased OBPS. We believe increased patient motivation and education around bowel preparation regimen plays an important role in the success of bowel preparations. Colonoscopy is essential for the diagnosis of many colonic diseases including colorectal cancer and inflammatory bowel diseases (IBDs). For adequate visualization of the bowel mucosa, patients must take a laxative preparation to wash the bowel of stool and digested food products. However, colonoscopy incompletion rates have been reported anywhere from 4% to 25% , and pooThere have been many different bowel preparations protocols assessed over the years in randomized controlled trials (RCTs) . For exa Randomized controlled trials are the gold standard in medicine to demonstrate intervention efficacy. However, the same characteristics that allow strong internal validity in RCTs may also limit their external validity and translation into a \u2018real-world\u2019 clinical setting. Randomized controlled trials are conducted in the ideal study setting of a controlled trial with strict exclusion criteria, with enhanced attention from research associates and often with medications provided through the trial. Factors such as compliance, education and patient motivation can significantly dampen the effect size seen in RCTs . EffectiBowel preparations studies may be particularly vulnerable to poor external validity because of the large role patient compliance plays in outcomes. Poor compliance risk factors include failure to follow instructions, gender and cognitive status . The latThis was a retrospective study comparing the effectiveness of colonoscopy bowel preparation regimens in clinical practice with the efficacy determined through prospective trials. Raw data were available from bowel preparation studies published over the last 10 years at the Gastrointestinal Diseases Research Unit of Queen\u2019s University in Kingston, Ontario. Therefore, data were collected from five prospective studies, including four randomized controlled trials (RCTs) , 36, 37 All studies collected the normal bowel habits as average number of bowel movements (BM) per day with the exception of Vanner and Hookey (2011), where the data were represented using categories <0.33 BM/day, 0.33 to 1 BM/day, 1 to 3 BM/day, >3 BM/day, or irritable bowel syndrome (IBS)/alternating.Studies were highly variable in reporting patient comorbidities. The observational studies by Vanner and Hookey (2011) and Arya et al. (2014) indicated patients were comorbid if they were managing diabetes mellitus, chronic obstructive pulmonary disease or chronic kidney disease. Hookey and Vanner (2009), Flemming et al. (2012) and Fowler et al. (2009) only included data on the comorbidity of diabetes mellitus. No data were available on patient comorbidities in the studies conducted by Melicharkova et al. (2013) and Flemming et al. (2015).Clostridium difficile infection , inflammatory bowel disease (IBD), bowel habit change not yet determined, anemia, diarrhea, bright red blood per rectum, known polyp, nausea/vomiting, abdominal pain and in study classifiThe primary outcome was the difference in efficacy of colon cleansing assessed using the Ottawa bowel preparation score between One prospective study focused on patients over the age of 70. Anticipating that this study will skew the prospective trials patient demographic, data analysis was performed both including and excluding this study.t test to compare the Ottawa bowel preparation scores between treatment arms within trials. The prospective trials and clinical practice studies were pooled using a linear mixed effects model with a random intercept and random treatment effect to account for potential heterogeneity in the Ottawa bowel preparation scores and the treatment effects between studies. Finally, the Ottawa preparation scores obtained from the prospective trials were compared with those obtained from the clinical observational studies using a linear mixed effects model with random intercepts and treatment effects and controlling for age, sex, indication and bowel preparation method. We used restricted maximum likelihood with denominator degrees of freedom estimated by the Satterthwaite method, as implemented in the mixed procedure of SAS Version 9.4.We used a two-sample Clostridium difficile infection or change in bowel habit. Data were collected and analyzed from 715 patients from the five prospective trials, and 657 patients from the two observational prospective studies. Patient ages ranged from 19 to 98 years and were instructed to use a variety of bowel preparation types. Indications for colonoscopy ranged from asymptomatic indications such as screening or a positive fecal occult blood test (FOBT) result to symptomatic indications such as irritable bowel syndrome, Crohn\u2019s disease, ulcerative colitis, anemia, diarrhea, bright red bleeding per rectum (BRBPR), known polyps, nausea/vomiting, abdominal pain, We then pooled prospective trial data and the clinical practice data . After eSince PEG traditional and split-dosing bowel preparations were only studied in one prospective study and one observational study, we concentrated instead on the effect of P/MC bowel preparations. Finally, using a multivariable mixed effects model, we found that greater age, male sex, and results of observational clinical studies were all associated with worse (higher) Ottawa bowel preparation scores see . Each deP=0.23), respectively. Furthermore, split-dose P/MC regimens were not selectively chosen for morning versus afternoon cases, suggesting time from completion of bowel preparation and procedure start did not differ between split-dose and traditional P/MC regimens in clinical practice.Further analyses of the split-dose versus traditional P/MC populations in clinical practice showed no significant difference in baseline characteristics. The average ages for split-dose and traditional P/MC populations were 59.2 and 59.3 years (P=0.91), respectively; the average bowel movements per day were 1.37 and 1.46 than prospective trial patients after controlling for patient age, sex, indication of colonoscopy and bowel preparation method. In our study, prospective trial patients tended to be younger than real-world patients, possibly had fewer comorbidities and had higher average daily bowel movements, probably related to significant constipation being an exclusion criterion for many trials. An additional interesting finding was that scope incompletion rates were higher in prospective trials, 6.3% versus 1.7%. This may be explained by the greater proportion of asymptomatic screening indications in the pooled prospective trials when compared with clinical practice, 61.4% versus 49.9% . While pWe postulate that the superior bowel cleansing seen in prospective trials when compared with clinical practice can be explained by the difference in study design. Prospective trials, exemplified by RCTs, are conducted in a controlled setting designed to study one major hypothesis. This requires strict inclusion and exclusion criteria, involves a clinical research associate giving personalized teaching to each subject and being available for questions after the fact, and, in our study and others, often results in a study population that is younger and healthier than the general population . The curP=0.86), though our study was not specifically powered to detect a difference between these split-dose and traditional dose bowel preparations. While split-dose bowel regimen has been shown to be superior across both high- and low-volume bowel preparations in meta-analyses of randomized controlled trials , where real life versus prospective trials were compared. This study showed that while obesity presents as a risk factor for poor bowel preparation in trials of retrospective clinical practice, it is not reflected in prospective controlled trials . They suThe nature of the data collection and collation in a mixed group of studies such as these leads to limitations in the study. Our results showed that a greater proportion of clinical practice patients have comorbidities than the pooled prospective trials data, but the significance of this result is limited by the difference in the way comorbidities were defined across the studies and incomplete data. Three of the five prospective trials only recorded diabetes mellitus , 19, 38;In conclusion, we have shown that patients in clinical practice have poorer bowel preparations than patients enrolled in a prospective trial or RCT. The split-dose P/MC regimen was not associated with a better OBPS when compared with the traditional dose in the observational studies. This brings to light the need for further observational studies on the effectiveness of split-dose regimen in clinical practice. We believe that this disparity can largely be explained by the increased motivation and compliance in RCT patients, stressing the importance of patient education when prescribing bowel preparations for colonoscopies."} +{"text": "Clinical internships are a critical transition period for nursing students to develop into nursing professionals and are essential for the solidification of their professional attitudes. Undergraduate nursing students face multiple sources of severe anxiety that affect their nursing career development during this period. This study aimed to understand the anxiety experienced by undergraduate nursing students in the later period of their internship periods in a Chinese educational context.. Data were collected through semi-structured interviews and analyzed using the Colaizzi seven-step method for data analysis.A descriptive qualitative study was conducted. A purposive sample of 17 undergraduate nursing interns with experiences of anxiety was selected from three teaching hospitals in Hunan Province, ChinaThree themes emerged: the sources of perceived anxiety, the psychological journey of coping with anxiety, and suggestions for nursing management and education. The sub-themes for the first theme included career decision dilemmas, competitive pressures for employment, worries about progress of the graduation projects, challenges of the nursing licensure examination, and low self-confidence in clinical competence. The second theme consisted of two sub-themes: negative avoidance and retreat, and positive preparation and response. The third theme consisted of policy support to create more employment opportunities, comprehensive guidance from nursing schools and hospitals, and psychological assistance and counseling.This study revealed that undergraduate nursing students were prone to anxiety during the later period of their clinical internships. Specific sources of anxiety encompassed career decision-making, job pursuit, graduation, and licensure examination. Undergraduate nursing students adopted both positive and negative approaches in coping with anxiety during the later period of their clinical internships. Nursing educators and administrators should develop appropriate instructions and support systems to reduce anxiety among undergraduate nursing students. A shortage of nurses affects hospital stability and quality of patient care . As the Undergraduate nursing education in China is a four-year program. According to the three-stage training model of public courses, professional courses, and clinical placement, students are required to complete a 10-month clinical internship in their fourth year . The intAnxiety is a complex emotional state involving tension, entanglement, panic, and irritability caused by confrontation with a potentially dangerous situation . CertainNumerous studies have revealed that nursing students who undergo internships experience various degrees of anxiety. The reasons are multifaceted, and the main stressors might be lack of knowledge and skills, and fear of medical error resulting from inadequate preparation . In addiLi et al. , 16, 17 Drawing on the principles of naturalistic inquiry, a descriptive qualitative study was conducted to explore the experience of anxiety among Chinese undergraduate nursing students and strategies to alleviate anxiety during the later period of their clinical internships. Descriptive qualitative research is valuable for exploring the experiences of a phenomenon when a comprehensive summary is desired . The aimPurposive sampling was used to recruit participants from three teaching hospitals in Hunan Province, China . After oThis study is based on Jiang\u2019s theory of \u201cPsychological Stress System\u201d , in whicThe study data were collected from March 2021 to April 2021. Data were collected through face-to-face, semi-structured interviews at three teaching hospitals in the Hunan Province. Interviews were held in quiet and accessible rooms to enable participants to speak confidently. All interviews were conducted by the first author, who had 26\u2009years of clinical training experience and was proficient in qualitative research. Participants signed the consent forms and were briefed on the purpose, procedures, and significance of the study before the face-to-face interviews. Before the interviews, the first author sought the informed consent of the participants through phone calls. Interview dates and times were also set through phone calls prior to the interviews. All participants completed a form seeking demographic information according to the instructions of the first author. The form contained questions about gender, place of residence, month of internship, graduation, or employment intention and self-assessment of thesis progress and clinical competence. All questions were open-ended except for the self-assessment portions, and were assigned a scale measuring satisfaction from 0 (very dissatisfied) to 5 (very satisfied). None of the participants dropped out during formal interviews. The researcher also tried to avoid verbal or non-verbal signs of bias to allow participants to speak freely. Interviews averaged between 20 and 30\u2009min and were audio-recorded and then transcribed verbatim.After the interview, descriptive statistical analysis was applied to the participants\u2019 demographic characteristics using SPSS 23.0 . Two nurTranscripts were reviewed numerous times to obtain a general picture of students\u2019 perceptions and experiences of anxiety.We first underlined the significant statements from the transcription that could be attributed to the three interview questions . The questions were developed based on a psychological stress system theory .Each underlying meaning was coded and typed in the code system of Nvivo 11.0.We compared the determined codes and organized formulated meanings into themes such as career decision dilemma and competitive pressure for employment which could be attributed to the \u201csources of perceived anxiety.\u201dIn this step, the significant statements, formulated meanings, and sub-themes were integrated into exhaustive descriptions of each question.We narrowed the themes and explanations of their fundamental structures. We merged similar or meaningfully similar subthemes into the main categories and reached a consensus.A follow-up appointment was conducted between the researchers and each participant via telephone call. Each participant was given the chance to respond to the analyzed data, and alterations were made according to their feedback. Eventually, all of them agreed that the results reflected their real experiences.We followed Denzin and Lincoln to ensure credibility, transferability, dependability, and confirmability to verify the trustworthiness of the data . The intn\u2009=\u200917), sleep disturbance (n\u2009=\u200915), unfortunate premonitions (n\u2009=\u200913), fatigue (n\u2009=\u200912), fear (n\u2009=\u200912), frequent urination (n\u2009=\u200911), excessive sweating (n\u2009=\u200910), facial flushing (n\u2009=\u200910), dizziness (n\u2009=\u20099), hand and foot tremors (n\u2009=\u20098), panic (n\u2009=\u20098), dyspnea (n\u2009=\u20098), nightmares (n\u2009=\u20096), somatic pain (n\u2009=\u20096), palpitations (n\u2009=\u20096), and stomach pain and indigestion (n\u2009=\u20096). The main themes and subthemes were categorized, as shown in Table\u00a0Anxiety experiences included nervousness \"Read and study for two hours a day, complete the predetermined small goals, communicate with friends, and communicate with parents to obtain support and comfort.\"\u2014N7Moderate anxiety is protective and can improve the body\u2019s ability to respond to environmental stimuli, which can have positive effects on work and learning. In this study, anxiety enabled some nursing students to respond actively, by paying attention to employment information, adjusting employment expectations, and preparing for employment in advance.\u201cBig hospitals have bigger development platforms with better benefits, so they attract people who are desperate to squeeze in. If county- and city-level hospitals can increase recruitment positions as well as improve job benefits and future career development security, I am happy to change my direction of employment.\"\u2014N8Some nursing students also suggested the establishment of an employment platform to provide employment information. \u201cThere is a lot of job information on the internet, but it feels like the information is scattered. A platform dedicated to providing employment information could increase our chances of getting information on employment.\u201d\u2014N16.Considering the fierce competition in finding jobs, nursing students seek policy support at the government level that could provide more employment opportunities. For example, raising wages in county and municipal hospitals, and promoting specialized training in geriatric nursing, community nursing, rehabilitation nursing, hospice care, and traditional Chinese medicine nursing.\"I hope that teachers will inform students of the employment situation through continuous lectures from freshman year and by holding mock recruitment scenarios and mock job application competitions in junior year.\u201d\u2014N6\u201cThrough meetings or information, systematically analyze the employment situation, inform students what they need to prepare for the nursing exams, how to balance subjects, nursing exams and work, so that nursing students are not so confused.\u201d\u2014N13\u201cI hope that the school can help broaden professional knowledge and provide skills training for skills such as first aid skills, cardiac monitoring, indwelling needle usage, and other operations to enhance professional skills.\"\u2014N16Regarding the hospital, nursing students believed the hospital could provide more employment information in the later period of their internships by providing guidance on the employment scenario, simulations and competitions to further improve their clinical skills, and give students sufficient time to work on their graduation projects and prepare for licensure exams.\u201cI hope that hospitals will provide more recruitment information in the middle and later period of the internship, so that nursing students can think more and prepare early according to the recruitment requirements and their own conditions.\"\u2014N14\"Training on the process and form of job applications and simulating job interviews so that students can be prepared by being familiar with the form of job applications.\"\u2014N4\u201cI hope the skills training room can be expanded to provide opportunities for nursing students to practice operational skills.\"\u2014N16With respect to nursing schools, nursing students expressed their desire to have career planning education immediately after they entered the school and to receive guidance on graduation projects, on how to balance the graduation project with other commitments, on licensure exams, on employment, and on how to improve clinical competency.\"Sometimes I feel like I need a place to breathe or someone to talk to. However, in the internship hospital, we are not familiar with each other, so I hope there could be a special psychological studio.\u201d\u2014N3Another nursing student\u2014N13\u2014also said, \u201cWhen I feel stressed and can\u2019t bear it alone, I hope to find a place like this.\u201dThe participants mentioned that they would like clinical teachers to pay more attention to the psychological state of students, especially in the later period of their internships, and to give positive guidance and comfort to students when they ask questions or are emotionally tense. Some nursing students suggested setting up a counseling room or establishing activity groups to carry out psychological guidance activities so that nursing students can have an organization to rely on when they need it, which can effectively relieve anxiety.The later period of the clinical internship is a decisive period for nursing students considering a career in nursing . Our stuIn the process of career selection, undergraduate nursing students face various psychological pressures and conflicts. For example, some nursing students desire to pursue postgraduate studies but are worried about not being shortlisted or have difficulty deciding; some nursing students wish to transfer to civil service or start their own business because they do not identify enough with nursing work or because of its physical nature, but do not want to give up their 4-year professional education. In summary, they are very confused about career decisions, and this dissonance between ideals and reality makes nursing students feel lost, confused, and anxious when choosing a career in the later period of their internships .The shortage of human resources for nursing professionals is a global challenge according to the World Health Organization , especiaOur study found that undergraduate nursing students who were affected by anxiety in the later period of their internships exhibited poor cognizance of their professional identities and showed negative professional attitudes toward nursing. Such negative attitudes by nursing students include them considering changing majors in graduate school, changing careers, and being reluctant to engage in clinical nursing; they were even ready to give up clinical nursing work, which would result in the loss of nursing professionals.To strengthen nursing students\u2019 professional identities, schools can introduce ideological elements in the curriculum design and find ways to inspire nursing students and guide them in their career planning . AdditioOur study revealed that nursing students need targeted training and support from the government, schools, and hospitals during the later period of their internships. For example, a sound employment service platform should be provided to undergraduate nursing students. Internship hospitals should provide employment guidance and group-based mock interviews on recruitment examination content, procedures, and skills, and help students analyze the employment scenario to improve their self-identity and mental health . InternsIn the later period of their internships, support from family, institutions, hospitals, and friends can help nursing students make a smooth transition out of nursing school . As showStudents should also seek help from their supervising teachers for psychological guidance to relieve them from their anxiety. Meanwhile, clinical preceptors should pay attention to the psychological condition of nursing students during the post-internship period, conduct regular assessments, and provide group or individual psychological counseling to students with different personality traits, such as relaxation training, breathing training, guided reflection, positive meditation training, music therapy, and aromatherapy to help nursing students regulate their own emotions . When seOur results, combined with the literature review, suggest that nursing students in the later period of their internships faced multiple stressors that caused anxiety. Nursing educators and nursing administrators should pay attention to the factors that cause anxiety among nursing students in the later period of their clinical internships and take appropriate measures to reduce the anxiety conditions of nursing students to help them transition smoothly to nursing positions. More particularly, we recommend that nursing schools provide specific career planning courses, psychological support, and employment guidance for nursing students throughout their study. In addition, the government could set up an employment service platform that provides timely employment information. Internship hospitals should also pay attention to the psychological condition of nursing students during the post-internship period and provide psychological counseling services regularly.There were some limitations to our study. First, we used a relatively small sample size with samples from only three hospitals; hence, the results may not be applicable to all nursing students at different levels and in different regional hospitals in China. Further research is necessary with students working in different institutions and regions to obtain more conclusive evidence. Another matter to consider is that the interviews were conducted in Mandarin. The verbatim data presented in this study are directly translated from Chinese, and in places the text may be difficult to understand due to cultural differences. In spite of these limitations, we believe that the study adds to the knowledge of the experience of anxiety among nursing undergraduate students in the later period of their internships and could serve as a reference for future research.This study suggests that undergraduate nursing students are prone to anxiety during the later period of their clinical internships. Specific sources of anxiety are career decision-making, job pursuit, graduation, and licensure examination. Our study revealed both positive and negative approaches adopted by undergraduate nursing students in coping with anxiety during the later period of their clinical internships, and provided specific suggestions for nursing educators and administrators in developing appropriate instructions and support systems to reduce anxiety among undergraduate nursing students."} +{"text": "Cinenurducation, a film-based approach to nursing education that incorporates student-centered, problem-solving, experiential, and reflective learning strategies, allows students to experience a variety of indirect experiences and improves critical thinking and self-reflection through discussion.The aims of this study were, first, to employ a cinenurducation approach to help instill a proper professional nursing identity in second-year nursing students and, second, to examine the effects of this approach on the perception of nursing, satisfaction with major, and professional nursing values of the participants.Me Before You , Testament of Youth , Girl, Interrupted , Hungry Heart , Iris (nursing knowledge and problem solving), and Chronic (nursing knowledge and cooperation). The experimental group (n = 14) participated in the 8-week educational program, and the control group (n = 15) did not.An experimental, pretest-and-posttest design was used to test the primary variables, including perception of nursing, satisfaction with major, and professional nursing values. The nursing educational program was developed based on the learning concepts of cinenurducation and the core concepts of nursing. The program, which included six films, addressed the following concepts: Perception of nursing, satisfaction with major, and professional nursing values all improved significantly more in the experimental group than in the control group, with large effects observed.Cinenurducation is an effective approach to promoting professional nursing identity in nursing students. Educators should incorporate films into nursing education. In addition, nursing education should incorporate a variety of educational materials to provide students with opportunities for reflective learning. Professional nursing identity refers to the awareness of the functions and roles that professional nurses are expected to perform in clinical contexts . DevelopPerception of nursing refers to beliefs regarding the traditional, social, and professional impressions of nurses and nursing prospects . NursingSatisfaction with major refers to students' satisfaction with meeting their expectations related to their academic major. A good understanding of the career associated with one's major increases this satisfaction . In partProfessional nursing values are the overall beliefs that encompass views on nurses' activities and roles as well as opinions on nurses as professionals . As the Positive professional values are developed continuously in nursing students through their education and learning experience . These vFilms have been reported to be effective in helping students experience a variety of learning including emotions, feelings, knowledge, actions, skills, and attitudes . When seCinenurducation is an approach to nursing education that has recently attracted significant attention . CinenurResearch related to cinenurducation has focused on understanding patients and health promotion in nursing , multicuSome studies have indicated that third-year nursing students with clinical practice experience tend to have a negative perception of nursing, low satisfaction with their major, and inadequate professional nursing values . These tThe conceptual framework in this study is shown in Figure To promote professional nursing identity by improving perception of nursing, satisfaction with major, and professional nursing values, the contents of nursing education were organized using the nursing core concepts proposed by An equivalent control group was used in this study. A pretest/posttest, nonsynchronized design was used to examine the effect of the intervention on participants' perception of nursing, satisfaction with major, and professional nursing values Figure .The participants in this study were nursing students in their second year of a 4-year undergraduate program of a university located in Iksan City. In undergraduate nursing programs in South Korea, first-year students take an introductory course on nursing, second-year students take a basic major course and laboratory practicum, and third- and fourth-year students take a major course and clinical practicum. Nursing students are expected to establish their professional nursing identity before completing their nursing major course . TherefoThe educational intervention in this study was formatted as an extracurricular class that was not compulsory and was not graded. The researchers publicly posted the syllabus, overall purpose of the research, and participant criteria, and 40 students applied as volunteers to participate in the class and research.G*Power 3.1.3 was usedMe Before You , Testament of Youth , Girl, Interrupted , Hungry Heart , Iris (nursing knowledge and problem solving), and Chronic . For blinding purposes, the first author and second author were separately involved, respectively, in collecting the study data and conducting the intervention. Using SPSS, the participants were randomly assigned to the experimental and control groups in two classes of 20 students. Only the data collector knew the designation of each group to ensure that the instructor and the participants remained blinded.Data were collected from August 28 to December 13, 2017. Pretest and posttest data were collected first from the control group and then from the experimental group. The control group took the pretest between August 28 and 30, 2017, with the posttest conducted between October 18 and 20, 2017, after conclusion of the intervention. The experimental group took the pretest on October 25, 2017, with the posttest conducted on December 13, 2017, after conclusion of the intervention.Informed consent forms, general information on the study, study research aims, and information on voluntary consent/withdrawal were provided to potential participants. After providing informed consent, the participants were enrolled and completed the questionnaires, which took about 5 minutes. A small gift was presented to each participant upon questionnaire completion.The control group had received no film teaching or other supplemental teaching interventions. For ethical considerations, the intervention was given to the control group after all study data had been collected.t test. To identify the effects of nursing education using films, an independent t test was used, and the effect size was calculated using Cohen's d formula. Dependent variables that were not homogeneous in the pretest were controlled by covariance, analyzed using analysis of covariance, and were assessed for effect size using the eta-squared formula.The collected data were analyzed using IBM SPSS Statistics 24.0 . The general characteristics, perception of nursing, satisfaction with major, and professional values were estimated by number, percentage, mean, and standard deviation. The skewness of these variables was between \u22120.73 and 0.52, and kurtosis was between \u22120.57 and 0.33. As the skewness and kurtosis were both < \u00b11.965, the collected data were interpreted as normally distributed. Homogeneity between the experimental and control groups was verified using a chi-square test and an independent No significant difference was observed between the two groups in terms of gender, age, general satisfaction with major, perceived difficulty of their major, motivation for selecting major, satisfaction with major, or professional nursing values Table . Howevert test of the mean difference between posttest and pretest scores showed that the intervention had a significant effect on satisfaction with major and professional nursing values in the experimental group. Analysis of covariance, which controlled for the perception of nursing pretest scores by covariance, showed that the intervention had a significant effect on perception of nursing , with large effects observed (The effects of nursing education using films are shown in Table observed .This study was conducted to verify the effect in nursing students of a film-based nursing educational intervention on perception of nursing, satisfaction with major, and professional nursing values. To evaluate the effects of the program, the changes in pretest and posttest results were examined between the experimental and control groups.After the completion of the intervention, a larger increase in the mean score for perception of nursing was observed in the experimental group than in the control group, with a large effect size of 0.55 . This reFurthermore, satisfaction with major was found to have improved significantly in the experimental group, with a large effect size of 0.97 . As few In addition, nursing education using films was found to be effective in improving professional nursing values, with a large effect size of 0.93 . AccordiNursing education using films was developed based on the learning concepts of cinenurducation suggested by Although this program helped nursing students understand the content of the films and organize their opinions, one limitation was the length of time required for each session. Therefore, a teaching method that requires less time per session is needed. Educators may provide orientation and discussion topics on the films via online sources, and then students could watch the film in advance and discuss the topics offline. Meanwhile, the films in this study were selected from Western countries only. Thus, the selection lacked consideration of possible differences/discrepancies between Eastern and Western cultural settings. Therefore, further research is needed to account for cultural contexts in films. In addition, as this study was conducted on second-year nursing students, the nursing professional identity formed before the clinical practicum may conflict with the experience in the clinical setting. Therefore, longitudinal research should be conducted to confirm the persistence of intervention effects through graduation.This study was conducted to verify whether nursing education using films affected the perception of nursing, satisfaction with major, and professional nursing values in nursing students. The results showed that the intervention was effective in these three aspects. Therefore, it is suggested that the intervention developed in this study be used as a strategy to improve the professional nursing identity of nursing students.On the basis of this study, the implications of nursing education in undergraduate programs are as follows. First, nursing students' indirect experience through using films may promote learning of the core concepts of nursing. Therefore, educators should actively attempt nursing education using films. Second, this intervention was meaningful in terms both of using films as an educational resource and of promoting reflective learning through in-depth discussions. In the future, nursing education should be designed to provide students with opportunities for reflective learning using various educational materials. Finally, to help better establish the professional nursing identity of undergraduate students, educators should offer various supplementary courses such as the intervention attempted in this study in addition to the standard curriculum."} +{"text": "Children of parents with a mental illness (COPMI) are at risk of behavioral, emotional, and cognitive difficulties and diagnoses. Support and information about parents\u2019 mental illness may contribute to improve their lives, which is the purpose of the intervention Child Talks (CT). This study aimed to investigate the participation rate of CT, characteristics of participating patients and children, and themes in sessions with children.Data were collected from 424 electronic patient journals written by healthcare professionals (H) for patients admitted to a clinic for mental health and substance use disorders in the years 2010\u20132015. Both quantitative statistical analysis and qualitative thematic analysis were carried out.Eighteen percent of assessed parents with minor children received the CT intervention and children participated in half of them. Participating children more often knew about their parent\u2019s treatment and condition when initially assessed, and more often lived with the hospitalized parent. Three main themes were identified in sessions with children; communication about parental mental illness within the family, childrens\u2019 struggles, and healthcare professionals\u2019 (HCPs) evaluation of the child\u2019s situation and need for further support.Sessions with patients\u2019 children appeared to be relatively rare, and participating children did not necessarily receive appropriate information, support, or follow-up. To ensure that HCPs provide quality support and follow-up to COPMI, the routines and the training of HCPs need to be improved. In Norway, 12.2% of children have parents who are receiving treatment for mental illness and/or alcohol use disorder each year approximThe transmission of mental illness from one generation to the next is a complex process. Such transmission is influenced by the interaction of factors related to the mentally ill parent, the child, the family, and the social environment . ProtectKnowledge about mental health provides resilience against mental illness , 19. ChiChild Talks (CT) is a brief preventive intervention, developed in the Netherlands for COPMI aged 0\u201325 , 25. To The content of the CT intervention accords to \u00a710 a) of the Health Personnel Act . The CT Most HCPs have positive attitudes toward a family-focused practice in adult mental health services . Still, There is a lack of knowledge about how factors related to the parent and the child influence whether children are given information and support by the parent\u2019s HCPs. Little is known about the extent to which COPMI participate in psychoeducational interventions and whether the children who do participate are provided with support, information, and follow-up actions. In this study we aimed to address this knowledge gap by analyzing patients\u2019 health records.The main aim of the present study was to evaluate the performance of CT sessions, with a particular focus on sessions with participating children. More specifically, we aimed to investigate:(1)parents\u2019 participation rate in CT,(2)children\u2019s participation rate in CT, and reasons for their exclusion,(3)age, gender, and psychosocial differences between participating and non-participating children, and(4)HCPs\u2019 support and information to children.This is a retrospective study based on electronic patient journal data for the period 2010\u20132015. The approval from the data protection officer at UNN allowed us to extract the data from the electronic patient journals in 2015. The study has a mixed-methods approach since both quantitative data from electronic patient journal entries and written reports entered by HCPs to analyses qualitatively were used.The total HCPs workforce at the Division for Mental Health and Substance Use Disorders (DMHSD) at UNN was 436 in 2010 , whereasThe data material in this study is information extracted from the Family Assessment Forms and Logbooks from CT sessions, as recorded by HCPs in electronic patient journals. Over the course of the project, two different forms were implemented in the electronic patient journals at the DMHSD at UNN. These two forms were a Family Assessment Form and a Logbook from CT sessions. HCPs were instructed to fill out the Family Assessment Form for patients admitted to the DMHSD who had minor children. Secondly, the patients were to be invited to participate in CT, and HCPs were instructed to write a short report of the sessions in the electronic patient journal, labeled CT Logbooks. Information from these two forms was extracted from the electronic patient journals in 2015.The Family Assessment Form consists of five categories of questions: (1) general information about the child, (2) the child\u2019s network, (3) concerns for the child, and how the child is coping, (4) the child\u2019s knowledge and information about PMI, and (5) the family\u2019s need for support and follow-up.n = 39). In the CT Logbooks, background information such as date, duration of session, place, participants, and parents\u2019 diagnoses are requested. HCPs are also asked whether they have any concerns or issues regarding the family. In the following sections, HCPs are asked to respond to openly formulated questions about each session. Five sections are to be filled out for session one, two, and three about which topics and concerns were discussed, support options for the children and families, any questions regarding the child posed by the parents, any additional details, and agreements for the next session. For session three, there are additional sections for follow-up agreements and advice given to the child and parents, as well as for referrals and necessary follow-up actions that HCPs are to take. In our analyses, we used all the sections from sessions in which children participated. The amount of information and degree of details in the CT logbooks varied. In most logbooks HCPs had written a response in all sections. Some logbooks were several pages long, while others only contained a few paragraphs.Logbooks from cases with participating children were used in the thematic analysis children\u2019s participation rate and (2) number of non-participating children whose parent participated. For non-participating children, we used information from the Logbooks to detect and quantify reasons for their absence.Does your child know that you receive treatment/are hospitalized?\u201d and \u201cHas your child received information about your condition?\u201d The response categories for these two items are \u201cno\u201d, \u201cpartially,\u201d and \u201cyes.\u201d Descriptive statistics, t-tests, and chi-square testing were computed in IBM SPSS Statistics 25.Descriptive information of participating and non-participating children and parents was compared by analyzing information reported from CT sessions and Family Assessment Forms. For our analyses, we used information from the Family Assessment Form on parent gender, parent diagnosis, child age and gender, and where the children lived. We also used two questions about whether the children had received information about the parent\u2019s treatment/hospitalization and condition: \u201cd. The magnitude for all effect sizes was interpreted in accordance with Cohen for effect size measurement. To test for differences in the mean age for participating and non-participating children, we initially performed a Levene\u2019s test to determine if the variance of the groups was unequal or equal. The results from the Levene\u2019s test showed that the variance of the groups was unequal. Therefore, we performed a two-tailed t-test with unequal variance for the groups to test for age differences. We calculated the effect size of the mean differences using Cohen\u2019s th Cohen .Child Talks Logbooks were analyzed to identify the thematic content of the sessions in which children participated, and all CT sessions with participating children were imported into the qualitative analysis program NVivo 12 Pro. The logbooks are a secondary source of information of the CT sessions, written and processed by HCPs.For the thematic analysis of the CT sessions with participating children, the authors and researchers of the present study used the six-step phase guide by Braun and Clarke . This isThe first step in the guide by Braun and Clarke is gettiAround 5,500 patients were receiving treatment at the DMHSD each year during the project period . A totalIn 39 (50%) of the performed CT interventions all or some of patients\u2019 children participated. The 78 patients who received the CT intervention had 157 children in total. Of these children, 62 (39%) took part in the intervention, leaving 95 children (61%) not participating despite their parent receiving the intervention.Based on the Family Assessment Forms, a total of 864 children were identified. Of these children, 62 participated in CT, resulting in a total participation rate of 7% for the identified children. See n = 23). The reasons stated in records were: (1) the patient was soon to be discharged from hospital and therefore the task of talking to the children was postponed to a later occasion or transferred to personnel in other services(n = 7); (2) the patient had little contact with the child/children (n = 7); (3) the patient rejected the offer of CT with participating children (n = 5); and (4) the other parent of the child did not consent to the child participating, or personnel had not been given a response from the family (n = 4).Healthcare professionals provided information about the reasons for children\u2019s absence in some of the records = 3.805, p = 0.051 and had a small to medium effect size (\u03c6 = 0.18). Information about parents\u2019 gender was missing for 44 of parents\u2019 children.The difference between participating and non-participating children in terms of parent\u2019s gender was not significant at Children participating in the intervention were between 3 and 22 years of age. We observed that the proportion of participating children increased with age see . Two chiM (51) = 11.69, SD = 3.78] and non-participating children , we performed a t-test. The results from Levene\u2019s test for difference of variance between the groups were significant at p < 0.05: F = 5.764, p = 0.018. We therefore preformed a two-tailed t-test for which unequal variance for the groups was assumed. Cohen\u2019s effect size value (d = 0.34) implied a small to medium magnitude of difference between the two groups, but the difference was not statistically significant at p < 0.05: t (111) = 1.83, p = 0.07.To test for difference in the mean of age of participating children [p < 0.05, X2 = 0.335, p = 0.563 and had a very small effect size (\u03c6 = 0.05).Of the participating children, 33 were girls and 23 were boys. Of the non-participating children, 36 were girls and 31 were boys see . Informap < 0.05 between the groups, X2 = 10.62, p = 0.001, with a medium magnitude of difference (\u03c6 = 0.36).Participating children knew that their parent was receiving treatment or was being hospitalized more often than children who did not participate in the sessions, measured at assessment point. Of the participating children, 97.7% (42 out of 43) were aware of their parent\u2019s treatment or hospitalization, whereas 72.5% (29 out of 40) of the non-participating children were aware of this. However, answers to this question were missing for 19 participating children and 55 non-participating children. We compared participating and non-participating children by performing a chi-square test and found a significant difference of p < 0.05 level, X2 = 6.54, p = 0.011, with a small-to-medium effect size (\u03c6 = 0.28).Participating children had also received information about their parent\u2019s condition more often than non-participating children, at assessment point. Of the participating children, 85.7% (36 out of 42) were aware of their parent\u2019s condition, compared to 60.5% (23 out of 38) of the non-participating children. Answers to this question were missing for 20 participating and 57 non-participating children. The results from the chi-square test comparing the two groups showed a significant difference at p < 0.05, X2 = 11.93, p = 0.001, with a medium effect size (\u03c6 = 0.36).For participating children, 94% (47 out of 50) lived with the hospitalized parent. In the case of the non-participating children, 65.9% (29 out of 44) were registered as living with the patient. Answers to the question of where the children lived were missing for 12 out of 62 participating children and 51 out of 95 non-participating children. A chi-square test conducted to assess the difference between participating and non-participating children in terms of living with the hospitalized parent showed a significant difference at The thematic analysis of the written reports from CT sessions involving children resulted in three main themes and ten sub-themes. The main themes were communication about PMI within the family, children\u2019s struggles, as well as HCPs\u2019 evaluation of the child\u2019s situation and identification of further support. See Healthcare professionals frequently explained to parents why talking openly about mental health within the family is important. Some of the parents were open about their illness, and many said they wanted to be even more open. In many families, however, there was little to no communication about the parent\u2019s illness. Hence, for some children the CT session was the first time they received information about their parent\u2019s illness. The children usually expressed that they were glad to receive information from HCPs. However, some children did not want information and did not want to visit or have contact with the mental health services.In some cases, the parents were unwilling to talk about mental illness and prevented their children from receiving information or being given the opportunity to talk about their situation. Reasons were that they did not want their children to be unnecessarily worried or to focus on the negative characteristics and psychiatric diagnosis of the sick parent. A mother who was diagnosed with depression, anxiety, and an eating disorder protected her 10-year-old daughter (ID 35), from words such as \u201cmental illness\u201d and \u201cpsychiatric hospital.\u201dThe information that children received was not always correct or clarifying. Proper explanations about parents\u2019 illness, symptoms and behavior were often missing. Some children knew that something was wrong or that their parents were struggling, but not what or how. Often parents and HCPs used word such as \u201cexhausted\u201d or \u201chaving headache\u201d when explaining parents\u2019 mental illness.Questions from children appeared in a few records. The questions reported concerned the care of younger siblings, heredity, and the home situation. Children did not usually ask directly, but rather expressed their curiosity about a theme in the session.Children reported missing their hospitalized parents and being worried about them. Two boys aged 10 and 11 years (ID 78) missed their father while he was hospitalized and reported being worried about him. This was also given as the reason for the youngest boy having trouble concentrating at school. Some children kept in contact with the hospitalized parent by using video calls and some had also visited their hospitalized parent on several occasions. Some children missed the way things used to be before their parent became ill, like a 15-year-old girl (ID 17) who said that she was missing her \u201chealthy\u201d mother. She missed spending time with her mother in the evenings, lighting candles, and watching movies. She also missed tasty and healthy meals.Several children in the records were worried about, and had great responsibilities for, the care of their sick parent and/or younger siblings. A 12-year-old girl (ID 42) had to physically stop her mother from dying by suicide. The girl was worried about what might happen once her mother was discharged from the hospital. HCPs emphasized the importance of making the daughter aware that it was not her responsibility to take care of and look after her mother, yet they advised her to contact the police if her mother did anything like that again. The girl also expressed her concerns for the care of her younger siblings while her mother was hospitalized, especially the one-year-old. She was not sure her father would be able to take care them. She had trouble sleeping at night. The girl was advised to contact help services if things became difficult or if she needed someone to talk to.Some children were overinvolved in the illness of their parent, such as a 12-year-old boy (ID 18) whose mother had been diagnosed with anxiety:The boy said he was going to look after his mother until he became an adult. He was worried about his mother when she got her anxiety attacks. When she got the anxiety attacks, he massaged her.Another child, a 16-year-old boy (ID 60), frequently had to participate in his mother\u2019s doctor appointments and translate letters from the Ministry of Foreign Affairs. He was also involved in the conflict between his parents and described how he had to stop his father from being violent with his mother:In the beginning, after they came to Norway, his father was physically violent toward his mother. One night he got up and told his father that if he ever beat her again, he would call the police. The father went out and did not return until much later. Since the incident the father has never beaten her. The boy cried while telling this. He said this was the very first time he had cried in front of his sister.Many children had a difficult home situation, living in families with severe, long-lasting problems. Many of the children had experienced frightening episodes at home. The children\u2019s situations at home were often described as unpredictable, stressful, and characterized by high conflicts levels and violence between family members. In one case a 16-year-old girl (ID 39) who was living with her mother, who had been diagnosed with a psychotic illness, was physically abused:There was general concern for the family because of the mother\u2019s mental state. There was also concern for the daughter\u2019s situation and whether she was given help for her own mental health problems, which she had had for several years. Her mother was unstable and had on several occasions pushed and thrown things after the girl. The mother had also called her names. It was difficult for the daughter because her mother was suspicious and seemed to be in a paranoid state of mind.The patient in the example above was discharged from the hospital and sent home to her daughter. A report of concern was sent to The Child Welfare and Protection Services (CWPS). The daughter was advised to contact the school nurse when she returned to school at the end of the summer.Several children had more or less concrete thoughts about dying by suicide. For one of these children, HCPs stated that the child\u2019s mental health problem was taken care of by their general practitioner. In another case a 16-year-old boy (ID 60) was invited to call HCPs if he wanted to talk, after he had told them that he had thought about cutting his main artery if he was sent back to Afghanistan. He had even looked in the kitchen drawer for a knife. For a child who had attempted to die by suicide earlier, HCPs were concerned for the child\u2019s mental health problems and whether appropriate help was provided.In many of the records, HCPs observed and evaluated the situation of the children and the parent\u2019s caring abilities. In some cases, HCPs explicitly wrote down the agenda for evaluating the children, for example to \u201clook at the interaction between the mother and the daughter\u201d or to \u201cobserve the relation and the interaction between the children and parents.\u201d In one case (ID 26), the HCPs evaluated the attachment and how the child acted around his parents. HCPs even talked with the four-year-old boy while his parents were waiting outside.Healthcare professionals explored the children\u2019s network and support options as part of the intervention. Many sessions led to referrals to CWPS and The Child and Adolescent Mental Health Services (CAMHS), but in some cases where children described severe problems, no referrals were made. HCPs frequently encouraged parents and children to contact their general practitioner, their teacher, or the school nurse. Responsibility for establishing contact with help services or professionals was often left with the child. Some families were already in contact with CWPS and/or CAMHS. School nurses were frequently recommended as a support option. In the case of the 10-year-old girl (ID 35) who was being shielded from words such as \u201cmental illness\u201d and \u201cpsychiatric hospital,\u201d the school nurse was recommended as a support option because the parent and the child knew of her. Likewise, in a case with a 17-year-old boy (ID 09), the school nurse was emphasized as a support option as a neutral person the boy could talk to about everyday life and other relevant topics of conversation for adolescents. In addition, HCPs often gave children and parents the opportunity to have several sessions and to get in touch with them outside of the sessions if they needed to talk. The HCPs also gave children the opportunity to call them if they had any questions. In some cases, there was a mutual agreement that the family would benefit from staying in touch with the ward.Of the thousands of patients at the DMHSD at UNN during the period 2010\u20132015 , 37 and Seven percent of children identified in the Family Assessment Forms participated in CT. However, the number of children identified in the Family Assessment Form does not represent all minor children of patients at the DMHSD. In fact, around 5,500 patients were receiving treatment at the DMHSD each year during the project period , and likAccording to the CT manual, children should participate in the second session and optionally in the third session . HoweverThe main reasons for children not participating reported in this study were reluctance of one or both parents, little contact with the children or ending of the parent\u2019s treatment. In cases where the parent does not have custody or contact with the children, inviting the children to participate in a session is not appropriate, thus these patients would not have been invited to participate in the CT intervention. Large demographical distances between the clinic and the childrens home might have made childrens participation difficult in some cases, especially for the youngest children. In cases in which parents were reluctant to bring their children to a CT session, HCPs are in a good position to argue in favor of child participation. The reasoning behind including children is available in the CT manual and motivating parents and planning how to inform the children is the core activity in session one . AlignedIn terms of factors relating to the parent, differences between participating and non-participating children regarding parents\u2019 diagnoses could not be tested in reliable ways because of the sample size. However, the parent\u2019s gender might be a factor influencing child participation, with a difference between participating and non-participating children close to our chosen significant level, with a small to medium effect size. COPMI more often lived with their mothers as a sole caregiver and therefore were in the care of relatives while their mother was hospitalized . A more Mostly older children participated in CT. Child participation increased with child age, and the difference between participating and non-participating children in mean age was close to our chosen significance level, with a small to medium effect size. In earlier studies HCPs have reported feeling insecure about who has the responsibility of children visiting patients and how to have age-appropriate conversations . FurtherIn terms of knowledge about PMI, there was a significant difference, of a small to medium magnitude, between participating and non-participating children. Participating children more often already knew about parents\u2019 treatment/hospitalization and condition. Families that are more open about PMI might be more willing to have children participating in a conversation with HCPs. This is in coherence with earlier studies in which families\u2019 fear of involving children was perceived as an important hindering factor for a family-focused practice by HCPs .The main themes in the CT sessions with children reflected the objectives of the CT intervention : communiChildren were glad to receive information, which is in line with earlier studies which show that children appreciate support and information from HCPs , 23. ParHealthcare professionals evaluated and explored children\u2019s situations but were reluctant to refer to other services or provide further support. High conflict levels within the family, domestic violence, physical and mental abuse, mental health problems and suicide thoughts among children were described. However, few appropriate actions were taken by HCPs. Despite HCPs\u2019 obligation by law to refer children to the CWPS when concerned with their living situation, HCPs did not take appropriate actions in all cases. In previous studies HCPs have reported hesitation against referrals because of insecurities of whether there were grounds for referral, whether a referral would benefit the child and whether a referral would harm the family and their relationship with the patient . The lacOne limitation of this study is missing data for several variables, particularly for the non-participating group of children. The results must therefore be interpreted carefully. The small sample size may be a factor contributing to the lower sensitivity of the t-test, resulting in less reliable results.The journal data were a secondary source of information of the CT sessions, written and processed by HCPs, based on their perception of what is important and of interest. What was written in the logbooks was partially decided and influenced by the CT logbook and the questions HCPs were to answer. However, the questions were openly formulated and did invite HCPs to give detailed descriptions of the conversations and share a range of information. Despite this, it varied how much and how specific the written information about the conversations were. By focusing on the themes that were discussed in sessions, rather than looking for meanings behind the text, the data material was suitable to answer the associated research aim in the present study. The benefit of the research design is that it enabled a reduction of the disturbance and influence of an observing researcher. A researcher present in sessions might have made participants more hesitant to speak openly about sensitive and personal subjects. In addition, HCPs should be able to perform the sessions at a time they found appropriate in respect of the patient\u2019s course of treatment and time management. Having to plan for a third person\u2019s participation would have made the feasibility of the project weaker.Only CT Logbooks in the electronic patient journal were assessed; hence information written elsewhere was not available and not included in the analysis. What is logged from the sessions is partially prearranged from the Logbook forms. Since the Logbook forms are directly based on the manual\u2019s description of the intervention, the data may incorrectly confirm the HCPs\u2019 adherence to the manual. The HCPs were aware that the reports were going to be used in a quality-assurance project and they might therefore have reported the session more in line with the guidelines of the manual. There was, however, sections in the Logbook form with open formulated questions, which gave HCPs the opportunity to share a wide range of information.To facilitate and strengthen the degree to which children are given information and support they are entitled, more research is needed to gain detailed knowledge about factors influencing children\u2019s participation. Future research should identify reasons why HCPs are not including children and investigate whether it is due to lack of consent from parents, institutional constraints, or unfulfilled professional needs. It would be useful to know whether certain characteristics of the parent\u2019s illness, such as a sudden onset or a significant change in the parent\u2019s functional level and behavior, influence the need to give and receive information among HCPs, parents, and children. Whether child participation is influenced by parent gender also needs to be explored in future studies after adjusting for where the child usually lives. In addition, the difference between participating and non-participating children in terms of received information about PMI should be investigated when adjusting for confounding factors, such as the child age.Child Talks is an intervention that seeks to reinforce COPMI\u2019s ability to cope with their family situation by the provision of age-appropriate information about their parent\u2019s illness and treatment. The intervention also aims to provide additional support and follow-up for the children who require it. Of patients who were registered as having minor children, less than one fifth received the intervention, and only half of the patients who participated also had their children participating. Of the registered minor children, less than one in ten received CT. Ideally, children who participate in the intervention emerge better informed, supported and are, when necessary, provided with follow-up. However, this study shows that even participating children were not always followed-up or judged to have been adequately informed. Routines and training of HCPs to support parents with mental illness and their children need improvement. Initial identification of children of patients is important, and subsequent support and provision of adequate services to the identified children always needs to follow.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Data Protection Officer at the University Hospital of Northern Norway (UNN). The regional ethics committee (REK) categorized the project as a quality assurance project. Written informed consent from the participants\u2019 legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.KK made the analysis and the draft of the manuscript. CR was the primary investigator in the main project. CR and CL collected the data in collaboration with the project coordinator, Lisbeth M\u00f8rch, at UNN. All authors contributed to the writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "An estimated 1% of endovascular aneurysm repair (EVAR) devices become infected, carrying a high mortality rate. Surgical explantation is recommended and prognosis is guarded. This retrospective cohort analysis focuses on the role of outpatient parenteral antimicrobial therapy (OPAT) in the management of aortic vascular graft infections following EVAR.Patients who received OPAT for aortic graft infections (AGI) following EVAR from 2014 to 2018 inclusive were identified using the OPAT database. Clinical, microbiological and radiological data were collected. Survivors were followed up for a median of 36\u2009months (range 25\u201360) after first presentation with infection. Outcomes were assessed.Eleven cases with 20 OPAT episodes were identified: 10/11 male, median age 76 (IQR 71\u201381). Median time to presentation was 7\u2009months (range 0\u201381\u2009months) after EVAR. OPAT lead to a 55% reduction in length of hospital stay. One patient had graft explantation; four others had temporising measures. Eight of 11 were alive a median of 36\u2009months after presentation with infection, having had a median of 2 re-treatments on OPAT (range 1\u20133). Seven of the eight survivors were on continuous suppressive oral antimicrobials; three were also intermittently on intravenous antibiotics for flares of infection. Patient/ infection outcomes were cure (1/11), improved (7/11), failure (3/11).AGI following EVAR usually presents in the first year after graft deployment. OPAT has an important peri-operative role in patients suitable for curative surgery. OPAT followed by oral suppressive antimicrobial therapy can be a feasible long-term treatment for non-curative management of AGI. Survival in our cohort was longer than expected, and OPAT was feasible despite the complexity of these infections. OPAT can avoid multiple and lengthy hospital admissions and maximise time at home and quality of life in this cohort with life-limiting infection. Aortic graft infection (AGI) can follow open aortic aneurysmal repair or endovascular aneurysm repair . An estiThis study was performed in a tertiary referral hospital with a vascular surgery department, an infectious diseases (ID) department and an OPAT programme all on the same site. The majority of the OPAT programme in our hospital is run by the ID department. Joint decisions are made by the vascular surgeons and ID physicians regarding suitability of patients with AGI for OPAT. Once accepted onto the OPAT programme, patients are reviewed by face-to-face weekly visits with an ID physician and OPAT clinical nurse specialist in the ID department. There is a direct telephone line for virtual review of any issues falling outside of weekly visits, and extra in-person review can be organised as needed. Though there is no joint ID/ vascular clinic, review by the vascular surgeons can often be organised on the same or following day if necessary. Standard weekly laboratory testing includes full blood count, renal and liver profiles and C-reactive protein. Once patients complete the course of OPAT and are switched to oral suppressive treatment, the frequency of clinical review is decreased. While on the OPAT programme patients can call the OPAT nurse specialist directly. Once discharged from the OPAT programme patients are followed up in the general ID clinic, which can also be contacted directly if issues arise between visits. Once discharged from care, patients can still phone the ID clinic to be seen if any new clinical issue arises.The hospital Outpatient Parenteral Antimicrobial Therapy (OPAT) database was used to identify all patients who received at least one course of OPAT for infected EVAR over a 5-year period from January 2014\u2013December 2018. Only AGIs following EVAR were included (referred to here as AGI for brevity). Patient medical and electronic records were accessed to collect data. Management of Aortic Graft Infection Collaboration (MAGIC) case definitions were applied to stratify infections as early or late, suspected or definite . AGI wasEleven patients, with a total of 20 OPAT episodes, were identified. Eight patients (72%) met MAGIC criteria for definite infection, and 3 (27%) for suspected infection . Ten of Patients presented with infection a median of 7\u2009months range 0\u201381\u2009months) after EVAR. Symptoms at presentation with infection included fever in 6/11(55%), anorexia in 6/11(55%), abdominal pain in 5/11 (45%), back pain in 3/11(27%) and bleeding in 2/11(18%) (Table\u00a0\u2009months aPseudomonas aeruginosa (2/11) and Candida albicans (2/11) and Salmonella species (1/11) (Tables\u00a0Causative organisms were identified in 7/11 (64%) cases, 4 of whom had polymicrobial infections. Three patients 27%) had positive blood cultures, one had positive vertebral biopsy, three a positive culture from sac aspirate and two a positive sinus culture. Five patients had Gram-negative organisms isolated; 3/5 had suspected aorto-enteric fistulae. Organisms isolated included staphylococci and streptococci species(6/11), anaerobes (4/11), enterococci (2/11), 7% had poTwenty OPAT episodes were analysed, with patients having a median of two range 1\u20133) episodes. Most patients (10/11) received more than one intravenous antimicrobial agent at some stage during the course of their treatment, including their inpatient stay. Six patients received dual IV antimicrobial therapy on OPAT. All patients received oral antibiotics for a minimum duration of 6 months after OPAT and some received concomitant oral antibiotics during their OPAT treatment. Antibiotics received on OPAT can be found in Table\u00a0\u20133 episodThree patients received more than two OPAT courses to treat clinical flares of infection. Patient 9 had recurrent abscess formation at the site of axillobifemoral graft; this patient had multiple temporising measures (sac drainage) and three OPAT episodes, before eventually having elective graft explantation. The patient had a stormy post-operative course with a long admission to intensive care but was alive and on oral antimicrobials 25\u2009months after diagnosis of AGI and 15\u2009months after surgical explantation of the graft. Patient 8 successfully completed 12\u2009weeks of OPAT, and later presented with fatigue and rising inflammatory markers while on oral suppression therapy; the patient was switched back to intravenous antibiotics until symptoms and markers improved. This was done on two occasions and on one occasion was achieved without a hospital admission. Patient 4 had increasing abdominal pain and fevers on oral suppressive therapy and was admitted for switch to intravenous (IV) antibiotics and repeat surgical opinion. The decision was made that the patient was still not for surgical intervention and he was discharged within a week via OPAT. At the time of writing, he was on oral suppressive therapy, clinically stable, but with deteriorating radiological appearances.A total of five adverse events occurred in three patients during OPAT; all were antimicrobial-related. One patient presented with a daptomycin-induced eosinophilic pneumonitis for which they required oxygen therapy and antibiotics were changed to vancomycin (Patient 9). He made a full recovery from the pneumonitis. A second patient was admitted with transfusion-requiring anaemia during therapy with linezolid and caspofungin (Patient 8). Three other adverse events occurred which did not require re-admission; piperacillin/tazobactam-induced skin rash, cefuroxime-induced skin rash, and asymptomatic eosinophilia on daptomycin.Four patients had to be re-admitted for infection-related reasons at least once while on OPAT; patient 9 was admitted 3 times ; patient 8 was admitted twice (once for failure of therapy and once for complications of antibiotics). Patient 4 was re-admitted for failure of therapy and a repeat surgical opinion and patient 7 was re-admitted electively for repeat imaging Tables\u00a0 and 2.Eight (72%) patients were alive at a median of 36\u2009months of follow up (range 25\u201364\u2009months) Table\u00a0. OPAT ouThe median number of days on OPAT was 46.5 (range 14\u201391\u2009days). Patients were on IV antibiotics for a total of 1560\u2009days; 696 in hospital and 864 on OPAT. OPAT facilitated a saving of 864 total hospital days, representing a 55% reduction in length of stay.Our patients had similar demographics to those in previously published literature on aortic vascular endograft infections, insofar as they were mostly male, older and with multiple co-morbidities, and these latter factors correlated with mortality , 9, 10. Expert consensus favours surgical removal of an infected graft where possible , 8. One However, this small selective cohort cannot be used to compare surgical versus medical therapy for AGI, but only to describe outcomes in those who survive and are considered candidates for outpatient management. There is an inherent bias in the selection of patients for analysis from an OPAT registry, as those who presented in extremis or died during the first presentation of infection are not included. By definition, the patients in our cohort were well enough for hospital discharge at least once with OPAT, skewing overall survival as compared with cohorts including all patients who present with AGI. In a series including all presenting AGI patients, 17/22 (77%) presented with an urgent surgical indication (rupture or bleeding), as compared with only two of our patients presenting with bleeding and none with rupture [To the best of our knowledge there is no other published data on OPAT outcomes for AGI. As with other complex infections, OPAT can deliver significant savings in inpatient bed-days and healthcare costs . This stOPAT has multiple roles in the setting of AGI, and both patients who undergo surgery and those who do not may benefit. OPAT may be used as a temporizing measure in those awaiting radical surgery, as induction therapy in those AGIs for medical management, and for treatment of clinical \u2018flares\u2019 in patients otherwise well-maintained on oral suppressive therapy. In each of these instances, OPAT can avoid multiple and lengthy hospital admission, reduce costs and maximise time at home and quality of life in this cohort with life-limiting infection. Importantly, we have also demonstrated that long-term outpatient medical management of patients with AGI for whom cure is not intended is feasible. No deaths attributable to vascular graft infection occurred during the follow-up period, but the prognosis and unpredictability of AGI warrants early setting of treatment goals by a multi-disciplinary team comprised of vascular surgeons, infections specialists and, where appropriate, palliative care practitioners. Patients with early AGI and frailty who are not considered candidates for curative surgery should be counselled about their prognosis and consider criteria forre-admission and preparedness for potential vascular catastrophes in the community if appropriate. OPAT followed by oral suppressive therapy can be a suitable long-term non-curative treatment option for certain patients with AGI."} +{"text": "Registration of Laparoscopic Ultrasound (LUS) to a pre-operative scan such as Computed Tomography (CT) using blood vessel information has been proposed as a method to enable image-guidance for laparoscopic liver resection. Currently, there are solutions for this problem that can potentially enable clinical translation by bypassing the need for a manual initialisation and tracking information. However, no reliable framework for the segmentation of vessels in 2D untracked LUS images has been presented.We propose the use of 2D UNet for the segmentation of liver vessels in 2D LUS images. We integrate these results in a previously developed registration method, and show the feasibility of a fully automatic initialisation to the LUS to CT registration problem without a tracking device.We validate our segmentation using LUS data from 6 patients. We test multiple models by placing patient datasets into different combinations of training, testing and hold-out, and obtain mean Dice scores ranging from 0.543 to 0.706. Using these segmentations, we obtain registration accuracies between 6.3 and 16.6 mm in 50% of cases.We demonstrate the first instance of deep learning (DL) for the segmentation of liver vessels in LUS. Our results show the feasibility of UNet in detecting multiple vessel instances in 2D LUS images, and potentially automating a LUS to CT registration pipeline. Liver resection is the standard treatment with curative intent for patients with liver tumours, and is increasingly performed laparoscopically . This apThe LUS to CT registration problem is particularly challenging and poorly constrained as the field of view of LUS is substantially smaller than that of CT. To mitigate this issue, the majority of proposed methods rely on an accurate manual initialisation to the registration along with electromagnetic (EM) tracking information to compose a LUS volume , 7. HoweSeveral authors have approached the vessel segmentation problem in ultrasound (US) across a range of applications. Vessel segmentation of 3D US has been performed by thresholding and filtering of Power Doppler images as well et al. [Other authors have proposed segmentation methods for 2D US, mainly for liver and musculoskeletal US. For musculoskeletal imaging, approaches have mainly focused on using temporal information across multiple images in a Kalman filter framework, while under the assumption that the imaged vessels have an approximately elliptical outline , 13. Howet al. have seget al. , and filWith recent advances in parallel computing, large-scale labelled datasets and advances in deep neural network architectures, deep learning based approaches using convolutional neural networks (CNNs) have been applied to 2D US hepatic vasculature segmentation with growing success. A patch-based CNN method with post-processing using k-means clustering was proposed to detect vessels in abdominal 2D US , but UNeThe first LUS vessel segmentation results on clinical data using DL.An evaluation of performance bounds for UNet for LUS vessel segmentation on clinical data.A novel measure to evaluate image-wise multiple vessel detection.The first results of a fully automatic initialisation to the untracked LUS to CT registration problem.In this paper we report the first instance of 2D LUS liver vessel segmentation using a UNet and integrate it in a registration pipeline. Our contributions include:LUS images with 668 mentclass2pt{minimmentclass2pt{minimA modified 2D UNet with batIn addition to the model, we describe the measurements used to quantify vessel segmentation performance.L and associated prediction P, we measure the binary Dice score D:Dice score is commonly used as a global performance metric for segmentation methods and has P, we define the success L with centroid L, through the following condition:We propose to assess the individual vessel detection rate per image by comparing prediction and ground truth vessels with a criterion based on a closest distance matching and a vessel size ratio. Given a prediction N ground truth vessels, we define the detection rate as the percentage of vessels that are successfully detected in P:Considering that each image has To further validate our segmentation, we integrate our results in our previously developed untracked LUS to CT registration framework , 9. In sSince the vessel matching problem between LUS and CT is ambiguous, we employ a probabilistic optimisation to combine the retrieval results of multiple LUS images in time. Considering that images that are close in time should also be close in rotation and translation, we use a discrete Hidden Markov Model (HMM) to estimate the most likely sequence of database poses to represent the LUS acquisition in CT space. We refer the reader to for furtWe perform three sets of experiments to evaluate the performance of our framework. All of our UNet models were trained on NVIDIA Quadro P5000 GPUs for 1000 epochs, using an Adam optimiser with a learning rate 10In a first experiment, we trained separate models for the segmentation of each of the six patient datasets and assess their performance using the Dice score as in Sect.\u00a0In a third and final experiment, we perform untracked registrations using segmentations from the best mean Dice scoring models. To mitigate the effect of segmentation noise, we remove segmentations that amount to less than 120 pixels. Liver surface and vessel models are extracted from contrast enhanced CT scans using a commercial service.We report two sets of results for our first experiment. Firstly, we present Dice score distributions from each of the 6 outer fold test set as boxplots in Fig.\u00a0p-value under the Bonferroni corrected In order to assess the model variability, we report the combinations of significantly different Dice score performing models for each outer fold in Table\u00a0Vessel detection rates for the best performing Dice score models are reported in Table\u00a0Results of the effect of ground truth vessel size in the detection rate are shown in Fig.\u00a0 see Eq.\u00a0, we measentclass1pt{minimaRegistration accuracy measurements of 5 sweeps of 6 LUS images for 4 patients are presented in Fig.\u00a0Visual results of two registration examples are displayed in Fig.\u00a0Our results show that UNet is suitable for liver vessel segmentation in LUS images. Our Dice score performances, the first reported for LUS segmentation, are comparable to other DL-based US vessel segmentation methods in the literature , 18. EveThe ANOVA reveals that models trained with larger patient datasets achieve higher Dice scores with lower variances. Specifically, models trained without the larger datasets from patients 4, 5, and 6 which hold 134, 704 and 539 images respectively, showing differing performance with statistical significance. An exception was observed for Patient 1 (see Fig.\u00a0The ground truth distribution of vessel sizes could explain the varying performances for larger vessels in Fig.\u00a0Our registration results are in agreement with previous trends in Dice score and detection rate\u2014the best performing segmentations of Patient 3 result in the best registration accuracies with values below 10\u00a0mm, and the worst performing segmentations of Patient 6 do not result in any clinically usable registration. These results also suggest that our detection rate measure is more informative than Dice score for registration performance. In almost all of the clinically usable registrations, detection rate showed high values, whereas Dice shows less distinct values across different registration accuracies. A few exceptions are observed mainly for Patient 5, where high detection rates have resulted in poor registrations. This may be explained by the poor segmentation of larger size vessels see Fig.\u00a0\u2014even if For future work, we propose three directions to improve segmentation performance. Firstly, we intend to include a larger cohort of patient data with a wider distribution of vessel sizes across each patient. Secondly, given the performance variability across different vessel sizes, we aim to refine our loss to specifically penalise mis-segmentations in larger vessels that are likely to be more relevant for registration. Lastly, we aim to incorporate time-series sequential constraints on the 2D segmentation problem. By using a recurrent model such as Mask-RCNN , the segWe present the first DL framework for the segmentation of vessels in untracked 2D LUS liver images, and integrate them in a untracked LUS to CT registration pipeline. Despite the size of our sample, our segmentation shows mean Dice scores ranging from 0.543 to 0.706, results that are comparable with current state-of-the-art approaches. Additionally, we observed that performance improves by considering larger training datasets with a wider distribution of 2D vessel shapes. Using these segmentations, we are able to obtain untracked registrations with accuracies ranging between 6.3-16.6\u00a0mm. Therefore, we also demonstrate for the first time fully automatic pipeline for the coarse registration of LUS to CT, potentially enabling the clinical translation of this image-guidance technique in the future."} +{"text": "Introduction: As an occupational group, medical providers working with victims of trauma are prone to negative consequences of their work, particularly secondary traumatic stress (STS) symptoms. Various factors affect susceptibility to STS, including work-related and organizational determinants, as well as individual differences. The aim of the study was to establish the mediating role of cognitive trauma processing in the relationship between job satisfaction and STS symptoms among medical providers.Procedure and Participants: Results were obtained from 419 healthcare providers working with victims of trauma (218 nurses and 201 paramedics). Three questionnaires, namely the Secondary Traumatic Stress Inventory, Work Satisfaction Scale, and Cognitive Trauma Processing Scale, were used in the study, as well as a survey developed for this research. Correlational and mediation analyses were applied to assess relations between variables.Results: The results showed significant links between STS symptoms and both job satisfaction and cognitive processing of trauma. Three cognitive coping strategies play the intermediary role in the relationship between job satisfaction and symptoms of secondary traumatic stress. However, this role varies depending on preferred strategies.Conclusion: Nurses and paramedics are significantly exposed to the occurrence of STS. Thus, it is important to engage health care providers in activities aimed at preventing and reducing symptoms of STS. The term secondary traumatic stress (STS), or secondary traumatic stress disorder (STSD), was adopted by The literature confirms the high occurrence of STS symptoms among medical providers, especially nurses . For exaOne study found that nearly 13% of American paramedic personnel meet STSD criteria . SymptomAlthough many studies have investigated the link between type of work and STS, relatively few have assessed the connections between job satisfaction and secondary traumatic stress symptoms. Furthermore, among those that have, the majority focused on satisfaction gained from helping, which may be treated as an opposition to CF , rather Some negative associations of secondary traumatic stress symptoms and job satisfaction has been showed among other groups of professionals as well. For example, job satisfaction turned out to play a role of mediator in the relationship between secondary traumatic stress and work engagement among specialists who consult people addicted to substances . The autAnother important factor for the development of STSD is cognitive trauma processing . Since SThe purpose of cognitive trauma processing is to make meaning of the events experienced and to adapt to a new reality altered by the trauma experienced. In order to make meaning, restore or extend the level of previous psychological functioning, the individual makes certain cognitive efforts. Williams, Davis, and Millsap indicated as manifestations of effective trauma processing the reduction of levels of negative emotions , the incorporation of information about the traumatic event and its acceptance, the perception of some positive sides of the trauma, and desensitization, which involves the reduction of negative emotions and stress. This cognitive processing often takes the form of cognitive coping strategies: downward comparison, positive cognitive restructuring, resolution/acceptance, regret, and denial.To date, very few studies have assessed the relationship between cognitive processing in the form of cognitive strategies and STS. One such study was conducted in 5 groups of specialists who worked with victims of trauma; findings confirmed the relationship between cognitive coping strategies and STS symptoms . Regret The current research aim was to examine the links between job satisfaction, cognitive trauma processing, and symptoms of secondary traumatic stress among healthcare providers exposed to secondary trauma, as well as the mediating role of cognitive processing of trauma in the relation betwixt job satisfaction and STS. The indicators of cognitive trauma processing were cognitive coping strategies.The adopted model referred to cognitive theories of trauma, in particular to the model of PTSD developed by M = 39.6, SD = 11.03); 137 (32.7%) males and 282 (67.3%) females. The study group consisted of paramedics (n = 201), of whom 60.2% were men, and nursing personnel (n = 218) of whom 92.7% were women. Most of the paramedics helped people who experienced various types of accidents, mainly car accidents (57.2%) and after injuries such as heart attacks and strokes (42.8%). The nursing team consisted of individuals who worked with oncology patients (87.7%) and car accident victims (18.3%). Work experience ranged from 1 to 43 years , the number of hours per week devoted to assisting injured patients ranged from 2 to 90 , the workload in the form of percentage of work devoted to assisting patients directly in relation to all work, ranged from 2 to 100% . Three standardized questionnaires were used in the study.The study included 430 participants who mostly work with injured individuals. Their participation was anonymous and voluntary. The participants were recruited mainly from polish hospitals: emergency, intensive care, oncology wards, and hospice units between 2019 and 2020. The study obtained approval from the bioethics committee from Opole (no 81/P1/2019). Participants were initially required to provide verbal consent; completion of the study questionnaire evidenced formal written consent. Authors or individual designated by the authors delivered the questionnaires to the participants\u2019 workplace, where they were completed while participants were on duty. Practice as a nurse or paramedic and regular interactions with people experiencing traumatic events, such as an accident or a life or health threatening illness were the inclusion criteria. The analysis excluded 11 subjects due to missing data, leaving 419 individuals aged 19\u201365 years (The Secondary Traumatic Stress Inventory (STSI) is a Polish adaptation of PCL\u20135 developed by The Work Satisfaction Scale (WSS) is a The Cognitive Processing of Trauma Scale (CPOTS) developed by Data analyses were conducted using 25 version of IBM SPSS. Initially we calculated means, standard deviations, and Pearson\u2019s r to check the relationships between the variables. Then we used the PROCESS by M = 24.14, SD = 16.11) obtained from specialists who work with victims of trauma , although slightly higher, did not differ significantly from the STS score obtained by paramedics . The percentage of individuals at high risk for STSD was similar across groups: 43.3% among paramedics and 43.6% among nursing staff. Gender did not influence STS symptom severity . Gender also did not differ the severity of STS in particular groups of professionals.2 There was a positive, although weak, association between participants\u2019 age, and STS symptoms . STS symptoms were negatively correlated with number of hours worked per week and workload , indicating that the more hours worked per week and the greater the workload, the lower the severity of STS symptoms. Seniority as a paramedic/nurse did not significantly correlate with overall STSI score.The STS score obtained by nurses , seniority, or gender. They were, however, positively correlated with age, and negatively, although weakly, correlated with workload and the number of hours worked per week.Analyses confirmed relationships between STS symptoms and both job satisfaction and cognitive processing of trauma, as well as between job satisfaction and cognitive trauma processing. The negative relation between job satisfaction and STS symptoms is consistent with previous studies and confGenerally, cognitive processing of trauma allows individuals who have experienced traumatic event, either directly or indirectly, to revise their assumptions about themselves and the world. It is also associated with the ability to give the experienced event sense and meaning and thus adapt to a new, changed reality. The negative strategies of regret and denial were positively correlated with STS symptoms, while positive strategies were negatively correlated, which is consistent with previous findings from professionals working with trauma victims . HoweverJob satisfaction was positively correlated with all cognitive coping strategies, more strongly with positive coping strategies. This means that the perceived job satisfaction is conducive to medical professionals undertaking remedial activity, particularly the search for a solution to a problem, its acceptance, and positive cognitive restructuring, as well as, although to a lesser extent, the use of regret and denial. An inverse relationship between the variables is also possible, in that competence in cognitive trauma processing, expressed primarily as using of positive coping strategies, promotes job satisfaction.The findings showed that cognitive processing of trauma plays a role of mediator in the relation between job satisfaction and symptoms of STS; however, this role varies depending on the coping strategies used. The positive strategy of resolution/acceptance decreases the strength of the correlation between the variables, while the negative strategies of regret and denial increase, albeit slightly, the strength of the relation, acting as a partial suppressor.Based on these findings we conclude that when medical providers use positive forms of cognitive trauma processing, it decreases the importance of job satisfaction for reducing STS symptoms. In contrast, using negative forms of cognitive trauma processing increases the role of job satisfaction in preventing the negative consequences of indirect trauma exposure.It is worth mentioning that among the cognitive remedial strategies, positive restructuring, and downward comparison did not play a mediating role. The former correlated, albeit weakly, with STS symptoms, while the latter was not correlated with them. Furthermore, the strategy of downward comparison was generally less important in the process of coping with trauma, and it had the weakest psychometric properties as described in the original version of the CPOTS .A mediating role of cognitive remedial strategies is consistent with the literature. For example cognitive processing of trauma played a role of mediator in the correlation between empathy and secondary traumatic stress among therapists, social workers, therapists, and probation officers , as wellThe present findings confirm the importance of cognitive activity related to trauma and that models of PTSD may also apply to STSD. However, it is important to note that cognitive trauma processing after secondary exposure to trauma may depend on a number of factors, such as the type of traumatic events experienced by clients, the level to which the helpers are cognitively engaged in trauma processing, and the coping resources they possess. Moreover, the negative effects of secondary exposure to trauma do not preclude the possibility of positive consequences revealed by vicarious post-traumatic growth (VPTG). Ongoing research in this area confirms the co-occurrence of STS and VPTG . PerceivThere are certain limitations to our research. The nature of the study was cross- sectional, and it does not allow concluding about causation effects. Although the study group was large and heterogeneous, there were more men in the paramedic group, while the nursing staff was mostly represented by women. The study did not examine the significance of a traumatic event experienced a direct way, whether work-related or personal. Subjective indicators of indirect trauma exposure were also not included. Furthermore, the Work Satisfaction Scale measures cognitive aspects of job satisfaction but excludes emotional aspects. Finally, we did not examine other coping strategies, including self-care practices, the role of which is stressed as important . Thus, aThe findings we describe may inspire further research, which could include emotional aspects of job satisfaction expressed as positive and negative effects, as well as other cognitive trauma processing index, such as disruptions in core beliefs or ruminations about traumatic events happening to patients. It also seems important to examine the role of health professionals\u2019 personal resources, such as self-efficacy or spirituality, which may reduce the severity of STS symptoms. Longitudinal studies with the aim of capturing changes in the intensity of secondary traumatic stress symptoms may also prove valuable.The current results can be used for practical purposes in the construction of preventive programs aimed at reducing the symptoms of STS. These programs could potentially increase job satisfaction and expand competency in coping with trauma, for instance by using primarily positive coping strategies, especially resolution/acceptance. Moreover, the importance of self-care should not be overlooked as a factor that supports the ability of helping professionals to effectively assist others, as well as potentially improve the quality of their work and personal lives .Paramedics and nurses are significantly exposed to the occurrence of STS. The results showed significant links between STS symptoms and work satisfaction as well as cognitive processing of trauma. Three cognitive coping strategies play the role of mediators in the relationship between job satisfaction and STS symptoms. The role of individual strategies as mediators in the relationship between job satisfaction and STS varies depending on the strategy used.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Opinion of the Bioethics Committee of the National Medical University of Opole No. 59/PI/2020. The patients/participants provided their written informed consent to participate in this study.PG: study design, data collection, interpretation of data, manuscript preparation, and sourcing of funding. NO-B: study design, statistical analysis, interpretation of data, manuscript preparation, and literature review. PM: manuscript preparation and literature review. EK and AS: data collection. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Safety climate is an essential component of achieving Universal Health Coverage, with several organisational, unit or team-level, and individual health worker factors identified as influencing safety climate. Few studies however, have investigated how these factors contribute to safety climate within health care settings in low- and middle-income countries (LMICs). The current study examines the relationship between key organisational, unit and individual-level factors and safety climate across primary health care centres in Ghana, Malawi and Uganda.A cross-sectional, self-administered survey was conducted across 138 primary health care facilities in nine districts across Uganda, Ghana and Malawi. In total, 760 primary health workers completed the questionnaire. The relationships between individual , unit , organisational-level and safety climate were tested using structural equation modelling (SEM) procedures. Post hoc analyses were also carried out to explore these relationships within each country.\u03b2\u2009=\u20090.56, p\u2009<\u20090.001), supportive supervision , and district managerial support . In Ghana, safety climate was positively associated with job satisfaction , teamwork , and supportive supervision , whereby the model explained 43% of the variance in safety climate. In Uganda, the total variance explained by the model was 64%, with teamwork , supportive supervision , and perceived district managerial support all found to be positively associated with climate. In Malawi, the total variance explained by the model was 63%, with teamwork and supportive supervision significantly and positively associated with safety climate.Our model including all countries explained 55% of the variance in safety climate. In this model, safety climate was most strongly associated with teamwork (Our findings highlight the importance of unit-level factors\u2014and in specific, teamwork and supportive supervision\u2014as particularly important contributors to perceptions of safety climate among primary health workers in LMICs. Implications for practice are discussed. Recent decades have seen progress towards Universal Health Coverage (UHC) across many low- or middle-income countries (LMICs), mostly through an expansion of basic health services and strengthening of primary health care. Changing health needs and growing expectations from residents of LMICs however, suggests that increasing physical and financial access to, and coverage of, health care remains insufficient, with a need to also improve the quality of existing health systems . Consequclimate , in reference to the more readily measurable aspects of safety culture, and as opposed to other aspects of culture such as behaviour and values. In this way, safety climate acts as an emergent property; a social construct, characterising groups of individuals based on their shared perceptions of enacted policies and practices that serve as an indicator of the true priority of safety against other organisational goals \u2009=\u200912.51, p\u2009<\u20090.001. Individual factors explained 46% of the variance for safety climate, with only job satisfaction associated with safety climate. Step two of the model, which included unit-level factors, explained up to 63% of the variance in safety climate . In this step, job satisfaction , teamwork , and supportive supervision emerged as strong predictors of safety climate. The final step, which included organisational factors did not explain any additional variance in safety climate. Overall, at step three the total variance explained by the model was 63%, F \u2009=\u200914.5, p\u2009<\u20090.001). In this model, teamwork and supportive supervision remained significant contributors to safety climate variance. District managerial support was not found to predict safety climate.Each step of the hierarchical regression model was significant managerial support . In Malaperceived safety climate, we cannot reliably ascertain whether higher levels of unit-level factors are associated with more objective accounts of safety climate. Third, other known correlates of climate safety, such as work engagement, safety behaviour, health worker knowledge and motivation [The current study is not without limitations. Firstly, the cross-sectional nature of our study design does not allow for inferences of causality. While teamwork and supportive supervision may contribute towards better safety climate, it is also likely that within health facilities with positive or favourable safety climate, health workers are more likely to work as a team and have supervision mechanisms in place encouraging them to perform their duties to a high standard. More likely however, the relationships between teamwork, supportive supervision, and climate safety are likely multidirectional and mutually reinforcing, rather than unidirectional in nature. Second, and as safety climate was measured using a self-report measure, as an indicator of tivation , availabtivation , were noTogether, our findings resonate with previous studies conducted by Yu and Liang and Vinc"} +{"text": "Ooceraea biroi, a species that is amenable to laboratory studies but for which no pheromones have been identified. During an alarm response, ants quickly become unsettled, leave their nest pile, and are sometimes initially attracted to the source of alarm, but ultimately move away from it. We find that the alarm pheromone is released from the head of the ant and identify the putative alarm pheromone as a blend of two compounds found in the head, 4-methyl-3-heptanone and 4-methyl-3-heptanol. These compounds are sufficient to induce alarm behavior alone and in combination. They elicit similar, though slightly different behavioral features of the alarm response, with 4-methyl-3-heptanone being immediately repulsive and 4-methyl-3-heptanol being initially attractive before causing ants to move away. The behavioral response to these compounds in combination is dose-dependent, with ants becoming unsettled and attracted to the source of alarm pheromone at low concentrations and repulsed at high concentrations. While 4-methyl-3-heptanone and 4-methyl-3-heptanol are known alarm pheromones in other more distantly related ant species, this is the first report of the chemical identity of a pheromone in O. biroi, and the first alarm pheromone identified in the genus Ooceraea. Identification of a pheromone that triggers a robust, consistent, and conserved behavior, like the alarm pheromone, provides an avenue to dissect the behavioral and neuronal mechanisms underpinning chemical communication.Ants communicate via an arsenal of different pheromones produced in a variety of exocrine glands. For example, ants release alarm pheromones in response to danger to alert their nestmates and to trigger behavioral alarm responses. Here we characterize the alarm pheromone and the alarm response of the clonal raider ant\u00a0The online version contains supplementary material available at 10.1007/s10886-023-01407-4. Many animals use alarm signals to alert conspecifics to danger, yet the modality of these signals varies. Some animals use auditory alarm calls, while others use visual or chemosensory signals or a combination of signals from multiple modalities in one of the two nest chambers , 30 additional ants from the same stock colony were kept in a separate Petri dish with a plaster of Paris floor. These ants were used as the stimulus during experiments. Ants were fed every 1\u20132\u00a0days with ers Fig. , we begaOn each day of behavioral experiments, the acrylic lid of the stimulus chamber on the same side as the ants\u2019 nest was removed. Alarm arenas were placed into an enclosed container with controlled light-emitted diode lighting and videos were taken using webcams . Images were taken at a rate of 10 frames per second and 2,592\u2009\u00d7\u20091,944-pixel resolution. Prior to adding a stimulus, baseline behavior was recorded for 5\u00a0min. Behavior was recorded for another 5\u00a0min after exposure to the stimulus.For the live alarmed ant experiments, 4\u00a0min and 30\u00a0s into the recording, an ant from a separate dish was agitated by repeatedly picking her up and putting her down with forceps. This \u201calarmed ant\u201d was then added to the open stimulus chamber at 5\u00a0min into the recording. In control experiments, a folded piece of filter paper was added into the stimulus chamber instead.For the body part experiments, the head of an ant was removed using forceps. After recording baseline activity for 5\u00a0min, the head or headless body of the ant was crushed using forceps and then quickly added to the open stimulus chamber.S. invicta brood. The following day, the remaining food was removed.After each assay, the arena was removed, and the behavioral recording box was left open for 1\u20135\u00a0min prior to adding the next arena. The total number of ants in the arena was manually counted. Behavioral assays were performed every other day to allow ants to re-settle into a nest after being alarmed. After each behavioral assay, ants were fed with frozen O. biroi heads, mesosomas, gasters, or full bodies in a glass-wool-packed thermodesorption tube and added it in the thermodesorber unit . The thermodesorption tube was heated up to 260\u00a0\u00b0C for 10\u00a0min. The desorbed components were transferred to the cold trap (5\u00a0\u00b0C) to focus the analytes using N2 flow in splitless mode. The cold trap was rapidly heated up to 310\u00a0\u00b0C at a rate of 60\u00a0\u00b0C per minute, held for 5\u00a0min, and connected to the gas-chromatography/mass-spectrometry unit via a heated transfer line (300\u00a0\u00b0C). The GC was equipped with a HP-5MS UI capillary column . Helium was the carrier gas using 1.2874\u00a0ml/min flow. The initial GC oven temperature was 40\u00a0\u00b0C for 1\u00a0min, then raised to 300\u00a0\u00b0C at 5\u00a0\u00b0C per min, where it was held for 3\u00a0min. The transfer line temperature between GC and MS was 300\u00a0\u00b0C. The mass spectrometer was operated in electron impact (EI) ionization mode, scanning m/z from 40 to 650, at 2.4 scans per second. Chemical compounds were first identified using the NIST library and later confirmed with co-elution of synthetic 4-methyl-3-heptanone and 4-methyl-3-heptanol (Sigma Aldrich M48309). Compounds eluting after 30\u00a0min were excluded from the analysis due to lack of volatility.To identify candidate alarm pheromone components from the head, we placed 5 dissected O. biroi heads was purchased from Pfaltz and Bauer (Item #: M19160) and\u2009\u2265\u200999% 4-methyl-3-heptanol (mixture of stereoisomers) was purchased from Sigma-Aldrich (Item #: M48309). Compounds were freshly diluted on each day of behavioral experiments. Dilutions were made using 100% pentane purchased from Sigma-Aldrich (Item #: 236705) as the solvent and diluted compounds were kept in glass vials with a silicone/PFTE magnetic screw cap (Gerstel 093640\u2013079-00) to prevent evaporation.O. biroi was excised and inserted into a glass capillary filled with Ringer solution and attached to the reference silver electrode. The tip of the antenna was inserted into the recording glass electrode, which was also filled with Ringer solution. The antennal signal was 10 times amplified, converted to a digital signal by a high input impedance DC amplifier interface and recorded with GC-EAD software . Synthetic pheromone candidates (4-methyl-3-heptanone and 4-methyl-3-heptanol) were applied at 1, 10 and 100\u00a0\u03bcg doses on a square filter paper (1\u2009\u00d7\u20091\u00a0cm), which was inserted into a Pasteur-pipette. Stimulus air (2 L/min) was led into a constant, humidified and charcoal filtered air stream (2 L/min) using a Stimulus Controller CS-55 (Syntech). Each stimulation was given for 0.5\u00a0s. N-pentane, the solvent of the components, was used as a control stimulus. Three different doses of the pheromone candidates and the control were randomized and tested on 9 antennae.For the electroantennographic (EAG) recordings, the head of Statistical analyses were performed in GraphPad Prism 9.0. To account for differences between individual antennae, we analyzed data using mixed-effects analysis with a Geisser-Greenhouse correction which treats each antenna as a random factor in the model. This method uses a compound symmetry covariance matrix and is fit using Restricted Maximum Likelihood (REML), and results can be interpreted like repeated measures ANOVA in cases with missing values. We then compared each compound/amount to the pentane solvent, using Dunnett\u2019s multiple comparisons test.2) using a syringe. The pentane was allowed to evaporate for 30\u00a0s and then the paper was folded and placed into the open stimulus chamber 5\u00a0min into the recording.The alarm behavioral assay was performed as described above. Four minutes and thirty seconds after starting the experiment, 50\u00a0\u00b5l of the compound diluted in pentane or pentane alone (vehicle control) were added onto a small square of filter paper number of ants outside the nest pile, (2) number of ants outside the nest chamber, and (3) number of ants touching the mesh wall of the stimulus chamber. A single frame of the recording was scored according to these metrics every 30\u00a0s for the duration of the 10-min recording. Ants were scored as being outside the nest pile if they were not touching any other ant within the region of the nest pile Fig. . Ants weStatistical analyses were performed in GraphPad Prism 9.0. We limited the formal statistical analyses to the time window starting 1\u00a0min before the addition of the stimulus and ending 2\u00a0min after the stimulus had been added, because this was when relevant behavioral changes occurred. These analyses are fully consistent with behavioral dynamics across the entire time course Fig. &3. To evO. biroi by quantifying features of the behavioral response of colonies to a live alarmed ant and 4-methyl-3-heptanol (16.3% of the head contents) Fig.\u00a0, Table 1ts) Fig.\u00a0, Fig. S4O. biroi alarm pheromone, then ants should be able to detect both compounds with their antennae. To test this, we utilized EAG recordings and found that both compounds were detected Supplementary file2 (MP4 14363 KB) Video S1. Representative videos of the behavioral response to control (top) and a live alarmed nestmate (bottom)\". The initial nest pile is in the left nest chamber, and baseline activity was recorded for 5 minutes. 5 minutes into the recording, the stimulus is added to the stimulus chamber on the right side. The video is sped up 8x, and addition of the stimulus is indicated by a red circle in the top right cornerSupplementary file3 (MP4 6079 KB) Video S2. Representative videos of the behavioral response to a crushed body (top) and a crushed head (bottom). The initial nest pile is in the left nest chamber, and baseline activity was recorded for 5 minutes. 5 minutes into the recording, the stimulus (crushed head or crushed body) is added to the stimulus chamber on the right side. The video is sped up 8x, and addition of the stimulus is indicated by a red circle in the top right corner.Supplementary file4 (MP4 19691 KB) Video S3. Representative videos of the behavioral response to the vehicle control (top) and two amounts of 4-methyl-3-heptanone, 260 \u03bcg (middle) and 2600 \u03bcg (bottom). The initial nest pile is in the left nest chamber, and baseline activity was recorded for 5 minutes. 5 minutes into the recording, the stimulus is added to the stimulus chamber on the right side. The video is sped up 8x, and addition of the stimulus is indicated by a red circle in the top right cornerSupplementary file5 (MP4 19990 KB) Video S4. Representative videos of the behavioral response to the vehicle control (top) and two amounts of 4-methyl-3-heptanol, 260 \u03bcg (middle) and 2600 \u03bcg (bottom). The initial nest pile is in the left nest chamber, and baseline activity was recorded for 5 minutes. 5 minutes into the recording, the stimulus is added to the stimulus chamber on the right side. The video is sped up 8x, and addition of the stimulus is indicated by a red circle in the top right cornerSupplementary file6 (MP4 16929 KB) Video S5. Representative videos of the behavioral response to the vehicle control (top) and two amounts of a blend of 90% 4-methyl-3-heptanone and 10% 4-methyl-3-heptanol, 260 \u03bcg (middle) and 2600 \u03bcg (bottom). The initial nest pile is in the left nest chamber, and baseline activity was recorded for 5 minutes. 5 minutes into the recording, the stimulus is added to the stimulus chamber on the right side. The video is sped up 8x, and addition of the stimulus is indicated by a red circle in the top right cornerSupplementary file7 (PDF 173 KB) Figure S1. Alarm arena design. The alarm arena had two areas separated by a tunnel. Each area consists of a small rectangular stimulus chamber and a large square nest chamber, separated by a mesh wall (denoted by a purple dashed line in the figure). These chambers have separate clear plastic acrylic lids, allowing access to the stimulus chamber without disturbing ants in the nest chamber. The brown circle represents the nest pile, where ants and their eggs are tightly clustered prior to starting the experiment. Created with BioRender.comO.biroi. Quantification of features of the behavioral response of O.biroi colonies to a live alarmed ant (a-c) and crushed body parts of an ant (d-f). Each datapoint indicates the mean and error bars indicate the 95% confidence interval. Sample sizes represent replicate colonies testedSupplementary file8 (PDF 729 KB) Figure S2. Full time course of characterization of alarm behavior and localization of alarm pheromone in O.biroi colonies to 4-methyl-3-heptanone (a-c), 4-methyl-3-heptanol (d-f), and a blend of 90% 4-methyl-3-heptanone and 10% 4-methyl-3-heptanol (g-i). Each datapoint indicates the mean and error bars indicate the 95% confidence interval. Sample sizes represent replicate colonies testedSupplementary file9 (PDF 1268 KB) Figure S3. Full time course of behavioral response to candidate alarm pheromone components. Quantification of features of the behavioral response of Supplementary file10 (PDF 827 KB) Figure S4. Chemical compounds in the ant body. Gas-chromatographic representation of one sample of 5 pooled workers (a), 5 mesosomas (b) and 5 gasters (c). Compounds found in the head are numbered and can be found in Table 2p<0.05, **p<0.01, ***p<0.001, ****p<0.0001Supplementary file11 (PDF 393 KB) Figure S5. Antennal detection of candidate alarm pheromone components. Results from EAG recordings in response to 1 \u03bcg, 10 \u03bcg, and 100 \u03bcg of 4-methyl-3-heptanone or 4-methyl-3-heptanol and the solvent control pentane. In total, 9 antennae were tested, except for the 1 \u03bcg 4-methyl-3-heptanone condition where 8 antennae were tested. Statistical comparisons were made using a mixed-effects analysis with a Geisser-Greenhouse correction and Dunnett\u2019s multiple comparisons test to compare the response to each compound with the solvent control. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001Supplementary file12 (PDF 192 KB) Figure S6. Comparison of behavioral responses to candidate alarm pheromone components and the synthetic alarm pheromone blend. Area under the curve the first 2 minutes after adding the stimulus for ants outside the nest pile (a), ants repelled from the compound(s) (b), and ants attracted to the compound(s) (c). The two compounds and blend were tested in a separate set of experiments and a vehicle control (in grey) was run for each set of experiments. Each datapoint indicates the mean, and error bars represent the 95% confidence intervals. Statistical comparisons were performed using a 2-way ANOVA with Tukey\u2019s multiple comparisons tests to compare the different compounds and blend across concentrations. *Below is the link to the electronic supplementary material."} +{"text": "Barleria prionitis is known for its medicinal properties from ancient times. Bioactive iridoid glycosides and phenolic compounds have been isolated from leaves of this plant. However, other parts of a medicinal plants are also important, especially roots. Therefore, it is important to screen all organs for complete chemical characterization.B. prionitis, including leaf, root, stem and inflorescence in search of bioactive compounds, with a rapid and effective metabolomic method. X500R QTOF system with information dependent acquisition (IDA) method was used to collect high resolution accurate mass data (HRMS) on both the parent and their fragment ions . ESI spectra was obtained in positive ion mode from all parts of the plant. A comparative analysis of antioxidant and antibacterial activity was done and their correlation study with the identified compounds was demonstrated. Principal component analysis was performed.All parts of B. prionitis. Antioxidant and antibacterial activity was revealed in all organs, root being the most effective one. Some of the iridoid glycoside and phenolic compounds found to be positively correlated with the tested biological activity. Principal component analysis of the chemical profiles showed variability in distribution of the compounds.Iridoid glycosides and phenolic compounds were identified from all parts of the showing variability in presence and abundance. Many of the compounds are reported first time in B. prionitis are rich source of bioactive iridoid glycosides and phenolic compounds.All parts of Barleria, a member of Acanthaceae family, is a spiny shrub and known for its medicinal properties from ancient times is considered to be the most suitable technique for chemical characterization of natural extracts. Although the necessity of the technique in natural product science is unquestionable, however, it is time consuming and cannot meet the demand arising due to increase in number of samples. Therefore, it is required to find a more efficient technique which can perform high-throughput analysis by overcoming the time constrains of the conventional method. Application of direct mass measurement through techniques, such as electrospray ionisation (ESI-MS) have proven useful in characterizing crude extracts, such as medicinal plants essentiaB. prionitis and B. cristata were collected from the Ayurvedic Garden of Banaras Hindu University, Varanasi, India, in the month of December, 2021. The plant material was washed thoroughly under running tap water and dried in air. Different parts of the plant, such as root, stem, leaves and inflorescence were separated and kept in oven with temperature not exceeding above 50\u00b0C. Dried plant material was grinded into fine powder. About 50 g of plant powder was extracted by absolute ethanol with sonication at 45\u00b0C for 15 min with frequency 40 kHz, power 100W, the procedure was repeated thrice. The decoction was filtered using the Whatman paper, the plant material was again extracted thrice with 70% ethanol; both the filtrates were pooled together and dried at 45\u00b0C. The obtained residue was extracted with hexane, to remove non-polar compounds, the remaining residue was used for further analysis.Total free radical scavenging capacity of the extract was estimated using the stable DPPH radical, as per the method described by Where control was 3 ml methanol with 1 ml DPPH and sample was methanolic plant extract.50 value was determined from the plotted graph of scavenging activity against the different concentrations of extracts, which is defined as the total antioxidant necessary to decrease the initial DPPH radical concentration by 50%.Antioxidant activity of the extracts was compared with standard antioxidant (Ascorbic acid). IC0C. Now a 0.5 ml of methanolic extract was taken in a test tube and diluted by 0.5 ml methanol, followed by addition of 2 ml frap reagent, the mixture was kept for 15 minutes in 37\u00b0C. Absorbance of this reaction mixture was measured at 593nm using UV/VIS spectrophotometer. Antioxidant activity was calculated by comparing with standard antioxidant (ascorbic acid).Frap assay was performed according to the method of Escherichia coli, Staphylococcus aureus sub sp. aureus, Bacillus subtilis, Pseudomonas aeruginosa and Klebsiella pneumonia. Plant extract of 10mg/ml concentration was prepared in distilled water. Nutrient agar plates were prepared and 0.5 cm wells were made on the solid media. Plates were inoculated by bacterial culture and wells were filled with 20 \u00b5l of the extract. The plates were incubated for 24h at 37\u00b0C. Antibacterial activity of the compound was determined by measuring the diameter of zone of inhibition of microbial growth. Antibiotic, Streptomycin at concentration of 1 mg/ml was used as positive control.Antibacterial activity was performed by well diffusion method, according to the method decribed in literature , on the B.prionitis and B.cristata, prepared in methanol, were directly analyzed by mass spectrometer. The SCIEX X500R QTOF system with the Turbo V\u2122 source was operated in positive electrospray ionization (ESI) mode. The TOF MS scan was conducted over a range of 100-1000 m/z. Following MS parameters were selected; ion source gas one 60 psi, ion source gas two 60 psi, curtain gas 40 psi, source temperature 400-500\u00b0C, ion spray voltage 5500 V, accumulation time 0.25 s, declustering potential 60-100 V and collision energy 7 V. An automated information dependent acquisition (IDA) approach was chosen for data collection. The TOF-MS data were acquired with high resolution mass measurement (HRMS) and isotopic resolution. Ion intensity signal less than 1000 cps (count per second) was not considered for evaluation. The resulting ion intensity matrices were normalized to be expressed as a percentage relative to the most intense ion in the spectrum (taken as 100%). Using the X500R\u2019s high-resolution mass spectrum TOF-MSIDA, with a single sample injection, TOFMS and TOF-MS/MS mass spectra were obtained. Using primary high-resolution mass numbers and the MS/MS database for comparison , published database searches and structural verification were performed for identification of the compounds.Plant extract (10 mg/ml) of root, leaf, stem and inflorescence of 50 values. Pearson\u2019s correlation coefficient was calculated and statistical significance was determined with one way ANOVA test. A statistical significance of p\u2009<\u20090.05 was considered to be significant. The principal component analysis (PCA) was used to show the variation of multivariate data set in terms of components. This study includes ion intensity signals of components present in root, stem, leaf, and flower. The visual presentation PCA has been done through R software. In R software biplot function and library(ggfortify) has been used to plot PCA. Heatmaps were created using the statistical software Python. Ion intensity data peak were normalized and an average value of five replicate was used for construction of heatmap.All the assays were repeated five times and results were shown as mean\u2009\u00b1\u2009standard deviation. Linear regression analysis was used to calculate the ICBarleria species. A total of 102 ions were detected from the mass spectra of leaf, root, stem and inflorescence of the plants. We were able to identify 58 ions, belonging to phenolic and iridoid group. A clear difference was observed in the chemical profile of both the plants as well as within different parts of the plant, showing variation in distribution and abundance of the compounds group at the C-4 position, generating product ions at m/z 233 and 215.Shanzhiside methyl ester was observed as sodium adduct [M+Na]f 10:3:1 Figure\u00a02+ at m/z 471, potassium adduct at m/z 487, sodium dimer [2M+K]+ at m/z 919 and potassium dimer [2M+K]+ at m/z 935, with a normal isotopic distribution pattern. MS/MS analysis of barlerin at m/z 471 [M+Na]+ gave fragment ion at m/z 411 formed by cleavage of methylacetate (COOCH3) group from the parent ion and at m/z 249 formed by removal of glucose group. Loss of water molecule gives a peak at m/z 231, whereas loss of methanol (32 Da) give rise to a peak at m/z 217. In barlerin also, a hydroxyl group is linked at C-6 position, so it losses a methanol molecule to form a lactone with the COOCH3 group at the C-4 position. Loss of CO group (28 Da) from 231 ion give rise to peak at m/z 203. Replacement of sodium ion with hydrogen from 231 followed by loss of water gives peak at 191. Loss of water from 217 and replacement of sodium ion with hydrogen, yields a peak at m/z 177 Da. Further loss of water followed by removal of CO, yields a peak at m/z 159 and 131 respectively group from the parent ion. MS2 signal for Gossypetin 3-methylether was also observed due to its higher abundance (heat map). Fragment with m/z 340 was generated by loss of water molecule and 353 by loss of CH3OH group from the parent ion. A flavonoid glycoside, quercetin rutinoside was identified by its diagnostic fragment ion 285 and 325 formed by breakage of sugar group from the parent ion.Flavonoids detected in bundance Table\u00a01.B. cristata (Heat map). It was observed as sodium adduct [M+Na]+ at m/z 647 and potassium adduct [M+K]+ at m/z 663. Fragment ions of acetoside at m/z 663 [M+K]+, generated characteristic ion of caffeoyl group (m/z 163). Ion at m/z 517 was produced by loss of caffeoyl group and addition of water molecule to the parent ion [M-caffeoyl+H2O+K]+. Ion at m/z 495 was generated by loss of rahmnose group from the parent ion with replacement of potassium ion with water molecule as adduct [M-Rha+H2O-K]+. Cleavage of hydroxyltyrosol group was observed, as indicated by appearance of ion at m/z 177 [hydroxyltyrosol+K]+. Dissacharide sugar group was cleaved from the parent molecule to generate fragment at m/z 365 [sugar+K]+ and addition of water generated fragment at m/z 383 from the parent ion (m/z 793). Acetylpoliumoside was detected by presence of its ions at m/z 893 [M+Na]+ and 914 [M+K]+ formation of poliumoside ion by loss of acetylgroup , indicating them to be a phenolic compound forming sodium and potassium adduct. Similarly, ions with m/z 604 and 630 observed in B.prionitis were also observed as signal of a similar compound indicating loss of water molecule (18Da) and glycosyl group; showing their probability to be a phenolic glycoside . The results showed that PC1 explains 48.26 percent, PC2 explains 29.29 percent, PC3 13.21 percent and PC4 explains 4.42 percent variation of dataset Table\u00a03.B.prionitis and B.cristata. X500R QTOF system uses information dependent acquisition (IDA) method to collect high resolution accurate mass data (HRMS) on both the parent and their fragment ions. IDA performs a non- targeted screen of MS1 fingerprint and separates MS1 signals into small ion currents, further selecting the most abundant ion current from the TOF MS total ion chromatogram to perform MS/MS fragmentation (A rapid metabolomic approach was used in the present research work for chemical profiling of active compounds in entation . The whoentation . Most ofentation . Many ofBarleria species (B. prionitis had high relative abundance of iridoid glycosides, particularly shanzhiside methy ester, barlerin and acetylbarlerin, whereas B. cristata showed higher relative abundance of acetoside. These metabolites are active compounds with broad spectrum of biological activities. Extracts enriched with iridoid glycosides have shown glutathione S-transferase inhibitory activity, acetylcholinesterase inhibitory activity, free radical scavenging, antimicrobial, anti-inflammatory, immunomodulatory and gastroprotective activities (in vivo studies (O-acetylshanzhiside methyl ester and its cis isomer have shown activity against respiratory syncytial virus (Barleria sps. have shown to possess glutathaione S transferase and acetylcholinesterase inhibitory activity (Barleria species is known for its antioxidant, analgesic, anticancer, anti-inflammatory and photoprotective activity (Iridoid glycosides and phenolic compounds have been reported from aerial parts of species , however species Figure\u00a02 species , showed tivities . Shanzhi studies . Shanzhi studies . Iridoidal virus . Similaral virus shown inactivity . Verbascactivity . IdentifBarleria sps. (B.cristata to be more effective against tested bacterial species. As we can see that most of the phenolic acid and flavonoids showed a positive correlation with the antioxidant activity. Phenolic compounds are widely distributed plant substances and have been considered as significant contributors to antioxidant activity (Staphylococcus aureus (Some studies have demonstrated antioxidant and antibacterial activity of ria sps. , howeveractivity . Some ofactivity . Acetosis aureus . None ofB. prionitis and B. cristata led to the identification of iridoid glycosides and phenolic compounds in different parts of the plant. Most abundant iridoid glycosides were barlerin, acetylbarlerin and shanzhiside methylester, whereas acetoside and acetylacetoside were the important phenolic compound present in the plants. Many of the chemical compounds are reported for the first time in Barleria species, such as ferulic acid, caffeic acid, kaempferol, acetlyacetoside. Most of the compounds detected in root, stem and inflorescence have not been reported earlier. Chemical compounds were found to be distributed in all parts of the plant with variation in their presence and abundance. They were shown to be correlated with antioxidant and antibacterial activity.Phytochemical investigation of The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication."} +{"text": "Follicular regulatory CD4+ T cells (TFR) limit TFH numbers and function in vitro and in vivo. We evaluated the hypothesis that TFR suppress HIV replication in TFH using a well-established model of ex vivo HIV infection that employs tonsil cells from HIV uninfected individuals spinoculated with CXCR4- and CCR5-tropic HIV-GFP reporter viruses. Both CXCR4 and CCR5-tropic HIV replication were reduced in TFH cultured with TFR as compared to controls. Blocking antibodies to CD39, CTLA-4, IL-10, and TGF-beta failed to reverse suppression of HIV replication by TFR, and there were no sex differences in TFR suppressive activity. TFR reduced viability of TFH and even more so reduced HIV infected TFH as assessed by total and integrated HIV DNA. Exogenous IL-2 enhanced TFH viability and particularly numbers of GFP+ TFH in a concentration dependent manner. TFR reduced productively infected TFH at low and moderate IL-2 concentrations, and this was associated with decreases in extracellular IL-2. Both IL-2 expressing cells and larger numbers of FoxP3+CD4+ cells were detected in follicles and germinal centers of lymph nodes of people living with HIV. TFR may deplete TFH in vivo through restriction of IL-2 and thereby contribute to decay of HIV expressing cells in B cell follicles during HIV infection.Follicular helper CD4 They faollicles , 12.+ T cells found in B cell follicles and germinal centers that directly impair TFH function and proliferation and limit germinal center responses are a population of CD4esponses \u201322. TFR T cells , althoug and were transfected with X4-tropic pNLENG1-IRES (293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS and Primocin (NG1-IRES or R5 trNG1-IRES virus pl+ T cells were enriched from disaggregated tonsil tissue using negative selection (STEMCELL Technologies). CD4+ T cells were labeled with CD3-AF700 , CD8-BV510 , CXCR5-PE , CD25-PECy7 , and 7-AAD (Tonbo) and sorted using a FACSAriaIII cell sorter equipped with an 85 \u03bcm nozzle into TFR (CD3+CD8-CXCR5+CD25+7-AAD-) and TFH (CD3+CD8-CXCR5+CD25-7-AAD-) populations. A minimum of 4 x 106 TFH were spinoculated with X4-tropic or R5-tropic GFP reporter virus and labeled with violet proliferation dye 450 (VPD) (Becton Dickinson). Spinoculated VPD+TFH were cultured 1:1 with uninfected, unlabeled TFH or TFR at a final concentration of 2x106 cells/ml for five days in Advanced RPMI (Gibco) supplemented with 10% heat inactivated FBS, 1X nonessential amino acids (Gibco), 1X Glutamax (Gibco), 1X Primocin (In vivogen), (Advanced R-10) in the presence of 5 \u03bcM saquinavir (Sigma). IL-2 (NIH AIDS reagent program) (Tonsil CD4program) , IL-6 , 1 \u03bcM estrogen receptor antagonist ICI 182,780 (Sigma), or 300 pg/ml \u03b2-estradiol (Sigma) where indicated.+TFH (VPD+7-AAD-) were sorted after 5-day coculture with unlabeled, uninfected TFH or TFR. DNA was isolated using a Puregene kit (Qiagen). Cellular DNA was quantified by QPCR for cell equivalents of DNA per volume as described previously were added and used for cell count determination. For phenotyping, the following antibodies were used: CD3-AF700 , CD8-BV510 , PD-1-APC or BV785 , CXCR5-PE , CD25-PECy7 , CTLA-4-PerCP-eFluor710 , GITR-PE-eFluor610 , LAG-3-Superbright 702 (Clone 3DS223H), and CD39-BV711 . Cells were analyzed on a Fortessa flow cytometer, and data analyzed using FloJo v10.Cells were washed in PBS and resuspended in PBS and Ghost Dye Red 780 (Tonbo). After 2 minutes, antibodies in PBS, and where appropriate, Brilliant Violet Stain Buffer (BD) were added and incubated for 30 minutes in the dark at room temperature. Cells were washed in PBS and resuspended in 2% paraformaldehyde in PBS. A minimum of 2.55 x 10For intracellular staining, in place of 2% PFA, cells were fixed and permeabilized with FoxP3/Transcription factor staining buffer kit (Tonbo) according to manufacturer\u2019s instructions. Labeling of intracellular FoxP3 and CTLA-4 was performed using the following antibodies: Foxp3-APC and CTLA-4-PerCP-eFluor710 for 30 minutes. Cells were washed, suspended in PBS and analyzed on a Fortessa flow cytometer and data analyzed using FloJo v10.Cryopreserved tonsil cells were thawed and cultured in Advanced R-10 overnight. 2ml PBS was added and samples were centrifuged at 300xg for 10 minutes. Cell pellets were resuspended in Advanced R-10 supplemented with 50 ng/ml PMA (Sigma), 1 \u03bcg/ml ionomycin (Sigma), and monensin (eBioscience) and cultured 5 hours. Samples were washed and stained as indicated for extracellular proteins, then fixed and permeabilized with Cytofix/Cytoperm (BD) according to manufacturer\u2019s instructions. Samples were stained for 30 minutes with either IL-10-AF647 or TGF-\u03b2 , and analyzed on a Fortessa flow cytometer. Gates for IL-10 and TGF- \u03b2 were determined using unstimulated, monensin treated samples as controls.On day 5 of infection, cell cultures were centrifuged at 800\u00a0g for 10 minutes and supernatant was collected and stored at -80\u00b0C. IL-2 ELISA (Sigma) was performed according to the manufacturer\u2019s instructions using diluted supernatant. Samples were analyzed on a plate reader at 450 nm and IL-2 concentrations were calculated using IL-2 standards provided.in situ hybridization (ACDbio). Following in situ hybridization, sections were incubated overnight with antibodies to FoxP3 and either CD20 or Ki67 on adjacent sections. Appropriate fluorescent secondary antibodies (Invitrogen) were added and all sections were counterstained with DAPI. Whole sections were imaged on an Aperio slide scanning system (Leica) at 40X.Six micron sections of frozen inguinal lymph nodes were thaw mounted onto glass slides and fixed in 4% paraformaldehyde for 30\u00a0min. After blocking with hydrogen peroxide, sections were stained for IL-2 and CD4 RNA using multiplexed fluorescent Using Aperio Imagescope , total, follicular (based on CD20 staining) and germinal center (based on Ki67 staining) areas were defined. IL-2 RNA positive cells were counted and categorized as extrafollicular, follicular, or germinal center IL-2 positive cells and their frequency determined by quantitative image analysis. Percentages of IL-2 positive cells that co-expressed CD4 were also determined. In follicles or germinal centers where there were IL-2+ cells, FoxP3+CD4+ cells were counted and the ratio of IL-2+ cells to FoxP3+CD4+ cells was determined. A final ratio was obtained by averaging data from each IL-2 containing follicle or germinal center for each lymph node.+CD8-CXCR5+CD25lo/-) and TFR (CD3+CD8-CXCR5+CD25hi) is shown in +) while very few TFH expressed FoxP3 compared to TFH , 30. FurAdditional experiments to evaluate the impact of TFR on CCR5-tropic HIV GFP reporter virus expression revealed a similar magnitude of TFR-mediated suppression of HIV replication as seen in CXCR4-tropic HIV GFP reporter virus infection , TFR reduced percentages of GFP+ cells similarly for both subsets (p=0.226). The TFH (CD3+CD8-CXCR5+CD25lo/-) population was thus used for all subsequent experiments.The defined TFH population consisted of both CXCR5+ compared to TFH . As shown previously in +VPD+TFH ; however, there was no statistically significant difference in frequencies of IL-2 positive cells between follicles and germinal centers, and follicles and extrafollicular areas inhibit the differentiation of CD4+ T cells into both TFR and TFH through induction of STAT5 , 53, whe+ T cells with Tregs resulted in decreased exogenous IL-2 concentrations and increased apoptosis of CD4+ T cells. Intriguingly, depletion of Tregs using anti-CD25 antibodies in rhesus macaques with natural virologic control resulted in increased plasma viremia despite augmentation of SIV-specific CD8+ T cell responses and supported by the NCI award (P30 CA023074) to the University of Arizona Cancer Center Flow Cytometry Shared Resource. Support for Andriana La Mantia was provided by T35HL007479 (Witte). Support for Matthew Ollerton was provided by the Moya-Teller Fund. Funding sources had no role in the experimental design, data collection, interpretation of data, or decision to submit the manuscript for publication.GFP reporter virus plasmid was contributed by Dr. David Levy. Dr. Una O'Doherty provided invaluable guidance regarding DNA quantification assays. Human rIL-2 was provided by Dr. Maurice Gately, Hoffmann - La Roche Inc. through the HIN AIDS Reagent Program, Division of AIDS, NIAID, NIH.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Asiatic acid (AA) is a pentacyclic triterpene isolated from Centella asiatica, holding great promise for treating a variety of skin disorders. However, the dermal application of AA is limited by its poor solubility and permeability. This study aimed to identify a hydrogel formulation for AA and improve its skin penetration by various penetration enhancement methods. Four kinds of hydrogel bases were selected to prepare the AA hydrogel, in which different organic amines and chemical enhancers were incorporated in combination with microneedle pretreatment. The results showed that AA had good release profiles in the presence of hyaluronic acid as the hydrogel base and organic amines as the counter-ions. Diethylamine and Span 80 could promote drug penetration into the skin, and pretreatment with microneedles could further increase the drug permeability. In conclusion, the optimized hyaluronic acid hydrogel has great potential for use in the topical delivery of AA, and its penetration via the skin can be further improved by different pharmaceutical approaches. Asiatic acid , chitosan (CS), poloxamer 407 (P407), and carbomer 940 (C940), were selected to construct the hydrogel bases, and their release profiles toward AA were compared to identify the optimal base for AA. Based on that, different enhancement strategies, including chemical enhancers, counter-ions of organic amines, as well as microneedle pretreatment, were investigated to improve the skin permeability of AA . The stuAA, diethylamine (DEtA), triethylamine (TEtA), and ethanolamine (ETA), N-Methylpyrrolidone (NMP) were all purchased from Aladdin Reagent Co., Ltd. . HA, CS, C940, P407, oleic acid (OA), polyethylene glycol 400 (PEG 400), glycerol (GL), 1,2-propylene glycol (PG), Azone, Span 80, isopropyl myristate (IPM), L-menthol, and menthyl Lactate (ML), were bought from Shanghai Macklin Biochemical Technology Co., Ltd. . Methanol and acetonitrile were chromatographically pure and supplied by Shandong Yuwang Chemical Reagent Co., Ltd. . All other reagents used were of analytical or chromatographic grade.To ensure the sufficient dissolution of AA in hydrogels and maintain the sink condition during the penetration experiments, we determined the solubility of AA in different solvents which might be used for dissolving AA in the preparation of hydrogels or as the receptor liquid in permeation experiments. In different solvents, an excessive amount of AA was added. The mixture was shaken vigorously and then placed in a constant temperature oscillator to equilibrate at 32 \u00b1 1 \u00b0C for 72 h. After that, the suspension was filtered through a 0.22 \u03bcm filter membrane, and the filtrate was obtained after the discarding of the initial filtrate. The drug content was analyzed by HPLC after appropriate dilution.w/w). Then, the drug solution was added to different hydrogel bases and stirred evenly to obtain the AA-loaded hydrogels with a drug content of 2.0%. For different types of hydrogel bases, they were respectively prepared as follows. HA: 0.0540 g HA was added in 1.35 mL deionized water and then placed at 4 \u00b0C overnight for HA swelling to form the hydrogel base. CS: 0.0900 g CS was added and wet in 1.2 mL deionized water, in which 20 \u03bcL of acetic acid was subsequently added to form the hydrogel base by stirring. P407: 0.4500 g P407 was added in 1.0 mL deionized water and then placed at 4 \u00b0C overnight for swelling to form the hydrogel base. C940: 0.0250 g C940 was added in 0.415 mL deionized water and placed at 4 \u00b0C overnight for swelling. After that, a 5% triethanolamine solution of 0.835 mL was added and stirred evenly to form the hydrogel base. Among them, CS and C940 require the addition of pH adjusters to form the hydrogels. All the obtained blank bases were colorless and transparent. After the hydrogel base swelled, the AA solution of PG was added to these hydrogel bases and then stirred evenly to mix well. All prepared AA-loaded hydrogels appeared to be opaque and white in color and could be easily applied to the skin. During the formulation investigations, different base types, HA content, and drug content were changed according to the prescriptions, as listed in Four types of polymers were selected to prepare the AA-loaded hydrogels, including HA, CS, P407, and C940. First, 0.0580 g AA or its mixture with organic amines and chemical enhancers were added and dissolved in 1.0000 g PG at 70 \u00b0C to get the AA solution were separately added to the AA solution and then mixed with the 3.5% HA hydrogel base to get the preparations. The AA-loaded hydrogel without any organic amine was used as the control to evaluate the effect of counterions on the skin permeation of AA. For preparing the AA-loaded hydrogels containing 5.0% different chemical enhancers, 0.1101 g L-menthol, Azone, OA, Span 80 or ML were separately added in the AA solution along with the DEtA as the preferred organic amine and then mixed with the HA hydrogel base to get the preparations. For investigation of the combined effect of different enhancers, 5.0% L-menthol and 5.0% Span 80 were added into the AA solution and mixed with the HA hydrogel base to get the preparations. Using the DEtA as the preferred organic amine, the AA-loaded hydrogel without any enhancer was used as the control to evaluate the effect of different chemical enhancers on the skin permeation of AA. As for the prescriptions for the microneedle experiment, it is that 9.21 \u03bcmol DEtA was added into the AA solution and mixed with a HA hydrogel base to get the formulation. Then, L-menthol as the preferred enhancer and the microneedles of the length of 0.5 mm and 0.75 mm were separately used in combination for experiments. All these prescriptions are listed in 2. PBS containing 3% Tween 80 (w/v) of 7.0 mL was added to the receptor cell, which was kept at 32 \u00b0C with magnetic stirring at 150 rpm. At 2, 4, 6, 8, 10, and 12 h, 3.5 mL receptor fluid was taken out, and an equivoluminal fresh receptor fluid was added. The sample was centrifuged at 15,000 rpm for 10 min for HPLC analysis. The cumulative release of AA was calculated by Formula (1):Q: the cumulative release of the drug; iC: the concentration of the released liquid of the i-th replacement sampling; iC\u22121: the concentration of the released liquid of the (i \u2212 1)-th replacement sampling; iV: displacement volume of receiving solution; V: total volume of release medium (V = 7.0 mL); n: number of times to replace receiving solution; A: effective diffusion area.Drug release experiments were carried out with a modified Franz diffusion cell. The cellophane dialysis membrane was placed between the donor cells and the receptor cells, and then AA-loaded hydrogels were added to the donor cells and totally contacted the membrane with a diffusion area of 1.76 cmw/v, i.p.). Then, the abdominal hair was successively shaved with an electric clipper and a razor. Full-thickness skin was excised after the rats were sacrificed, and the subcutaneous fat adhering to the skin was surgically trimmed. The obtained skin was cut to the appropriate size, wrapped in aluminum foil, and then immediately stored at \u221220 \u00b0C for a maximum of 2 weeks. Prior to each experiment, the skin was allowed to thaw slowly for subsequent use. The thickness of the rat skin used in our study was 680 \u00b1 50 \u03bcm. For microneedle pretreatment, the skin was pierced with an array of microneedles at the predefined depth and force for a certain time. After that, the penetration experiments were carried out accordingly to the above release method. According to the principles of animal ethics, all animal-related experimental procedures were carried out with the approval of the Department of animals of China Medical University.Skin penetration experiments were carried out similarly to the release method for the determination of drug penetration through the skin, and the difference was that the semi-permeable membrane was replaced by the excised rat skin. Male Sprague-Dawley (SD) rats (weighing 200\u2013230 g), purchased from SPF (Beijing) Biotechnology Co., Ltd., were first anesthetized with urethane of the skin was collected via successive tape striping (20 times) with QJMDM tapes . The remaining skin was weighed again, and the weight of the stratum corneum was obtained by the difference method. The drug in the stratum corneum and the remaining skin layers were separately extracted via immersion in 3.0 mL and 0.5 mL methanol overnight and ultrasound for 15 min. The obtained suspension was centrifuged at 15,000 rpm and 4 \u00b0C for 10 min, and the supernatant was filtered through the 0.22 \u03bcm membrane for HPLC analysis of AA content in each skin.3PO4) . The flow rate was 1.0 mL/min, the column was kept at 30 \u00b0C, and the detection wavelength was set to 205 nm.The content of AA was determined with a validated HPLC method. The analysis was performed on an HPLC apparatus equipped with an e2695 pump and a 2489 variable-wavelength UV detector . The separation was carried out on a Diamonsil C18 (2) column . The mobile phase consisted of a mixture of acetonitrile and purified water , equipped with Rheoplus software, and CTD 620 gas convection controlled thermostat circulating water bath, appended with cone and plate geometry . Firstly, to observe the rheological characterization of HA hydrogel when they were used in the skin, these samples were tested at 32 \u00b0C, which was the temperature of the skin. Then, the quality of the flow of these hydrogels was tested by a flow curve test. Viscosity (Pa\u00b7s) and shear stress (\u03c4) were assessed at the rate of 0.1\u2013100 rad/s. After that, the amplitude sweep test was used to determine viscoelastic behavior. Linear viscoelastic region (LVR) was observed with the measurement of the storage (G\u2032) and loss modulus G\u2033 by keeping the shear stress (Pa) between 0.1 and 100 Pa and the constant frequency of 1 Hz. Lastly, the tests of the storage (G\u2032), loss modulus (G\u2033), and complex viscosity (\u03b7) were carried out by frequency sweep measurements in the LVR by setting the angular velocity from 0.1 to 100 rad/s and at 1% constant strain amplitude.The surface structure of the HA samples was investigated with scanning electron microscopy . HA hydrogel samples were freeze-dried at \u221250 \u00b0C, extracted by liquid nitrogen, and then coated with a thin layer of gold for SEM observation. d6. Beyond that, 0.0050 g pure AA was dissolved in DMSO-d6 as a control.All NMR samples were tested on a 600 MHz Bruker ascend DRX 600 spectrometer, appended with standard Brucker pulse sequences. To obtain samples with that same concentration, 0.0500 g AA with 0.1 mol ratios of DEtA and 0.0050 g AA with equal molar ratios of DEtA were dissolved in DMSO-The surface of microneedle-treated skin was investigated with the H&E staining method. Firstly, the abdomen of the rats was shaved and then treated with the microneedle . At 0 min, 5 min, 10 min, 30 min, and 1 h, the rats were sacrificed, and the skin tissues were separately removed. Lastly, these skin tissues were fixed in 4% paraformaldehyde for 48 h. After paraffin embedding, sectioning, and the H&E staining, the effect of microneedle action on the structure of the skin was observed with an optical microscope.p were determined to assess the statistical significance. The value of p\u2009 >\u2009 0.05 indicated no significant difference, * p < 0.05 indicated a significant difference, and ** p < 0.01 indicated a highly significant difference.All experiments were carried out at least in triplicate, and the results were indicated as mean \u00b1 SD (n \u2265 3). The difference values of For passive skin penetration, drug solubility in vehicles is an important parameter related to drug transport via the skin . General2). It was speculated that the carboxyl groups in HA would provide a mild acid environment to make AA exist in the form of free molecules, which facilitated their diffusion and release [2), probably due to its H-bonding interaction with the drug [2), which might be attributed to the electrostatic interactions between the amino group in CS and the carboxyl group in AA. Therefore, it was speculated that the interaction of the drug with the hydrogel base would not benefit its release from the preparations. In addition, zero-order drug release kinetic profiles were noticed for each type of hydrogel with different polymers as the bases. It might be attributed to the relatively high drug loading, which avoided the dose depletion during the release and enabled the drug release rate independent of the drug concentration, and the non-chemical crosslinking structure of hydrogel bases, which also facilitated the drug release. Given the versatile benefits of HA for skin application, including the hydration effect, bioadhesive property, hydrophobic interaction with stratum corneum [The type of polymers could significantly affect the drug release and skin penetration from the hydrogels prepared from them . In this release . C940 co release . P407 cothe drug . By cont corneum , as well corneum , we seleHA is a natural starting material for constructing hydrogels, and it can form hydrogels via both physical and chemical approaches ,34. By cFor the hydrogels formed with 2.5%, 3.5%, and 5.1% HA, we investigated their rheological behaviors. As shown in SEM images were obtained to determine the microstructure of the freeze-dried hydrogels with different content of HA, and the result is shown in 2, 67.36 \u00b1 1.72 \u03bcg/cm2, 81.05 \u00b1 7.34 \u03bcg/cm2. This result suggested that the release of AA is a passive diffusion process. Even at 12 h of release, dose depletion was not observed, suggesting that HA was a suitable carrier of AA. We were also informed that as the increase of AA content, the release of AA was increasing gradually. However, a further increase of the AA content would affect the appearance of the drug-carrying hydrogel and cause the AA to be unevenly distributed, and thus the optimal drug loading was selected as 2.0%.The drug content in hydrogels is another important factor affecting drug release , and theThe human skin is the best choice for use in percutaneous studies; however, it is not always available and may also bring ethical problems. Additionally, the large variation in the permeability of human skin is also a point of concern. As a potential substitute, rat skin is the most commonly used to estimate the skin permeability of various drugs for the development of skin-related pharmaceutical formulations due to its accessibility and easy handling . TherefoThe skin permeability of ionizable drugs like AA is not only affected by their physicochemical properties but also by counterions . Unlike p < 0.01) and ETA (p < 0.05) could significantly increase the drug distribution in the stratum corneum, while all the organic amines improved drug retention in the underlying skin, among which the DEtA (p < 0.01) exhibited the greatest enhancement effect. We also investigated the effect of organic amines on the release of AA. According to the result shown in 1H-NMR investigation. The 1H-NMR spectra of AA, AA-0.1 mol eq DEtA and AA-1 mol eq DEtA are shown in The experimental permeation results are shown in p < 0.01). Last but not least, AA was not detected in the receptor cells of all chemical enhancer groups in our study. As a non-ionic and lipophilic surfactant, Span 80 was reported to be only deposited in the lipophilic stratum corneum after topical application [p < 0.01). The possible mechanism for L-menthol might be that it had strong hydrogen bonding capability and could compete for the hydrogen bonding sites present in the stratum corneum, making the drug get rid of the interaction with the stratum corneum components and transport into the deep skin layers [Chemical enhancers play an important role in promoting the absorption of transdermal drugs and are commonly used to promote the skin delivery of drugs ,46. The lication , thus imn layers ,49. AparAs the most effective components, respectively, on the stratum corneum and the remaining skin layers, Span 80 and L-menthol were also investigated for their combined enhancement effect. However, they did not provide a synergistic or combined efficacy, as the enhancement effect was not as good as that when they were used alone. Interestingly, a considerable amount of drug permeation through the skin was noted in the receptor fluid when Span 80 and L-menthol were used together, which was shown in p < 0.01), 5 N (p < 0.01), 6 N (p < 0.01), and 8 N (p < 0.01) groups were significantly better than the control group, while the drug content in the remaining skin layers was a little parabolic with the magnitude of the force. When the force was less than 5 N, the drug content in the skin rose with the increase of the force, while when the force was greater than 5 N, it decreased with the increase of the force. It might be because the skin was excessively pressed, making the state of the skin change, and it becomes difficult for the drug to penetrate. Furthermore, it can be seen from p < 0.01), and for the 5 min, 6 min, and 8 min groups, the differences were more pronounced (p < 0.01). It was possible because the longer the microneedle action time, the more obvious the holes left by the microneedles on the surface of the skin, which make it easier for the drug to enter the skin through the holes, thereby increasing the intradermal drug content. Lastly, it could be seen from p < 0.01). It indicated that the increase in the length of microneedles could promote the entry of AA into the skin to a certain extent. It was possible because the longer the microneedle penetrated the skin, the more favorable it would be for the drug to pass through the stratum corneum and into the deeper layers. In addition, we observed that AA was not detected in the receptor cells of all of the above experimental groups, probably due to the troublesome physicochemical properties of AA which was unbeneficial for its delivery through the micropores formed by the microneedles.As a novel enhancement technology, microneedles depend on single or multiple needle-like structures to pierce the stratum corneum and create multiple microchannels in a minimally invasive manner to improve the permeability of drugs . The micAfter that, the microneedle pretreatment was used together with the best enhancer, L-menthol, to further enhance the skin penetration of AA. Under the action of 0.5 mm microneedle and L-menthol, there was no improvement for the drug deposition in stratum corneum (228.09 \u00b1 40.54 \u03bcg/g) or underlying skin (37.50 \u00b1 7.15 \u03bcg/g). A further increase in the microneedle depth (0.75 mm) combined with L-menthol only significantly enhanced the drug retention in stratum corneum (344.81 \u00b1 49.33 \u03bcg/g) instead of underlying skin (40.26 \u00b1 3.39 \u03bcg/g). Disappointingly, we also found that the combination of 0.75 mm microneedle and L-menthol produced drug penetration into the fluid receptor, which was shown in In our study, we investigate the state of microneedle-treated skin using the H&E staining method in order to observe changes affected by the action of microneedles. The microneedle used in our experiment is shown in In this study, we developed a novel kind of hydrogel for AA and investigated different pharmaceutical approaches to improve the dermal delivery of AA. HA of 3.5% content was used as the preferred hydrogel base, according to the drug release profile, as well as morphological rheological properties. The counterion that provided the greatest penetration enhancement effect of AA was AA-DEtA, and the optimal chemical enhancer offering the highest drug penetration in deep skin layers was L-menthol. The optimized application scheme of microneedle pretreatment was determined as 6 min of treatment time, 5 N of force, and a needle length of 0.5 mm.In summary, all these approaches could increase drug retention in the skin when they are used alone. Different types of counterions and chemical enhancers may provide specific enhancement effects towards drug retention in stratum corneum or underlying skin layers. Interestingly, the study found that the combination of enhancer-microneedles or enhancer-enhancer could produce transdermal absorption of the drug. For topical delivery of AA, the most appropriate strategy might be the cooperation of DEtA and the best chemical enhancer of L-menthol, which provided the greatest drug content of 70.80 \u00b1 14.07 \u03bcg/g in the deep skin layers. The study could facilitate the design and development of the topical drug delivery system for AA and other drugs with similar physicochemical properties."} +{"text": "Goats are popular in China because of their superior meat quality, delicate flesh, and unique flavor. Long noncoding RNAs (lncRNAs) play important roles in transcriptional and post-transcriptional regulation of gene expression. However, the effects of lncRNAs on adipocyte differentiation in goat has not been fully elucidated yet. In this investigation, we performed RNA-Seq analysis of intramuscular and subcutaneous adipocytes from Jianzhou Daer goat before and after differentiation, including both intramuscular preadipocytes (IMPA) vs. intramuscular adipocytes (IMA) and subcutaneous preadipocytes (SPA) vs. subcutaneous adipocytes (SA). A total of 289.49\u00a0G clean reads and 12,519 lncRNAs were obtained from 20 samples. In total, 3,733 differentially expressed RNAs were identified by pairwise comparison. There were 135 differentially expressed lncRNAs (DELs) specific to intramuscular adipocytes, 39 DELs specific to subcutaneous adipocytes, and 8 DELs common to both adipocytes in these 182 DELs. Some well-known and novel pathways associated with preadipocyte differentiation were identified: fat acid metabolism, TGF-beta signaling pathway and PI3K-Akt signaling pathway. By integrating miRNA-seq data from another study, we also identified hub miRNAs in both types of fat cells. Our analysis revealed the unique and common lncRNA-miRNA-mRNA networks of two kinds of adipocytes. Several lncRNAs that regulate potentially goat preadipocyte differentiation were identified, such as XR_001918 647.1, XR_001917728.1, XR_001297263.2 and LNC_004191. Furthermore, our findings from the present study may contribute to a better understanding of the molecular mechanisms underlying in goat meat quality and provide a theoretical basis for further goat molecular breeding. Fat is generally divided into subcutaneous fat, intermuscular fat and intramuscular fat. The amount of intermuscular fat is small and its composition is similar to subcutaneous fat, so it is generally considered that fat is mainly divided into subcutaneous fat and intramuscular fat . The disAdipogenesis is a complex process regulated by various transcription factors, non-coding RNA and signal pathways . RNA seqHerein, we provided a comprehensive transcriptome profile on intramuscular and subcutaneous adipocytes in before and after differentiation of Jianzhou Daer goat. The results revealed the expression patterns of mRNAs and lncRNAs, which were important in the developmental stages of two different adipocytes. An integrated analysis of differentially expressed lncRNAs (DELs) and mRNAs (DEGs) was performed, and the lncRNA and its target miRNA/mRNA that could potentially regulate the differentiation of intramuscular and subcutaneous preadipocytes were screened through bioinformatics analysis. At the same time, a lncRNA-miRNA-mRNA interaction network was constructed based on miRNAs known to play a role in adipogenesis, which were identified by another study (manuscript in preparation). The lncRNA that may be combined with miRNA and mRNA was identified and verified by RT-qPCR technique. The results from the present investigation provided a theoretical basis for in-depth analysis of the regulation mechanism of goat fat cell differentiation and improvement of meat quality in goats.All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee, Southwest Minzu University. Also, all the experiments complied with the requirements of the directory of the Ethical Treatment of Experimental Animals of China.Capra hircus) (n = 5) was purchased from Sichuan Jianyang Dageda Aminal Husbandry Co., Ltd , being euthanized by bloodletting. The longissimus dorsi and subcutaneous fat were excised from the goats and minced. The isolation and culture of goat intramuscular and subcutaneous preadipocytes was performed as described assay by . RNA quality was determined using NanoPhotometer spectrophotometer and Agilent 2,100 bioanalyzer, which analyzes the integrity of the RNA \uff08RIN\uff09. The lowest RIN accepted for RNA analyses is 6.8. The lncRNA library is constructed using a chain-specific library. The method for synthesizing the first strand of cDNA by reverse transcription is the same as the normal method of NEB library construction. The difference is that when the second strand is synthesized, dTTP in dNTPs is replaced by dUTP. After that, cDNA end repair, A-tailing, ligation of sequencing adapters, and length screening were also performed, and then the second strand of cDNA containing U was degraded by USER enzyme, and then PCR amplification was performed to obtain a library. Finally, twenty libraries were sequenced at Novogene Co. Ltd. on Illumina HiSeq Sequencing System. The RNA-Seq dataset supporting the conclusions of this article is available in the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) under accession numbers GSE186988.After sequencing, the raw data were stored in fastq format . We remoTo ensure the quality of the obtained lncRNAs, three criteria were used to identify the desired lncRNAs in the transcriptome assemblies: 1) transcripts with length >200\u00a0bp and exon number \u22652 were selected; 2) Cuffcompare (v2.1.1) was used to calculate the read coverage of every transcript, and transcripts with an FPKM value (Cuffquant) of more than 0.05 were removed; and 3) the coding potential of the transcripts was predicted using the coding potential calculator (CPC <1) , Coding p value was used to screen the DELs and DEGs between two different adipocyte samples. When p < 0.05, the lncRNAs and mRNAs are considered to be differentially expressed.The FPKMs values of lncRNAs and mRNAs in IMPA, IMA, SPA and SA libraries were calculated by StringTie (v1.3.1), Ballgown and Cuffp-value < 0.05 was set as significant threshold. Go (gene ontology) can enrich and analyze the target genes of DELs. Goseq (Release2.12) (p < 0.05 is considered to be the significant enrichment of GEGs. KEGG is a database (http://www.genome.jp/kegg/) for understanding the advanced functions and utilities of biological systems such as cells, organisms, and ecosystems (The trans function describes the co-expression relationship between lncRNAs and mRNAs. Pearson correlation coefficient (R > 0.95 or R < 0.95) was calculated by custom scripts. David was used to cluster the target genes among 20 samples for functional enrichment analysis of lncRNA target genes . Adjustease2.12) is used osystems . We usedosystems softwarep-value <0.05. miRWalk, miRanda, RNAhybrid, and Targetscan were used for screening DEMs target DELs and DEGs. Then, the intersections of co-expressed lncRNAs, miRNAs, and mRNAs in the four programs were used to construct a lncRNA\u2013miRNA\u2013mRNA network.We have previously established small RNA libraries by RNA-Seq before and after differentiation of intramuscular and subcutaneous adipocytes (data not yet published). The raw reads were filtered to remove low quality reads and reads with connectors to obtain clean reads. Novoaligen software was used to match the clean reads with the miRBase database to identify known miRNAs, and MirDeep software was usedDELs and DEGs were combined with the target lncRNAs and mRNAs of DEMs respectively to obtain the miRNA-mRNA relationship pair and miRNA-lnRNA relationship pair. Finally, the lncRNA\u2013miRNA\u2013mRNA network was visualized using Cytoscape v3.7 software .UXT) was used as a housekeeping gene (\u2212\u0394\u0394CT) method (Primers were designed using Primer-BLAST on the NCBI website . 1\u00a0\u03bcg ofing gene . The qPC) method . The exphttps://www.ncbi.nlm.nih.gov/genome/?term = goat), and 44.56\u201387.33% uniquely mapped reads were obtained from the total mapped reads from the twenty samples (We drew Venn diagrams to visua < 0.05) . The DEL < 0.05) . Unique < 0.05) , and the < 0.05) , while t < 0.05) .p < 0.05). In addition, the analyzed genes also was enriched in fat acid metabolism and TGF-beta signaling pathway, which are related to adipocyte formation (Supplement 10). The unique DELs target genes of subcutaneous adipocytes before and after differentiation are most significantly enriched in biosynthesis of amino acids, glycolysis/Gluconeogenesis, carbon metabolism, valine, leucine and isoleucine degradation, histidine metabolism, fatty acid degradation, melanoma, fatty acid metabolism (adjusted p < 0.05), among which fatty acid degradation, fatty acid metabolism, TGF-beta signaling pathway, PI3K-Akt signaling pathway have a significant impact on the adipogenesis (p < 0.05) .As to miRNAs analysis, 210 known miRNAs and 95 novel miRNAs were found to have significantly different expression between intramuscular preadipocytes and adipocytes (Supplement 3), 175 known miRNAs and 67 novel miRNAs were found to have significantly different expression between subcutaneous preadipocytes and adipocytes (Supplement 4). Among them, we obtained 146 differentially expressed miRNAs (DEMs) unique to intramuscular adipocytes, 83 (DEMs) unique to subcutaneous adipocytes, and 159 (DEMs) shared by two adipocytes . After t32,029 lncRNAs (Supplement 8) were found to have potential targeted binding relationships with DEMs. Combined with the sequenced intramuscular adipocyte-specific 2,724 DEGs and 135 DELs, 6,572 miRNA-mRNA relationship pairs and 1,240 miRNA-lncRNA relationship pairs were obtained . The 1,1UXT was used as an internal reference for qRT-PCR analysis. The results showed that XR_001917557.1, and LNC_004191 were significantly down-regulated in expression during the differentiation of intramuscular adipose tissue. Whereas, the XR_001918647.1, XR_001917728.1 were significantly up-regulated in expression during the differentiation of intramuscular adipose tissue. XR_001295810.1, XR_001917637.1 and XR_001297263.2 were significantly down-regulated expression during the differentiation of subcutaneous adipose tissue, and the LNC_004191 was significantly up-regulated expression during the differentiation of subcutaneous adipose tissue, These results were consistent with the trend of RNA-seq, indicating the credibility of the RNA-seq results is the material basis of marbling, and an important factor affecting meat flavor. A large number of studies have shown that IMF is directly involved in the formation of meat tenderness, juiciness and flavor . Goat isR2) of each sample is greater than 0.8, which indicated that our experiment was reliable and the sample selection was reasonable.LncRNA is a kind of noncoding RNA longer than 200\u00a0nt, which has attracted substantial attention in the last few years. Studies have shown that lncRNAs regulate metabolic tissue development and function, including adipogenesis, hepatic lipid metabolism, islet function, and energy balance . DespiteLncRNA functions by regulating mRNA. At present, the mechanism of interaction between lncRNA and mRNA is not clear. We predict the biological function of lncRNAs through its co-expression with protein coding genes. Consequently, we found that many target genes of DELs were also differentially expressed in goat intramuscular and subcutaneous preadipocytes. This suggested that lncRNAs may function through complementary target genes, which can play critical roles in the differentiation of goat intramuscular and subcutaneous preadipocytes. For example, SMAD1 is a target gene of the differentially expressed lncRNAs LNC_009792, LNC_007731, LNC_000706, LNC_008467, LNC_006192 and LNC_004878, and it has been reported to regulate the differentiation of preadipocytes . These fTo explore the similarities and differences of different adipocytes, DELs target genes (IMPA vs. IMA and SPA vs. SA) were subjected to GO and KEGG pathway enrichment analyses. We found that few common term was found between the IMPA vs. IMA and SPA vs. SA comparisons. Several pathways involved in preadipocyte differentiation were previously identified, including the TGF-\u03b2 signaling pathway (IMF and subcutaneous fat) , PI3K/AKTo date, many genes have been reported to regulate the differentiation of preadipocytes. However, few studies have been conducted on the roles of lncRNAs in intramuscular and subcutaneous preadipocytes differentiation. The molecular and cellular mechanisms regulating goat preadipocytes differentiation are thus still poorly understood. Here, we constructed a miRNA-lncRNA-mRNA interaction network, and calculated the degree (the number of times each factor interacts with other factors) of each factor through Cytoscape (CentiScaPe ). We selIn conclusion, we first generated the expression profiles of lncRNAs from intramuscular and subcutaneous adipocytes of Jianzhou Daer goat (IMA vs. IMPA and SA vs. SPA) based on RNA-Seq technique. We found that the number of lncRNAs regulating IMF differentiation was more than that of subcutaneous adipocytes. Our results suggest that those lncRNAs might play important roles in adipocyte differentiation. Collectively, this study takes the first step toward understanding the molecular mechanisms underlying variations in goat adipogenesis. Also, our results provided a theoretical basis for molecular breeding to improve the meat quality in goats."} +{"text": "The motion of the train is replicated using the slip grid technique. The results indicate that when a fire breaks out on a moving train in tunnels, the piston wind leads the longitudinal movement of the smoke. If a fire erupts in the head or middle car of a moving train, the time of smoke backflow is delayed by 30 s or 17 s, respectively, compared to that for the tail car. The obtained results provide a theoretical basis for reasonably controlling the smoke flow in subway tunnels and reducing casualties in fire accidents.Scenario models of a moving subway train can help investigate the influence of different fire locations on smoke propagation characteristics in curved tunnels. To this end, this study adopts the three-dimensional Unsteady Reynolds Average Navier-Stokes equations method and the renormalization group Urban subways are characterized by a high ridership, safety and reliability; furthermore, they use fewer surface transportation resources. Although subways provide convenience to people, safety problems are becoming more apparent. Fires on subway trains are among the most serious disasters, causing casualties and property damage. For example, the Daegu subway fire in South Korea in 2003 claimed 192 lives and injured 148 people [Due to the topography and location of stations, many subway tunnels have some curvature. Limited by research objects, experimental conditions, and other factors, many researchers have used stationary fire sources to study the diffusion law of fire smoke in tunnel. Wang et al. investigWhen a train catches fire while moving, it tends to continue for a certain distance. Zhong et al. reportedCurrently, research on fires in curved subway tunnels mainly focus on stationary fire sources. Some researchers have also studied fires in moving subway trains, but the fire scenarios mainly focus on straight tunnels. Some studies have reported that the lateral locations of fire source from the tunnel wall affect the critical ventilation velocity and longitudinal temperature field, but these studies ignore the influence of the train blocking ratio , 16. AccIn this research, the scenario of a moving subway train on fire in a curved tunnel is simulated. Moreover, smoke dispersion characteristics are investigated with regards to the fire source locations on a subway train. The distribution characteristics of smoke movement under different fire scenarios are also determined. The study results can provide a theoretical basis for reasonably controlling the smoke flow in subway tunnels and for reducing casualties in fire accidents.This paper is generally divided into four sections. The first section mainly introduces the background of this research. The second section briefly explains the geometric model and the numerical calculation method used in this study. The third section discusses the influence of the locations of subway train fire on the characteristics of smoke movement in a curved tunnel. The fourth section generalizes the conclusions obtained in this study and establishes the scope for possible future research.2. The train equipment cabin is a cuboidal structure with a length, breadth, and height of 7.98 m, 2.49 m, and 0.63 m, respectively. For simulating a tunnel train fire, the train model includes detailed structures, such as the equipment cabin and the bogie of the subway train, which can reduce the impact of model accuracy on simulation results by refining train modeling.This study considers a six-group subway train as the research object. A geometric model of the train is displayed in 2. The dimensions of the platforms are 150 m long, 8 m wide, and 6 m high. The tunnels at the outer ends of the two platforms are extended by 50 m. The \"Code for Design of Metro\" states that the minimum curve radius of the A-type metro running line should not be less than 250 m [an 250 m . After cNumerical simulation calculations can use various methods to study problems related to trains/tunnels, including 1D/2D/3D, compressible/incompressible, and steady/unsteady flow methods \u201323. The k-\u03b5 model with buoyancy correction is used to improve the accuracy of the fire smoke dispersion process simulation [k-\u03b5 turbulence model are as follows.The propagation of fire smoke in subway tunnels is a turbulent motion that occurs under the combined effects of a train slipstream and thermal buoyancy. In this study, the Unsteady Reynolds Average Navier-Stokes equations (URANS) method is used to deal with the Navier-Stokes equations, which reduces the computational complexity of the simulation process while ensuring its accuracy . For themulation . In addik equation:Turbulent kinetic energy \u03b5 equation:Turbulent dissipation rate The finite volume method used in this study is a numerical methodology commonly used in numerical simulations. The computational domain is solved using the commercial general-purpose software Fluent 18.1. The software Fluent 18.1 has rich physical models and advanced numerical methods as well as powerful pre-processing and post-processing functions; moreover, it is widely applied in the fields of fluids, heat transfer, and chemical reactions.Qr of the Rosseland model is obtained by a theoretical formula [Radiation models are used to simulate the radiative heat transfer between heat sources, fluids, and solid walls during fires. The length of the tunnel in this study is 1000 m, and the fire smoke spreads along the tunnel widely, which is suitable for the Rosseland radiation model [ formula .In this study, the most commonly used volumetric heat source model in engineering simulation is used \u201327. The (1) Fire source power and smoke release rate.Qc is usually 0.7 times the power of Q. The smoke release rate is related to the height of the smoke relative to the tunnel floor. Therefore, the maximum smoke release rate for a fire power of 7.5 MW in the tunnel is 27.78 kg/s.The extent of fire in subway trains depends on various factors, and the power range is generally 5 MW to 10.5 MW . After a(2) Locations of the fire sourceTrain equipment compartments usually contain electrical equipment, such as traction transformers, traction converters, and auxiliary power supply units. According to the statistics of train fire accident, the failure of train electrical equipment is the leading cause of fires . CompareConsidering the influence of train blockages on smoke propagation, a refined subway train model is adopted. The local mesh is shown in 3 and 7850 kg/m3; thermal conductivities are 1.2 W/(m\u00b7K) and 3 W/(m\u00b7K); and specific heat capacities are 0.88 kJ/(kg\u00b7K) and 0.50 kJ/(kg\u00b7K). The initial environmental conditions of the entire computational domain are set as follow: the ambient temperature is 300 K, and the pressure is 101 kPa [The boundary conditions of the developed model are shown in 101 kPa .To monitor the characteristic parameters of the smoke in the tunnel after the train catches fire, some measurement points are established inside the tunnel. The arrangement of measurement points is shown in In this study, a moving model of train on fire is used to verify the numerical calculation method . The mov2, and the blocking ratio of the train to tunnel is 0.43. The speed of the model train is 60 km/h during the test.As described in For verifying the numerical calculation method, a geometric model of the same size as that used in the moving model test is adopted. The accuracy of the numerical calculation method is verified by the airflow velocity and the peak smoke concentration parameters at the measurement points in the experiment. In the experiment, the smoke concentration was measured by the concentration sensors, and in the simulation software the measurement points automatically collected the information of smoke concentration in the tunnel. The locations of all measurement points in the test are displayed in u/V)max parameter represents the maximum value of the dimensionless flow velocity at P6 measurement point. Cc represents the smoke concentration at P3 measurement point in the numerical simulation, and CExpt represents the maximum value of smoke concentration at P3 measurement point in the moving model experiment. (Cc/CExpt)max represents the maximum ratio of dimensionless smoke concentration at P3 measurement point. By comparing the peak values of dimensionless flow velocity and smoke concentration, the numerical simulation results of the medium- and fine-mesh scale schemes are verifiably in a good agreement with the results of the moving model test. In contrast, a relatively large error exists between the results of coarse-mesh scheme and the moving model test.At the same time, in order to verify the reliability of the numerical simulation method, a comparison between the numerical simulation results of different mesh scale schemes and the moving model test results are shown in From This section examines the distribution of smoke velocity, temperature, and concentration when a moving subway train catches fire in a tunnel with a curve radius of 300 m. The power of the fire is 7.5 MW in the equipment compartments of the head, tail, and middle cars (fourth car) of the train.2 from a stationary state. After a travel time of 20 s, the normal operating speed of the train is 20 m/s. Next, the train travels with a constant speed of 20 m/s for 5.9 s and then a fire breaks out. The train then immediately decelerates at a rate of 1 m/s2 and finally stops in the middle of the tunnel after 20 s. The movement scenario is displayed in Figs t = 250 s. In conjunction with the longitudinal airflow velocity distribution of the tunnel vault at different fire sources . When the smoke concentration exceeds 0.03, people experience dizziness, nausea, and other reactions, severely limiting their ability to escape. If the smoke concentration continues to increase, suffocation can occur. Therefore, smoke is said to be highly concentrated when the smoke concentration (C) is greater than 0.03.t = 100 s and 250 s when the fire source is at different locations. In In addition, the total amount of fresh air in the blocked area of the train is less than that in the unblocked area. When the fire source produces the same amount of smoke, the smoke concentration increases in the blocked area. Therefore, the peak smoke concentration is lowest when the head car is on fire. The results are shown in the distribution of smoke concentration in the tunnel vault see .When the middle car catches fire, the peak of smoke concentration is always higher than that when the tail car catches fire. After the smoke countercurrent occurs, the difference between them increases further. Compared with t = 250 s for different fire locations. Furthermore, the diffusion distance of highly concentrated smoke upstream is the longest when the middle car is burning. It is 30.19% and 60.47% longer than that for the head and tail car fires, respectively.During this research, a numerical methodology is adopted to investigate entire fire history in a curved tunnel with a moving subway train on fire. The influence of different fire locations on the spread of smoke flow velocity, temperature, and concentration is analyzed. The key findings are listed below:For a moving train with fire in a tunnel, the smoke propagation was affected by the moving air in the slipstream of the train and spread rapidly along the direction of the train. The distribution characteristics of smoke were complicated.When a fire breaks out on a moving train, the closer the fire is to the tail car, the longer the length of the countercurrent and the peak countercurrent velocity are. Simultaneously, the upstream diffusion distance of the highly concentrated smoke is larger. Compared with the scenario of tail car fire, the timing of smoke backflow is delayed by 30 s or 17 s when the head or middle car is burning, respectively.The location of the fire determines the blockage length of the train/tunnel upstream and downstream of the fire source, which substantially impacts the smoke dispersion of a moving train on fire.Due to the limitation of conditions, the research adopts some idealized models. The current work still needs further research in the following aspect: This paper only studies the condition of a single curve tunnel, and following research can be carried out in combination with other curved radius tunnels to form a systematic research theory."} +{"text": "Recent studies found that secreted protein acidic and rich in cysteine-like protein 1 (Sparcl1) could inhibit lipid droplets accumulation by peroxisome proliferator-activated receptor-gamma (PPAR\u03b3) signal pathway. However, the associations of serum Sparcl1 level with lipids profiles and other metabolic phenotypes remain unknown in human population study.We determined serum Sparcl1 using sandwich enzyme-linked immunosorbent assays among 1750 adults aged 40 years and older from a community in Shanghai, China. Generalized linear regression models were used to evaluate the association between Sparcl1 and metabolic measures. Multivariable-adjusted logistic regression analyses were performed to evaluate the relationship of serum Sparcl1 with prevalent dyslipidemia.With the increment of serum Sparcl1, participants tended to have lower level of triglycerides, and higher level of high-density lipoprotein cholesterol . No significant associations between serum Sparcl1 and glucose, blood pressure, or body size were observed. The generalized linear regression models suggested that per standard deviation (SD) increment of serum Sparcl1 was significantly inversely associated with triglycerides . The prevalence of dyslipidemia decreased across the sparcl1 quartiles (P for trend <0.01). After controlling the potential confounders, participants in the highest quartile of sparcl1 concentration had the lowest prevalence of dyslipidemia , compared with the lowest quartile. Per SD increment of Sparcl1 was associated with 20% lower prevalence of hypertriglyceridemia and 12% lower prevalence of dyslipidemia. The association between serum Sparcl1 and dyslipidemia were generally consistent across subgroups .Serum Sparcl1 was significantly associated with decreased risk of prevalent dyslipidemia in Chinese population. Further studies are warranted to confirm this association. Secreted protein acidic and rich in cysteine like protein 1 (Sparcl1), belonging to the SPARC protein family, is a secreted glycoprotein that was found to be involved in the regulation of many physiological processes, including cell adhesion, migration, and proliferation . Sparcl1Recently, Sparcl1 was reported to have a negative effect during preadipocyte differentiation and inhibits lipid droplet accumulation through regulating peroxisome proliferator-activated receptor-gamma (PPAR\u03b3), lipoprotein lipase (LPL) and insulin-like growth factor 1 , suggestThus, to reveal whether serum Sparcl1 is associated with metabolic phenotypes in human population, we systematically examined the levels of serum Sparcl1, metabolic measures, particularly, lipid profiles and dyslipidemia in a cross-sectional study among Chinese population.The study participants were from a community cohort in Shanghai, China. Details of the cohort could be found in previous studies , 9. BrieThe study protocol was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao-Tong University School of Medicine. All participates signed written informed consent and took a comprehensive physical examination and interview.Using standardized questionnaires, we collected detailed information on sociodemographic characteristics, lifestyle habits, and medical history. Current smokers or drinkers were defined as smoking cigarettes or consuming alcohol regularly in the past 6 months. Physically active was defined as moderate intensity physical activity \u2265150 minutes per week, or vigorous intensity aerobic physical activity \u226575 minutes per week, or a combination of moderate and vigorous intensity activity \u2265150 minutes per week. Height and weight were measured with participants wearing lightweight clothes and no shoes. Body mass index (BMI) was calculated as body weight in kilograms divided by height squared in meters. Waist circumference was measured using a non-stretch tape at the umbilical level in a standing position with normal breathing. After at least 5-min rest, blood pressure was measured using an automated electronic device at a non-dominant arm. The average of three measurements was used to analysis.All participants were asked to keep fasting overnight at least 10 hours. A standard 75-g oral glucose tolerance test were conducted. Fasting plasma glucose (FPG) and 2-hour postload plasma glucose (2h-PG) levels were measured using the glucose oxidase method with an autoanalyzer . Hemoglobin A1c (HbA1c) was measured using high-performance liquid chromatography in the central laboratory located at Shanghai Institute of Endocrine and Metabolic Diseases, which is certified by the College of American Pathologists. Serum insulin was measured using an electrochemiluminescence assay . The homoeostasis model assessment of insulin resistance (HOMA-IR) index was calculated as fasting insulin (mIU/mL)\u00d7fasting glucose (mmol/L)/22.5 or median (inter-quartile range) and categorical variables were presented as number (proportion). P for trend according to quartiles of serum Sparcl1 was performed using linear regression for continuous variables and \u03c72 testing for categorical variables.The generalized linear regression models adjusting for age, sex, education of high-school or above, smoking and drinking status, physically active and BMI were used to evaluate the association between per SD increment of serum Sparcl1 and metabolic measurements .Potential non-linear association between the level of serum Sparcl1 and prevalence of dyslipidemia was examined using restricted cubic splines . Three kMultivariable adjusted logistic regression analysis was used to evaluate the association between serum Sparcl1 and prevalence of dyslipidemia and individual components. The multivariable model was adjusted for age, sex, education of high-school or above, smoking and drinking status, physical active and BMI. We also performed subgroup analysis on the association of serum Sparcl1 and dyslipidemia according to sex, age, current smoker (yes/no), high education (yes/no), physically active (yes/no) and BMI. The P values for interaction were calculated by likelihood ratio tests comparing models with and without the interaction terms.Statistical analyses were performed using SAS or R (version 4.1.2). A two-sided P value<0.05 was considered as statistically significant.Among the 1750 participants included, the mean (SD) age of the study participants was 55.74 \u00b1( 8.37) years. The mean (SD) of serum Sparcl1 level was 701.36 \u00b1 (501.48) ng/mL. Baseline characteristics according to the quartiles of serum Sparcl1 were presented in We further evaluated the association between per SD increment of serum Sparcl1 and metabolic profiles using generalized linear regression models. After adjusting for age, sex, education of high-school or above, smoking and drinking status, physically active and BMI, Sparcl1 was found significantly inversely associated with TG Table\u00a02.The prevalence of dyslipidemia was 35.31% (618/1750) in this population. Participants with dyslipidemia have lower level of serum Sparcl1 than those without dyslipidemia . The prevalence of dyslipidemia . 2 .Stratified analysis for the association between per SD increment of serum Sparcl1 and dyslipidemia was presented in This community-based cohort study found that serum Sparcl1 was inversely related to TG. Moreover, per SD increment of serum Sparcl1 was associated with 20% decreased prevalence of hypertriglyceridemia and 12% decreased prevalence of dyslipidemia. The inverse associations between serum Sparcl1 and dyslipidemia were generally similar across all subgroups. To the best of our knowledge, the current study represents the first report in population concerning the metabolic profiles and dyslipidemia in association with serum Sparcl1. These findings suggest that Sparcl1 might play an important role in the lipid metabolism and the development of dyslipidemia.rs7695558 and rs9998212, which were both associated with lower brain Sparcl1 gene expression, showed accelerated memory loss or atrophy of brain volumes , Shanghai Outstanding Academic Leaders Plan (Grant No. 20XD1422800), Shanghai Medical and Health Development Foundation (Grant No. DMRFP_I_01), Clinical Research Plan of SHDC (Grant No. SHDC2020CR3064B), and Science and Technology Committee of Shanghai .The authors thank all participants in the study. We thank Dr Lu Yan for scientific discussion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "PD\u2010L1) expression in non\u2010small cell lung cancer (NSCLC) tumors guides treatment selection. PD\u2010L1 expression in circulating tumor cells (CTCs) may provide further information. We have explored PD\u2010L1 and marker of proliferation Ki\u201067 in CTCs in longitudinal samples of 47 advanced NSCLC patients receiving pembrolizumab. A triple immunofluorescence, against cytokeratin, PD\u2010L1, and Ki67, was performed on peripheral blood mononuclear cells, at baseline, post\u2010first cycle, post\u2010third, and primary resistance (PMR). Patients displaying PMR were classified as progressive disease (PD) and those with clinical benefit as disease control (DC). CTCs were categorized as PD\u2010L1high/low/medium/negative and Ki67+ or Ki67\u2212. CTC evaluation revealed a significant increase in the PD\u2010L1low CTC rate at PMR compared to baseline (2.5% at baseline vs. 36.5% at PMR), whereas a reduction in the PD\u2010L1high CTC rate was observed . Investigation of CTC status between PD and DC patients showed that PD patients more frequently increased total and PD\u2010L1low CTCs after first cycle compared to DC . Progression\u2010free survival (PFS) was longer in patients with decreased total and PD\u2010L1low CTCs after first cycle compared to those with increased CTCs (median PFS: not reached vs. 2\u2009months). PD\u2010L1+ patients presenting a high Ki67 index (% Ki67+ CTCs >\u200930%) before treatment had a shorter PFS compared to those with a low Ki67 (\u2264\u200930%), and overall survival (OS) was shorter in PD\u2010L1+ patients harboring Ki67+ CTCs compared to those not presenting . In sequential samples of patients with a durable benefit, a low Ki67 index was observed. Our results suggest that monitoring PD\u2010L1 and Ki67 expression in CTCs of NSCLC patients treated with pembrolizumab may be predictive for pembrolizumab efficacy.Programmed cell death protein ligand\u20101 ( + CTCs (>\u200930%) before treatment, or an increase in total number of CTCs or in PD\u2010L1low CTCs after the first cycle, seems to be related to resistance to pembrolizumab.The authors investigated the differential expression of PD\u2010L1 and Ki67 in circulating tumor cells (CTCs) of non\u2010small cell lung cancer patients treated with pembrolizumab. A high percentage of Ki67 CKcytokeratinCTCscirculating tumor cellsDCdisease controlFBSfetal bovine serumHRhazard ratioICIsimmune checkpoint inhibitorsMFImean fluorescence intensityNSCLCnon\u2010small cell lung cancerOSoverall survivalPBMCsperipheral blood mononuclear cellsPBSphosphate\u2010buffered salinePDprogression diseasePD\u20101programmed cell death protein 1PD\u2010L1programmed cell death protein ligand\u20101PFSprogression\u2010free survivalPMRprimary resistanceRTroom temperatureTBSTtris\u2010buffered saline/Tween 201Lung cancer is the leading cause of cancer\u2010related death affecting over 1.8\u2009million people worldwide every year. There is an estimated 18% survival rate beyond 5\u2009years for all stages combined, with poor outcomes largely related to late diagnosis [Immune checkpoint inhibitors have been designed to target inhibitory checkpoint molecules such as the programmed cell death protein 1 (PD\u20101) and its ligand, PD\u2010L1. The immune response to cancer involves a complex network of cellular interactions between cytotoxic T cells, helper T cells, natural killer, and tumor cells. However, metastatic tumors have acquired mechanisms to evade immune detection and one such example is through the overexpression of PD\u2010L1 .The promising therapeutic activity of ICIs in NSCLC has led the US Food and Drug Administration (FDA) to approve pembrolizumab, an antibody directed against the PD\u20101 immune checkpoint molecule for the treatment of NSCLC patients whose tumors express PD\u2010L1 . It has Unfortunately, responses to ICI treatment are not ubiquitous. Interestingly, many patients experience a primary or innate resistance at first evaluation to ICI treatment , 8 whilede\u2009novo and resistant genetic alterations, as well as real\u2010time monitoring of treatment responses [Liquid biopsies, such as CTCs shed from various locations of the primary and/or metastatic tumors allow a rapid and accurate identification of the esponses , 15. Theesponses , 18, 19.In the present study, we have explored CTC status in longitudinal samples of 47 metastatic NSCLC patients treated with pembrolizumab. We initially established a semiquantitative method to evaluate PD\u2010L1 expression levels and then assessed PD\u2010L1 and Ki67 on CTCs from the enrolled patients before treatment (baseline), at post\u2010first, post\u2010third cycle, and primary resistance. Subsequently, we compared total CTC counts, the number and percentage of PD\u2010L1 and Ki67 subpopulations between baseline and each timepoint. Moreover, we examined CTC changes before and after the first cycle, between patients presenting primary resistance and disease control. Finally, we investigated CTC status in serial samples of patients who experienced a durable benefit from treatment.22.1Patients with metastatic NSCLC who received first\u2010line chemotherapy with platinum doublet, entered the ICI pembrolizumab maintenance trial (NCT02705820) if they had not progressed after 4\u20136\u2009cycles of chemotherapy. The NCT02705820 trial compared maintenance pembrolizumab to best supportive care. All patients had signed written informed consent. The study methodologies conformed to the standards set by the Declaration of Helsinki and they were also approved by the Cyprus National Bioethics Committee (2019/76).During their treatment with pembrolizumab the patients were enrolled in the current prospective study where CTC evaluation and phenotypic analysis were performed at baseline, post\u20101st cycle (3\u2009weeks after baseline), post\u20103rd cycle (9\u2009weeks after baseline), and at primary resistance. Primary resistance was defiLiquid biopsy samples were also obtained at post\u20107th cycle (21\u2009weeks after baseline), post\u201010th cycle (30\u2009weeks after baseline), post\u201015th cycle (45\u2009weeks after baseline), post\u201018th cycle (54\u2009weeks after baseline), and at secondary resistance [In the present study, all patients who were categorized as progressive disease (PD) by RECIST criteria had experienced disease progression at first CT evaluation of treatment (primary resistance). Furthermore, patients who had achieved an initial clinical benefit such as CR, PR, or SD were categorized as disease control (DC). Among 48 patients, all but one were evaluable for response according to RECIST criteria Table\u2009. Twenty\u20102.2d\u2009=\u20091077\u2009g\u00b7mol\u22121) centrifugation at 670\u2009g for 30\u2009min. PBMCs were washed three times with phosphate\u2010buffered saline solution (PBS) and centrifuged at 530\u2009g for 10\u2009min. Aliquots of 106 cells were centrifuged at 700\u2009g for 2\u2009min on glass slides. Cytospins were dried up and stored at \u221280\u2009\u00b0C. A total of 6\u2009\u00d7\u2009106 PBMCs per patient were analyzed and the results are expressed as CTCs/6\u2009\u00d7\u2009106 PBMCs as shown in Table\u2009Twenty milliliters of blood was obtained from each patient at each time point. To avoid blood contamination by epithelial cells from the skin, all blood samples obtained after the first 5\u2009mL of blood were discarded. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll\u2013Hypaque density gradient for 24\u2009h, and cell lysates before and after IFN\u2010\u03b3 treatment were used for the evaluation of basal and induced levels of PD\u2010L1 protein by immunoblotting analysis. Cells were centrifuged on cytospins according to the procedure followed for patients' samples and cytospins of cell lines spiked into healthy volunteers' PBMCs (104/250\u2009000 PBMCs) were also prepared.The lung cancer cell lines A549, H460, and H1975 were obtained from the American Type Culture Collection . Cells were cultured in the appropriate medium supplemented with 10% fetal bovine serum (FBS) (GIBCO\u2010BRL) and 50\u2009\u03bcg\u00b7mL2.4Cell lysates and immunoblotting were prepared as previously described . Briefly2.5PBMCs' cytospins for each patient were triple stained with pancytokeratin, PD\u2010L1, and Ki67. Briefly, cytospin fixation and permeabilization were performed with ice\u2010cold acetone/methanol, 9/1 (V/V) for 20\u2009min at RT, followed by incubation with blocking buffer for 60\u2009min. Detection of cytokeratin\u2010positive cells was performed using the pancytokeratin mouse antibody, , 1\u2009:\u2009100 dilution overnight at 4\u2009\u00b0C. This was followed by incubation with the FITC antibody, diluted 1\u2009:\u2009400 for 50\u2009min. The cytomorphological criteria proposed by Meng and colleagues were used to characterize a CK\u2010positive cell as a CTC . To evalleica lasx software [To evaluate the specificity of the antibodies, cytospins of cell lines spiked into healthy volunteers' PBMCs were used as positive and negative controls. Negative controls were prepared by omitting the corresponding primary antibody and adding the secondary IgG isotype antibody Figs\u2009A. Moreovsoftware , 23.+/CD45\u2212) from a patient with high CTC number are shown in Fig.\u2009Furthermore, in patients presenting high CTC numbers, PBMCs' cytospins were double stained with pancytokeratin and the common leukocyte antigen CD45. For double staining experiments, PBMC cytospins were incubated with the pancytokeratin mouse antibody diluted 1\u2009:\u2009100, overnight at 4\u2009\u00b0C followed by the corresponding secondary FITC antibody for 50\u2009min and the Alexafluor 647 anti\u2010human CD45 diluted 1\u2009:\u2009300 for 1\u2009h. DAPI was added to each sample for nuclear staining. The specificity of the antibodies staining and a representative image of a CTC expressing different levels of PD\u2010L1 protein as determined by western blot and triple immunofluorescence analysis Figs\u2009 and S3B.lasx) software (Leica Microsystems). The intensity of PD\u2010L1 protein was measured among PD\u2010L1 expressing cells detected in the positive control of H1975, H460, and A549 cells of PD\u2010L1 expression for each cell was measured using the Leica Application Suite X and ibm spss statistics, version 25 . Fisher's exact test and Wilcoxon matched\u2010pairs signed rank test were performed and Kaplan\u2013Meier analysis was used to estimate survival curves; PFS was calculated from the day of the administration of the first cycle of pembrolizumab until the day of documentation of disease progression or death from any cause. Overall survival (OS) was calculated from the day of treatment initiation until the date of death from any cause. Cox regression analysis was performed to investigate the risk for progression and death. P values were calculated by two\u2010sided tests and were considered statistically significant at the 0.05 level.All statistical analyses were performed using 33.1+ or Ki67\u2212.For this purpose, the Mean Fluorescence Intensity per pixel (MFI) of PD\u2010L1 expression for each cell expressing PD\u2010L1 was measured by LASX and the mean intensity of PD\u2010L1 staining from 300 cells in each cell line was used to set\u2010up the thresholds for classifying CTCs as high, medium, low, or negative Fig.\u2009. The hig3.2n\u2009=\u200947), post\u2010first cycle (n\u2009=\u200943), post\u2010third cycle (n\u2009=\u200923), and progression/primary resistance (PMR), (n\u2009=\u200919) is depicted in Fig.\u2009A total number of 48 advanced NSCLC patients were enrolled at the Bank of Cyprus Oncology Centre (BOCOC). One patient was excluded from the study because RECIST response was not available. A flow chart showing CTC analysis of patients' samples at baseline (MR), n\u2009=\u20099 is depiMR), n\u2009=\u20099 is depi3.2.1+ CTCs were detected in 27 (61%) patients. The median % of Ki67+ CTCs were 20% (range: 0\u2013100%).At baseline, CTCs were evaluated in 47 patients and detected in 44 (94%) patients , PD\u2010L1\u2010positive CTCs were detected in 38 (86%) patients . PD\u2010L1high, med, low, and negative CTCs were detected in 24 (55%), 24 (55%), 23 (52%), and 22 (50%) patients, respectively. The median % of the respective subpopulations were PD\u2010L1high: 2%, med: 4.5%, low: 8%, negative: 1.5% (range: 0\u2013100%). Ki673.2.2+ CTCs were detected in 23 (64%) patients. The median % of Ki67+ CTCs were 20% (range: 0\u2013100%).At post\u20101st cycle, CTCs were evaluated in 43 patients and detected in 36 patients (84%), , PD\u2010L1\u2010positive CTCs were detected in 32 (89%) patients . PD\u2010L1high, med, low, and negative CTCs were detected in 17 (47%), 22 (61%), 19 (53%), and 21 (58%) patients, respectively. The median percentages of the respective subpopulations were PD\u2010L1high: 0%, med: 16%, low: 9.5%, negative: 17% (range: 0\u2013100%). Ki673.2.3+ CTCs were detected in 6 (35%) patients .At post\u20103rd cycle, CTCs were evaluated in 23 patients and detected in 17 (74%), , PD\u2010L1\u2010positive CTCs were detected in 13 patients (76%) . PD\u2010L1high, med, low and negative CTCs were detected in 4 (24%), 5 (29%), 9 (53%), and 7 (41%) patients, respectively. The median % of the respective subpopulations were PD\u2010L1high: 0%, med: 0%, low: 33%, negative: 0%, range: 0\u2013100%. Ki673.2.4+ CTCs were detected in 14 (82%) patients . The details (CTC number) of each patient at the selected time points are shown in Table\u2009For patients who experienced progression at first treatment evaluation (primary resistance), CTCs were examined in 19 patients and detected in 17 (89%) patients , PD\u2010L1\u2010positive CTCs were detected in 14 (82%) patients , PD\u2010L1high, med, low, and negative CTCs were detected in 6 (35%), 8 (47%), 13 (76%), and 9 (53%) patients, respectively. The median % of the respective PD\u2010L1 subpopulations were PD\u2010L1high: 0%, med: 0%, low: 33%, negative: 16%, range: 0\u2013100%. Ki67We subsequently, compared the CTC status between baseline and post\u2010first cycle, baseline, and post\u2010third as well as baseline and PMR. All comparisons were performed among CTC\u2010positive patients at both time points and analyzed by Wilcoxon\u2010matched pairs signed rank test. There were no significant differences in total CTCs, PD\u2010L1, and Ki67 subpopulations of patients between baseline and post\u2010first or baseline and post\u2010third cycle (data not shown). However, important changes in the PD\u2010L1 subpopulations were observed between baseline and primary resistance.n\u2009=\u200916), total CTCs were numerically increased; median number: 3 (range: 1\u2013150) at baseline versus 10.5 (range: 1\u20131094) at PMR, P\u2009=\u20090.4. The number of PD\u2010L1low CTCs was subsequently raised; median number: 0.5 (range: 0\u201335) versus 1.5 (0\u2013571), respectively, P\u2009=\u20090.1. Importantly, the percentage of PD\u2010L1low CTCs was significantly increased; median%: 2.5% (range: 0\u201360%) at baseline versus 36.5% (range: 0\u2013100%) at PMR, P\u2009=\u20090.03. Fig.\u2009Among CTC\u2010positive patients at both time\u2010points at baseline versus 0% (range: 0%\u201350%) at PMR, P\u2009=\u20090.008. No significant alterations were observed in the number and percentage of PD\u2010L1med, PD\u2010L1negative, and Ki67+ CTCs among CTC\u2010positive patients between the baseline and PMR.Furthermore, the number of PD\u2010L1high CTCs declined at PMR; median number: 1.5 (range: 0\u201349) at baseline versus 0 (range: 0\u201345) at PMR, Interestingly, different drifts of the PD\u2010L1 subpopulations were observed at primary resistance suggesting that changes in both PD\u2010L1high and PD\u2010L1low subpopulations may be involved.3.3To validate our results, we investigated CTC status between patients with primary resistance (PD patients) and DC, before and after the first cycle of treatment. Specifically, we examined changes in total CTCs, PD\u2010L1, and Ki67 subpopulations in PD and DC patients presenting CTCs \u2265\u20091 and CTCs\u2009>\u20091. CTC changes were classified as \u2018increased\u2019 and \u2018decreased or not changed\u2019. Specifically, if the CTC number increased by one or more cell after the first cycle, this was defined as an \u2018increase\u2019 in CTCs. Similarly, if the CTC number decreased by one or more cell after first cycle, this was defined as a \u2018decrease\u2019 in CTCs. However, significant changes were observed only among patients presenting CTCs\u2009>\u20091.P\u2009=\u20090.03 of PD patients versus 37% (4/12) of DC, .03 Fig.\u2009.P\u2009=\u20090.004, Fig.\u2009Interestingly, among the different PD\u2010L1 subpopulations, significant changes were observed only in the PD\u2010L1low CTC subset. Specifically, PD\u2010L1low CTC counts were more often increased in PD patients compared to DC: 67% (8/12) of PD patients versus 8% (1/12) of DC, P\u2009=\u20090.01; median number of PD\u2010L1\u2010positive CTCs: 2.5 (range: 0\u201333) versus 5 (range: 0\u2013998), respectively, P\u2009=\u20090.01; median number of PD\u2010L1 low CTCs:1 (range:0\u201310) versus 2.5 (range: 0\u2013184), P\u2009=\u20090.02, Fig.\u2009P\u2009=\u20090.7, and the percentage of PD\u2010L1low CTCs was numerically increased; median%: 14.5% (range: 0\u201360%) at baseline versus 19% (range: 0\u201371%) at post\u20101st cycle, P\u2009=\u20090.6.Furthermore, the number of total CTCs, PD\u2010L1\u2010positive, and PD\u2010L1low CTCs were significantly raised in PD patients at post\u20101st; median number of total CTCs: 3 (range: 2\u201334) at baseline versus 7 at post\u20101st cycle (range: 2\u20131022), P\u2009=\u20090.004, Fig.\u2009P\u2009=\u20090.09; median number of PD\u2010L1low CTCs: 4.5 (range: 0\u201335) versus 2 (range: 0\u20133), respectively, P\u2009=\u20090.007; median % of PD\u2010L1low CTCs: 25% (range: 0\u201367%) versus 3% (range: 0\u201350%) P\u2009=\u20090.049, Fig.\u2009On the other hand, DC patients more frequently decreased or not changed PD\u2010L1low CTC counts compared to patients with PD: 92% (11/12) of DC versus 33% (3/12) of PD, + subpopulations between PD and DC patients . Moreover, PD\u2010L1+ patients presenting a high Ki67 index (% Ki67+ CTCs >\u200930%) prior to treatment showed a significantly shorter PFS compared to those with a low , were PD\u2010L1P\u2009=\u20090.028; HR: 4.6, P\u2009=\u20090.04, Fig.\u2009P\u2009=\u20090.8, Fig.\u2009Furthermore, among patients displaying CTCs\u2009>\u20091, those with increased CTC counts at post\u20101st cycle had significantly shorter PFS compared to those with decreased CTC counts , Fig.\u2009P\u2009=\u20090.9, Fig.\u2009Interestingly, patients presenting a decrease in PD\u2010L1\u2010positive CTCs had a trend for longer PFS compared to those with increased PD\u2010L1\u2010positive CTCs , Fig.\u2009P\u2009=\u20090.2, Fig.\u2009Finally, patients showing a decrease in PD\u2010L1low CTC counts after the first cycle had significantly longer PFS compared to those with increased PD\u2010L1low CTC counts . The median PFS and OS of these patients were 13.3 and 44.7\u2009months, respectively. Figure\u2009Five of the above patients had achieved a stable disease at first CT evaluation, before experienced a subsequent disease progression or secondary resistance, whereas only two patients (N10 and N31) had shown partial response among all the patients who participated in the study. Patients N4 and N5 experienced disease progression at 17 and 24\u2009weeks, while patients N22, N16, and N3 experienced progression, at 40, 52, and 76\u2009weeks, respectively, after treatment initiation. Furthermore, patients N10 and N31 have not experienced any disease progression until recently.Interestingly, we observed that in patients with partial response (N10 and N31), CTC counts were immediately increased at week 3, whereas the PD\u2010L1low CTC rates were decreased Fig.\u2009. The perOn the other hand, in N4 and N5 patients, total CTC counts were peaked at week 21 (point of progression) and PD\u2010L1low CTC rates were also increased at this time point Fig.\u2009.In patients N3, N16, and N22 who continued to benefit from pembrolizumab treatment for more than 21\u2009weeks, fewer CTC counts were detected through the whole examination period Fig.\u2009 and the 4In the present study, we examined the differential expression of PD\u2010L1 and the proliferation marker Ki67 on CTCs in longitudinal samples of NSCLC patients treated with pembrolizumab. We validated our findings between patients with primary resistance and disease control before and after the first cycle of treatment. Furthermore, CTC changes were investigated in sequential samples of patients who obtained a long\u2010term benefit from treatment.We initially hypothesized that CTC evaluation in an early phase of pembrolizumab treatment, could reveal significant changes in PD\u2010L1 subpopulations which might be associated with resistance or response to immunotherapy. Interestingly in our cohort of patients, all cases who were characterized as progressive disease (PD) by RECIST had experienced primary resistance. Subsequently, the CTC status was examined at baseline, post\u2010first, post\u2010third cycle, and the point of primary resistance.6 PBMCs per patient, high CTC detection rates were observed. Several studies using different CTC isolation and detection methods have shown varied CTC detection rates, ranging from 50 to 100%. Specifically, in NSCLC, fewer CTCs can be detected with antigen\u2010dependent methods compared to antigen\u2010independent methods [In the current study, using the ficoll density gradient centrifugation, an antigen\u2010independent method, and evaluating 6\u2009\u00d7\u200910 methods . Krebs e methods utilizin methods , 27. WhiAnother interesting approach in our investigation was that CTCs were classified into four PD\u2010L1 subcategories . In general, cancer cells expressing PD\u2010L1 bind to PD\u20101 receptor on T cells and effectively inhibit T\u2010cell proliferation. However, it has been reported that engagement of different levels of both PD\u20101 receptor and its ligand PD\u2010L1 can modulate the strength of PD\u20101 signaling and affect a variety of T\u2010cell functions, such as cytokines and IFN\u2010\u03b3 production, TNF\u03b1, and IL\u20102 derivation as well as T\u2010cell expansion . ConsequIn the present study, no significant changes were found in the total CTC counts and PD\u2010L1 subpopulations between the baseline and post\u20101st (3\u2009weeks after treatment initiation) as well as between baseline and post\u20103rd cycle (9\u2009weeks after treatment). Our results are in line with a recent study of Ikeda et\u2009al. who repoInterestingly, we found a significant increase in the PD\u2010L1low CTC rate at primary resistance compared to that before treatment initiation Fig.\u2009, suggestPD\u2010L1 is a dynamic and inducible biomarker that can be induced or maintained by many cytokines of which interferon\u2010\u03b3 (IFN\u03b3) is the most potent. Activated T cells or innate immune cells can release IFNs and stimulate PD\u2010L1 expression. Moreover, the interaction between tumor\u2010infiltrating T cells, IFN\u03b3 signaling genes, and PD\u2010L1 expression suggests that activated T cells contribute to high levels of PD\u2010L1. Subsequently, we can assume that at the time of primary resistance due to the absence of activated T cells, no induction of PD\u2010L1 can be achieved. Therefore, it is logical to expect a decline in the PD\u2010L1 high CTC rate in patients with primary resistance. In support of our findings, Herbst et\u2009al., demonstrated a lack of PD\u2010L1 upregulation by either tumor cells or tumor\u2010infiltrating cells in tumor biopsies of early progressing NSCLC patients treated with ICI .Immune profile analysis of cancer cells categorized tumors into \u2018inflamed\u2019 and \u2018non\u2010inflamed\u2019 types. Recent clinical studies in patients with melanoma cancer suggest that the \u2018inflamed\u2019 but not the \u2018non\u2010inflamed\u2019 tumors are associated with response to PD\u20101 pathway blockade.Interestingly, PD\u2010L1low expression in cancer and tumor\u2010infiltrating cells has been associated with \u2018non\u2010inflamed tumors\u2019 that may express high EMT and stem\u2010like features and show no\u2010response to ICIs treatment . TherefoOn the other hand, it has been proposed that patients with \u2018inflamed tumors\u2019 display high levels of PD\u2010L1 expression on cancer and tumor\u2010infiltrating cells, harbor activated T cells and they are responsive to PD\u20101 pathway blockade. Consequently, it is logical to assume that these patients will be able to reduce PD\u2010L1low CTCs and may have a better clinical outcome.To validate our results, we further investigated CTC changes between patients with primary resistance (PD patients) and DC, before and after the first cycle of treatment. As PD\u2010L1 is an inducible dynamic factor that can change over time and treatment might affect its expression, it has been suggested that evaluation of PD\u2010L1 expression should be performed as close as possible to the initiation of treatment . TherefoInterestingly, after the first cycle, total CTC counts, PD\u2010L1\u2010positive and PD\u2010L1low CTCs more often increased in PD patients compared to DC Fig.\u2009. Our resAnother interesting finding in the present study was that PD\u2010L1low CTCs were decreased in almost all DC patients Fig.\u2009. This isOn the other hand, it has been supported that CTC detection rates as well as the harvested CTC subpopulations may be dependent on the methodology used for CTC collection . In thisAnother important point of our study was that PFS was significantly longer in patients with decreased total CTC counts after the first cycle, compared to those with increased CTCs. This is consistent with previous studies showing that a decline in CTCs after chemotherapy , 37 or IInterestingly, in another study, the authors reported that the presence of PD\u2010L1\u2010positive CTCs \u2265\u20091% of total cells in NSCLC patients before ICI treatment , 17 was Herein, we showed for the first time, that changes in the number of PD\u2010L1low CTCs at an early evaluation time\u2010point of pembrolizumab such as 3\u2009weeks after the beginning of immunotherapy, may have a predictive value for the treatment efficacy. This finding is further supported by our previous results that patients with disease control state and subsequently an active immune system can effectively reduce PD\u2010L1low CTCs. However, we found that decrease in the PD\u2010L1positive or PD\u2010L1low CTC rates was not associated with significant changes in the PFS of patients. This result could be explained by the changes in both PD\u2010L1negative and PD\u2010L1positive CTCs during treatment and by the fact that the rate of PD\u2010L1positive CTCs is dependent from the total number of CTCs detected .In order to investigate whether CTC changes were predictive for long\u2010term efficacy of treatment, sequential follow\u2010up samples of patients with a durable clinical benefit were examined. Our results revealed that partial responders (N10 and N31), who had a prolonged PFS and OS period , presented a rapid increase in CTC number just after the first cycle of treatment (3\u2009weeks after the beginning of treatment), while the total CTC counts declined in the following treatment cycles Fig.\u2009. In line+ cells or CTC clusters (2\u201350 cells) with increased metastatic potential [Accumulating evidence has also demonstrated that CTCs can mobilize from the tumor due to tissue disruption in an acute response to cancer treatment and isolotential .+ CTCs had no significant changes in the early phase of treatment compared to baseline. However, we demonstrated for the first time that PD\u2010L1+ patients presenting a low Ki67 CTC index (\u2264\u200930%) prior to pembrolizumab, had a significantly longer PFS compared to those with a high Ki67 (>\u200930%), and PD\u2010L1+ patients harboring Ki67+ CTCs had a significantly lower OS compared to those not presenting. Our results suggest that Ki67 may have a predictive and prognostic role in PD\u2010L1+ metastatic NSCLC patients prior to immunotherapy. This is further supported by the observation that PD\u2010L1+ patients who had a prolonged PFS period (PFS\u2009>\u200921\u2009weeks), displayed a low Ki67 CTC index during treatment showed a poor prognosis, whereas patients with a low Ki67 index (\u2264\u200930%) on tumor specimens, had a better clinical outcome , 44. In Another interesting observation was that PD\u2010L1low CTC rates of patients who continued to benefit from pembrolizumab were increased at a later phase of treatment Fig.\u2009 and thisOur results suggest that besides the evaluation of PD\u2010L1 expression by tumor biopsy, the differential expression of PD\u2010L1 and Ki67 on CTCs could provide further predictive information for NSCLC patients treated with ICIs. Nevertheless, our results and their interpretation, are subject to limitations. One limitation comprises the low CTC number detected in some patients and a further one, that significant changes between PD and DC patients were observed in patients presenting CTC number >\u20091. In addition, CTC detection was exclusively based on CK expression, eliminating the detection of mesenchymal CTCs. Our limitation of not using the hematopoietic marker CD45 in the antibodies panel, reflects the technical restriction of triple immunofluorescence assay in using up to three markers. Notwithstanding this, we confirmed the presence of CK\u2010positive/CD45\u2010negative cells, in patients presenting high CTC numbers.To verify the significance of the PD\u2010L1low subpopulation as a predictive factor, our hypothesis should be validated in a larger homogenous cohort of patients including different PD\u2010L1 antibodies and CTC enrichment methods. Moreover, it should be investigated whether changes in PD\u2010L1 expression on CTCs are related to changes in tumor biopsies. Although, it is not feasible to obtain serial biopsies due to the invasiveness of the procedure, the examination of tumor tissue at baseline and on disease progression is essential to be investigated for this issue.5+ patients.To summarize, this study investigates whether the differential expression of PD\u2010L1 and Ki67 on CTCs can provide further predictive information in advanced NSCLC patients treated with pembrolizumab. The quantification of PD\u2010L1 expression levels on CTCs and the evaluation of PD\u2010L1 subpopulations at different timepoints revealed that changes in PD\u2010L1low subpopulation at an early phase of treatment, are importantly related to disease control or resistance to pembrolizumab immunotherapy. Furthermore, the evaluation of Ki67 prior to immunotherapy and in sequential follow\u2010up samples of patients treated with pembrolizumab, can possibly have a predictive and/or prognostic value for PD\u2010L1Finally, our findings suggest that monitoring the PD\u2010L1 and Ki67 expression in CTCs of NSCLC patients treated with pembrolizumab, might improve the selection of those patients that would potentially benefit from this therapy, reduce the medical cost, tailor patient's treatment, and improve the quality of patient's life.HC declares research institutional funding from MSD, and also Advisory Board participation with MSD, Novartis, Pfizer, Ipsen, with all fees collected by his institution. HC also declares travel expenses in relation to Oncology meetings covered by MSD, Roche, and Pfizer in the last three (3) years. All other contributors declare no conflict of interest.MS participated in study design and coordination, performed PBMCs isolation, cell cultures, and CTCs characterization, immunofluorescence, analyzed the results, and drafted the manuscript. CMN participated in laboratory work and in the preparation of the manuscript. PV was involved in data analysis. IS participated in data analysis. GG performed immunostaining experiments. CD co\u2010supervised the work and was involved in the preparation of the manuscript. AIC coordinated and supervised the study and was involved in the preparation of the manuscript. HC designed, coordinated, and supervised the study and was involved in data analysis and interpretation.https://publons.com/publon/10.1002/1878\u20100261.13317.The peer review history for this article is available at Fig.\u2009S1. PD\u2010L1 expression in NSCLC cell lines before and after induction with IFN\u2010\u03b3 by western blot. Representative western blot of three independent experiments. Total lysate (40\u03bcg) of NSCLC cell lines was electrophoresed by SDS\u2010PAGE (7.5% gel) and immunoblotted for total PD\u2010L1. Western blot showing strong baseline expression of PD\u2010L1 in H1975 NSCLC cells, medium in H460 and low expression in A549 cells. Treatment with 100 ng/ml interferon\u2010\u03b3 (IFN\u2010\u03b3) for 24h upregulated PD\u2010L1 expression. The rabbit monoclonal antibody E1L3N was used to detect endogenous levels of total PD\u2010L1 protein and GAPDH served as a loading control.Click here for additional data file.Fig.\u2009S2. Expression of CK, PD\u2010L1 and Ki67 on H1975 (A) and H460 cells (B) spiked in peripheral blood mononuclear cells (PBMCs), by triple immunofluorescence staining. Cytospins of NSCLC cell lines spiked in PBMCs were used as positive and negative controls to evaluate the specificity of the antibodies used for the immunofluorescence staining. Cells were triple stained with pancytokeratin (CK) mouse antibody/secondary anti\u2010mouse FITC (green), anti\u2010PD\u2010L1 rabbit/ secondary anti\u2010rabbit Alexa Fluor 555 (red) and anti\u2010Ki67 rabbit 647\u2010conjugated antibody (grey). Cell nuclei were stained with DAPI (blue). The positive nuclear dotted staining (grey) was evaluated for Ki67 staining. Images were obtained using LASX, (x40).Click here for additional data file.Fig.\u2009S3. Expression of CK, PD\u2010L1 and Ki67 on A549 cells spiked in peripheral blood mononuclear cells (PBMCs) (A) and on NSCLC cell lines (B) by triple immunofluorescence staining. Cytospins of A549 cells spiked in PBMCs were used as positive and negative controls to examine the specificity of the antibodies used, whereas cytospins of H1975, A549 and H460 cells were used for the semi\u2010quantitative analysis of PD\u2010L1 expression. Cells were triple stained with pancytokeratin (CK) mouse antibody/secondary anti\u2010mouse FITC (green), anti\u2010PD\u2010L1 rabbit/ secondary anti\u2010rabbit Alexa Fluor 555 (red) and anti\u2010Ki67 rabbit 647\u2010conjugated antibody (grey). Cell nuclei were stained with DAPI (blue). The positive nuclear dotted staining (grey) was evaluated for Ki67 staining. Images were obtained using LASX, (x40).Click here for additional data file.Fig.\u2009S4. Double immunofluorescence staining with CK and CD45 antibodies on H460 cells spiked in peripheral blood mononuclear cells (PBMCs) and on PBMCs of NSCLC patients with high circulating tumor cell (CTC) number. (i\u2013iii) Cytospins of H460 cells spiked in PBMCs were used as positive and negative controls to examine the specificity of CK and CD45 antibodies. Cells were double stained with pancytokeratin (CK) mouse antibody/secondary anti\u2010mouse FITC (green) and anti\u2010CD45, Alexafluor\u2010647 conjugated (grey). (iv) Representative image of a CK+/ CD45\u2010 cell among PBMCs from a patient with high CTC number. Images were obtained using LASX, (x40).Click here for additional data file.Fig.\u2009S5. Setting\u2010up the thresholds in circulating tumor cells based on three NSCLC cell lines with different PD\u2010L1 expression levels. The mean fluorescence intensity per pixel (MFI) of PD\u2010L1 expression in A549, H460 and H1975 cell lines was determined by imaging and analysis system LASX . The mean intensity (m) of PD\u2010L1 staining in each cell line is used to define the thresholds for semi\u2010quantification and it was significantly different among the cell lines tested (*P<0.0001). The highest mean intensity was observed in H1975 cells , a medium intensity of PD\u2010L1 was observed in H460 cells (39 \u00b1\u20095.5) and the lowest intensity of PD\u2010L1 staining was observed in A549 cells (24 \u00b1\u20095.3).Click here for additional data file.Fig.\u2009S6. Expression of CK, PD\u2010L1 and Ki67 on circulating tumor cells (CTCs) of NSCLC patients by triple immunofluorescence assay. Representative images of phenotypically different CTC subpopulations. Different scenarios of PD\u2010L1 and Ki67 differential expression in CTCs are shown in panels: (a) PD\u2010L1high and Ki67+ CTC, (b) PD\u2010L1high and Ki67\u2010 CTC among peripheral blood mononuclear cells (PBMCs), (c) two PD\u2010L1med and Ki67+ CTCs among PBMCs, (d) PD\u2010L1med and Ki67\u2010 CTC among PBMCs, (e) PD\u2010L1low and Ki67+ CTCs, (f) PD\u2010L1low and Ki67\u2010 CTC beside PBMCs, (g) PD\u2010L1neg and Ki67+ CTC among PBMCs and (h) three PD\u2010L1neg and Ki67\u2010 CTCs. Cell nuclei were stained with DAPI (blue), Ki67+ CTCs are shown with grey. Images were obtained using LASX, a Leica imaging and analysis system (x63).Click here for additional data file.Fig.\u2009S7. Changes in circulating tumor cell (CTC) status before and after first cycle, according to PD\u2010L1high, PD\u2010L1med, PD\u2010L1neg and Ki67+ CTCs between progression disease (PD) and disease control (DC) patients. The increased vs decreased or not changed of each CTC status was compared between PD and DC patients. No significant differences were observed in (a) PD\u2010L1high (b) PD\u2010L1med, (c) PD\u2010L1neg and (d) Ki67+ CTCs between PD and DC patients. P\u2010value was calculated by Fisher's exact test.Click here for additional data file.Table\u2009S1. Circulating tumor cell (CTC) enumeration and characterization according to PD\u2010L1 and Ki67 in 47 NSCLC patients during pembrolizumab treatment. CTC evaluation according to PD\u2010L1 and Ki67 in NSCLC patients: (i) at baseline, n=47 patients (ii) post\u2010first cycle, n=43 (iii) post\u2010third cycle, n=23 and (iv) at primary resistance, n=19 patients with disease progression at first evaluation of treatment . Patient's response according to RECIST criteria . Results are expressed as CTCs/6x106 PBMCs.Click here for additional data file.Appendix\u2009S1Click here for additional data file."} +{"text": "The approval of monoclonal antibodies against programmed death-ligand 1 (PD-L1) and programmed cell death protein (PD1) has changed the landscape of cancer treatment. To date, many immune checkpoint inhibitors (ICIs) have been approved by the FDA for the treatment of metastatic cancer as well as locally recurrent advanced cancer. However, immune-related adverse events (irAEs) of ICIs highlight the need for biomarker analysis with strong predictive value. Liquid biopsy is an important tool for clinical oncologists to monitor cancer patients and administer or change appropriate therapy. CTCs frequently express PD-L1, and this constitutes a clinically useful and non-invasive method to assess PD-L1 status in real-time. This review summarizes all the latest findings about the clinical significance of CTC for the management of cancer patients during the administration of immunotherapy and mainly focuses on the assessment of PD-L1 expression in CTCs. Minimally invasive liquid biopsies allow the analysis of tumor elements, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) in body fluids, mainly blood. The liquid biopsy concept launched approximately 10 years ago, has opened new horizons in cancer prevention, diagnosis, early identification of tumor recurrence, molecular characterization of tumors, monitoring of response to treatment, and detection of resistance mechanisms . Indeed,The Food and Drugs Administration (FDA)-cleared CellSearch platform represents the gold standard for CTC detection and enumeration in the bloodstream . CTC enuPD-L1) expression, which is assessed by immunohistochemistry in tumor specimens [PD-L1 in both tumor and immune cells, complicate the accurate measurement of PD-L1 expression in tumor tissues [In recent years, the promising results yielded from the development of CTCs in targeted therapies have paved the way for the implementation of CTCs into the domain of immunotherapy. The most important biomarker for treatment decision-making in the era of immunotherapy is programmed death-ligand 1 , and metastatic colorectal cancer (MCC) using CellSearch technology enables the monitoring of cancer patients during therapy. Moreover, this technology captures and identifies tumor cells in the blood that are associated with poor clinical outcomes . C\u03a4C couAn additional channel in the CellSearch system allows the examination of a fourth molecule of interest beyond the detection of cancer cells of epithelial origin. Establishment of the B7-H1/PD-L1 CTC analysis was performed for the first time by Mazel et al. showing that PD-L1 is frequently expressed on metastatic cells circulating in the blood of hormone receptor-positive, HER2-negative breast cancer patients . This st+CTCs all experienced progressive disease, suggesting that the persistence of PD-L1+CTCs might mirror a mechanism of therapy escape [Nicolazzo et al. showed that in NSCLC patients treated with the programmed cell death protein (PD-1) inhibitor nivolumab at baseline and at 3 months of treatment, the presence of CTCs and the expression of PD-L1 on their surface is associated with poor patients\u2019 outcome. Moreover, 6 months after treatment, patients harboring PD-L1 negative CTCs obtained a clinical benefit, while patients with PD-L1y escape .+CTCs, while PD-L1 expression in tumor tissue failed to prove any prognostic significance [\u00ae technology, provides prognostic information and may improve the diagnostic accuracy of malignant pleural effusion (MPE) [Sinoquet et al. have shown similar results concerning the worse outcome of PD-L1ificance . Apart fon (MPE) .+ on CTCs in mPC patients during the administration of next-generation AR axis inhibitors is feasible and may enable monitoring of immunotherapy [In mPC, immunotherapy against immune checkpoint inhibitors (ICIs) seems to be effective. For this purpose, identifying suitable biomarkers could facilitate the selection of the best candidates for this therapy . Expressotherapy . The expotherapy .+CTC could also be applied in patients with Merkel cell carcinoma (MCC), which is a rare, aggressive skin cancer with increasing incidence and high mortality rates. Riethdorf et al. show that even though a high prevalence of CTC occurs at first blood collection that is associated with a worse prognosis, the overall frequency of PD-L1 production in CTCs is very low [The assessment of PD-L1very low .In recent years, immunotherapy has become the first-line treatment for patients with NSCLC, with excellent responses in many patients . HoweverGuibert et al. prospectively analyzed blood samples from 96 advanced-stage NSCLC patients obtained before nivolumab treatment and at the time of disease progression . PD-L1 ep = 0.0091) and OS (p = 0.0410) versus patients who did not demonstrate an increase in PD-L1 expression or the no ICI-treated population [The examination of PD-L1 status through sequential biopsies could provide significant prognostic and predictive information due to status changes over time. A longitudinal evaluation of PD-L1 expression of CTCs isolated from NSCLC patients treated with nivolumab was reported by Ikeda et al. CTCs were enriched from 3 mL of peripheral blood using a microcavity array system at baseline and weeks 4, 8, 12, and 24 or until progressive disease. According to this study, PD-L1 expression on CTCs at week 8 has a superior predictive value compared to that at the baseline . In thispulation . A longipulation .+ on CTCs could still benefit from ICI therapy, while co-identification of PD-L1+CTCs or PD-L1+ tissues may help to identify patients who would benefit from immunotherapy [PD-L1 expression presents heterogeneous expression in CTCs and tumor tissues from advanced NSCLC patients. Zhou et al. show that CTCs release a higher detection rate of PD-L1 expression than tumor tissues (53.0% vs. 42.1%). Moreover, NSCLC patients with PD-L1\u2212 on tissues but PD-L1otherapy .+CTCs, while no change or a decrease in PD-L1+CTCs is observed in responding patients [+CTCs might indicate resistance toward PD-1/PD-L1 inhibitors. Similar results were shown by Sinoquet et al., where OS was significantly worse in NSCLC patients with PD-L1-CTCs and particularly in patients with PD-L1+CTCs compared with patients without CTCs. Moreover, the presence of PD-L1+CTC correlated with the absence of gene alterations in tumor tissue and with poor prognosis-related biological variables [Enough data support the fact that upon disease progression, NSCLC patients demonstrate an increase in PD-L1patients . Additioogenase) .PD-L1 expression has also been studied in groups of patients receiving other types of treatment besides immunotherapy. Wang et al. studied gene expression of PD-L1 in CTCs isolated before, during, and after radiation or chemoradiation using a microfluidic chip. PD-L1 mRNA was highly expressed in patients who had disease progression within 9 months compared to those who had stable disease for 9 months or more, indicating that radiation therapy induces PD-L1 expression in CTCs .p < 0.0001) and OS than those without PD-L1-high CTCs [+CTCs might predict better survival for patients receiving ICI treatment but worse survival for patients receiving other therapies. In addition, post-treatment PD-L1+CTCs were correlated with worse survival in cancers [The dynamic probability of PD-L1 as a surrogate marker has also been analyzed in multiple basket studies. Tan et al., in a study involving one hundred fifty-five patients with different advanced cancers, showed that the reduction in CTC counts and ratios of PD-L1-positive CTCs and PD-L1-high CTCs reflect a beneficial response to PD-1/PD-L1 inhibitors. In this study, patients with PD-L1-high CTCs had significantly longer PFS , and the PD-L1 positivity in the CTCs was found to be significant [In head and neck squamous cell carcinoma (HNSCC), the administration of immunotherapies has led to a response rate equivalent to 15\u201320%. However, ICIs have been approved for recurrent and metastatic (R/M) HNSCC patients as a first- and second-line therapy ,62. Rece 0.0485) .PD-L1 mRNA expression in EpCAM(+) CTCs has been developed by Strati et al. for the detection of PD-L1 overexpression in CTC. This prospective study enrolled 113 locally advanced HNSCC patients treated with curative intent at baseline, after two cycles of induction chemotherapy (week 6), and at the end of concurrent chemoradiotherapy (week 15). The findings of this study showed that patients with CTCs overexpressing PD-L1 at the end of treatment had worse outcomes , while its absence was strongly associated with complete response [A highly sensitive, specific, and robust RT-qPCR assay for = 0.002) .Immunotherapy represents a promising therapeutic option for the cure of prostate cancer patients . In a phMazel and co-authors were the first to report the expression of PD-L1 on CTCs of patients with ER(+) HER2(\u2212) breast cancer (BC) . Interes+CD45\u2212CK+ was higher in TNBC compared to luminal patients. Furthermore, among TNBC patients, there was an association of the phenotype PD-L1+CD45\u2212CK+ with a shorter OS [Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that molecular targeted therapies are lacking. Immunotherapy has been included as standard care for stage II-III TNBC . Vardas R = 8.7) +CTCs had a higher response rate to pembrolizumab, as well as longer PFS compared with patients with PD-L1-CTCs [The high immunogenicity of melanoma cancer makes immunotherapy one of the most effective treatment strategies . Molecul= 0.018) .+, and low CD4 and CD8 T-cell counts had shorter survival [Chalfin et al. evaluated the T-cell counts and CTC morphologic features of metastatic genitourinary cancer patients receiving combination immunotherapy at baseline and on therapy at cycle 2 and cycle 3. Five distinct morphologic subtypes were identified by calculating the Shannon Index, and increasing CTC heterogeneity during therapy administration was associated with worse OS. Moreover, patients with CTCs > 4, specific CTC morphologic subtypes, PD-L1survival .Immunological response to bladder cancer is well conserved, and PD-L1 expression is differentiated between high-grade and low-grade cancers . Morelli+CTCs was found to be an independent predictive biomarker of OS, and the objective response was more likely to be achieved in patients with a dynamic decrease in PD-L1+CTC counts at 1 month after treatment [In hepatocellular cancer (HCC), Su et al. investigated the predictive value of PD-L1 expression on CTCs in patients receiving PD-1 inhibitors combined with radiotherapy and antiangiogenic therapy. The count of PD-L1reatment .CTCs acquire key properties required for metastatic spread and constitute an intermediate stage of metastasis . They ex8+ T-cells and activation of tumor-specific T-cells in peripheral blood, while at the same time, CTCs were diminished in 6/8 (75%) of baseline-positive patients [NK cells are of major importance in host immunity against cancer. Several different approaches to NK-based immunotherapy have been reported . Allogenpatients . A singlpatients . Recent patients .+GM\u2212CSF vaccination was applied in 16 patients with HER2/neu-expressing primary breast cancer, while thirteen of the 16 patients (81.3%) had at least one HER2/neu+CTC (mean: 2.1 \u00b1 0.1 CTC/20 mL) in the peripheral blood. After vaccination, a reduction in CTC/20 mL and HER2/neu+CTC/20 mL was demonstrated [TAMs are the most frequent immune cells within the tumor microenvironment . Sialic nstrated .In-vitro experiments have shown that immune activation of the monocyte-derived dendritic cells (Mo-DCs) using patients\u2019 own CTCs is feasible. Kolostova K. et al. performed a co-culture of mature Mo-DCs (mMo-DCs) and autologous non-target blood cells (NTBCs). The activation effect of mature Mo-DCs on T-cell activation was monitored using multimarker gene expression profiling. Moreover, mMo-DCs might play a significant role in the PD-L1/PD1 regulatory axis since an elevated gene expression of PD-L1 was observed .Liquid biopsy represents a novel, non-invasive approach for detecting and monitoring cancer through the analysis of its biological components, such as CTCs. The main challenge of the liquid biopsy era is the primary detection of minimal residual disease (MRD), where cancer cells, disseminated from the primary tumor, are non-detectable with conventional clinical or radiological tests, increasing the probability of new tumors formation of high metastatic potential. Sensitive and specific isolation and detection of CTCs is very important, especially in the case where surgical removal of the tumor is difficult. In this case, the information from the tumor cannot be available, and thus, oncologists do not have the proper guidance for the correct administration of targeted therapy to the patients.Immunotherapy activates the body\u2019s immune system to destroy cancer cells by enhancing the recognition ability of immune cells to the surface antigens of tumor cells, achieving their elimination. PD-L1 is a critical immune checkpoint protein that binds to PD-1 in T cells. ICIs are blocking the PD-1/PDL-1 interaction enabling immune system attack and sequentially destroying the cancer cells. That being said, it highlights the necessity of technologies that can accurately determine and assess the status of PD-L1 biomarkers and guide clinical oncologists as to whether cancer patients are suitable for immunotherapy. However, larger clinical studies are needed to be performed for the evaluation of the PDL1 status of CTCs and the integration of the PD-L1-CTC test into daily clinical practice."} +{"text": "Oral diseases have been associated with cardiovascular diseases, and persons with continuous positive airway pressure [CPAP]-treated obstructive sleep apnoea [OSA] have an increased risk for negative consequences for both oral and general health. CPAP treatment is often life-long and adherence to treatment is essential. Xerostomia is a common side-effect which can lead to treatment abandonment. Oral health is a changeable part of our general health and well-being and exploring the views of oral health determinants from persons with experience of CPAP-treatment is important to prevent adverse oral health outcomes. The purpose of this study was to explore what persons with experience of CPAP-treated OSA view as determinants for their oral health.Eighteen persons with long-term experience of CPAP-treated OSA were purposively selected. Data were collected by semi-structured individual interviews. A code book based on the World Dental Federation\u2019s [FDI] theoretical framework for oral health was developed and used to analyse the data using directed content analysis. The domains in the framework\u2019s component driving determinants were used as pre-determined categories. Using the description of driving determinants as a guide, meaning units were extracted from the interview transcripts through an inductive approach. Then, by employing a deductive approach the code book was used to categorise the meaning units into the pre-determined categories.The views on oral health determinants described by the informants were compatible with the five domains in the component driving determinants in the FDI\u2019s theoretical framework. Ageing, heredity, and salivation , influences from family and the wider society , location and re-localisation , oral hygiene habits, motivation, willingness to change, professional support , and availability, control, finances, and trust (access to care) were viewed as important oral health determinants by the informants.The study points to a variety of individual oral health-related experiences that oral healthcare professionals could consider when designing interventions to reduce xerostomia and prevent adverse oral health outcomes for persons undergoing long-term CPAP-treatment. Recently, the World Dental Federation [FDI] proposed a new definition and theoretical framework of oral health, intending to move dentistry from the traditional focus on treatments towards oral health promotion and support . The FDICharacterised by repeated episodes of physical obstructions in the upper airway, OSA is defined as a sleep-related breathing disorder. Risk factors for OSA include obesity/overweight, male sex, postmenopausal state in women, and older age , and it Untreated OSA can affect the quality of life as well as increase the risk for conditions such as systemic hypertension, stroke, and type II diabetes, which makes adherence to treatment essential \u201316. DiffBy using the FDI\u2019s framework to explore oral health determinants in a specific population with an increased risk (i.e. due to CPAP-treated OSA) for negative consequences regarding both general and oral health, we can explore how persons with experience of CPAP-treatment view factors that affect their oral health. Creating a common ground for communication could help oral healthcare professionals and CPAP-practitioners to provide person-centred care and could enable shared decision-making within dentistry \u201329.To explore what persons with experience of CPAP-treatment view as determinants for their oral health.A study with a descriptive and explorative design was adopted and employed, using a qualitative method for data collection and analysis. Data were collected by semi-structured interviews and analysed by directed content analysis [DCA] , 31. GuiGenerally, an improved oral health in the Swedish population during the last 40\u00a0years has been reported . Since tThe FDI\u2019s entire framework illustrates the components of oral health and how complex interactions among driving determinants, moderating factors, and overall health and well-being, together form our oral health, Fig.\u00a0N\u2009=\u2009121). The examinations were conducted by an experienced dentist and dental hygienist (H.A.), during November 2018 to June 2019. After participating in the initial dental examinations in the project, all hypertensive persons diagnosed with OSA in 2007\u20132009 with ongoing or previous experience of long-term CPAP-treatment (>\u20091\u00a0year) were asked to participate in the current study (n\u2009=\u200942). The informants were interviewed once by using an interview guide designed to capture two different perspectives on oral health in relation to CPAP-treatment and OSA; the present perspective, focusing on views on oral health determinants, and another perspective focusing on critical incidents related to oral health , I try to think that it is, (\u2026) both the CPAP and becoming older (\u2026). The informants described how the changing circumstances they experienced during life and their consequences affected their oral health in different life stages. From the prenatal stages to the effects of ageing, both consequences that appeared instantly and those that became evident later in life were described. By relating to tooth position and tooth development, the early life stage was described as an oral health determinant that could affect them throughout life. Ageing per se was also viewed as a determinant, which was exemplified with experiences of increased crowding, tooth wear, and xerostomia in older age. Some considered both their CPAP-treatment and becoming older as part of the cause for their xerostomia.(\u2026) there are those around me (\u2026), who are extremely careful with their teeth, and brush and keep going on, (\u2026) who have much worse teeth than I've ever had. So, it's not just how you take care [of your teeth] but a little bit of luck and what you have inherited as well. By comparing their own oral health to that of their immediate family and persons in their surroundings, the genetic predisposition was experienced as a determinant. In their experience, some risk factors were more easily modifiable than others, and protective factors such as oral hygiene and dietary habits were factors that could balance a disadvantageous genetic heritage. Based on different genetic predispositions, they assumed that some had a better heritage or were luckier than others.(\u2026) That you get more oral dryness [when using the CPAP], and it is not the same\u2026maybe it releases a bit more\u2026as everything dissolves. (\u2026) and then I wonder if they [the restorations] release more if they are drier or if they calm down and release more if they are wet? (\u2026) The informants considered saliva to affect their teeth and mouth, even if the views on cause and effect of for example xerostomia varied. Experiences of xerostomia during CPAP-treatment could make them contemplate about how saliva could affect their oral health. They also expressed ambivalent thoughts on the cause of their xerostomia; was it due to mouth-breathing during CPAP-treatment or part of becoming older? Their views on the influence of saliva as a determinant were also expressed by making assumptions or perceiving themselves to have a lack of knowledge.Views on the social environment from throughout life were described, even if most were derived during childhood. The informants also described close relationships such as the child-parent relationship and influences from the surrounding community in different stages in life as oral health determinants.(\u2026) you don\u2019t have to go further than to your own children or grandchildren and compare their oral health to mine. It\u2019s a big difference. (\u2026) But it [oral hygiene habits] can vary between families, I don\u2019t know, I only know how it is with us. (\u2026) Experiences from the informant\u2019s own upbringing and how it had affected their oral health in adulthood included descriptions of how beneficial or inappropriate oral health care habits were formed during their childhood. Habits obtained in childhood were often viewed to be of major importance for their oral health in adulthood. During adolescence, the social environment also included peers, but their families were still influential. They also described how the social context in their childhood neighbourhood affected their parent\u2019s views on oral health through values in the community, and they said that this contextual effect had changed over time. Becoming a parent or grandparent also made them acknowledge possible differences among families, and how the social environment has contributed to differences among family members. In adulthood, the family constitution had changed but was still viewed as influential, which was exemplified by buying candy from grandchildren or letting their partner make decisions regarding oral hygiene products.. (\u2026) It\u2019s not that easy today because we are overwhelmed by commercials for a lot of stuff that is not healthy. UnfortunatelyThe societal influence from oral health campaigns and advertisements, for example on social media or television, was viewed by the informants to be influential on their habits and choices. They viewed public oral hygiene or dietary campaigns (e.g. Saturday candy and tooth-brushing twice a day) as influential and described how advertisements could affect their choice of oral hygiene products, especially if the product was promoted by an oral health professional. They acknowledged the wider societal impact on habits such as advertisements of adverse health products in the society, and how making healthy choices nowadays could be difficult.Oral health determinants related to the physical environment were viewed to have been influential in different ways during their life. Both the actual location and the characteristics of the location were described as determinants, and even a small or inevitable change could be experienced as having a significant impact on their oral health.(\u2026) where I grew up, where I live now as well, there has always been a lot of fluoride (\u2026). It could have been good; it could strengthen the teeth. But then it has been a couple of years in between (\u2026). That it [dental caries] has developed\u2026I have gotten dental caries (\u2026) The informants identified different ways the local environment in their childhood could be viewed as an oral health determinant. Having natural high levels of fluoride in drinking water during childhood was viewed as positive, but also recognised as an increased risk factor for dental fluorosis. Moving away from that location was also considered a risk for development of dental caries. They also acknowledged the influence of growing up in a rural instead of an urban area and said that the differences regarding the local community\u2019s efforts to provide access to oral healthcare clinics and to offer fluoride rinses in school, depended on where they lived.Yes, but more or less [xerostomia]. But it can be worse some days, some mornings it can be worse (\u2026) for example, if you don\u2019t have the window open, one night, it can be the fresh air, that\u2019s not bad either, it comes in [the bedroom] in one way or another. (\u2026) Changes were described to occur both when various conditions changed in their immediate environment, and when they changed location. CPAP-related xerostomia could for example either increase or decrease in impact due to changes in the local physical environment, and even small changes could have a major impact on their experience. Changes in air humidity, due to seasonal changes or when changing location, were experienced as affecting their xerostomia. The descriptions included experiences of increased xerostomia when using the CPAP during autumn/winter due to decreased air humidity or to frequently having a blocked-up nose. This made it difficult to use the CPAP and thus the xerostomia increased. Circumstances in the surrounding environment were also experienced as varying between different locations, where locations with higher levels of air humidity could decrease the xerostomia. Changing location was also experienced as influencing their oral hygiene habits negatively and made it more difficult to find an oral healthcare clinic if needed.The subcategory Health behaviour in this context regarded specifically oral health behaviour and was described in several ways, from creation of oral health habits to maintaining them over time. This was related to the informant\u2019s willingness to make changes when needed and the support they experienced from oral health care personnel. They also described both facilitators and barriers regarding their oral health habits, and how they maintained or improved their oral hygiene during CPAP-treatment.(\u2026) But I think I heard it from someone that you either have one or the other [dental caries or calculus]. However, sometimes I have noticed that you have both, you [I] had both cavities and calculus (\u2026) The informants described that their oral health habits , were formed based on their understanding or knowledge of oral health, and some relied on their formal education within healthcare or oral healthcare. The views on how they gained and viewed their knowledge varied among the informants, but making healthy choices was mostly viewed as important. When they described how their everyday habits were formed, they also weighed previous experiences of negative outcomes against their experienced beneficial habits, such as being a non-smoker, using fluoride toothpaste or an electric toothbrush, and their dietary habits. Relying on hearsay from others in their surroundings sometimes led to increased uncertainty when their views based on hearsay from others contradicted their own experiences.(\u2026) if you [I] had received it [the information] during my 20s you know, it would probably have affected [my oral health] in a different way\u2026 (\u2026) (pause). Yes, it affected it [oral health], that I am convinced of, but then if you are receptive to it [the information] it\u2019s like that. Maybe I wasn\u2019t. It\u2019s possible that someone tried, one [I] was not receptive to it [the information] The received information or recommendations from oral health care personnel throughout their life was described as an oral health determinant. It was not always the content of the information, but the experience of getting help or how it was given and received that had influenced their oral health. Receiving support made them view themselves and the oral health care personnel as a team working together, or it could make them feel safe. Receiving feedback on their efforts was viewed as beneficial for their oral health. Support was also described as a feeling that the personnel would not give up on them or that they were thorough, but they also described their own role in the encounter. While some experienced that they had not been pushed enough, others experienced that they had done what they were told or their best, but it did not work. Some described how lack of support or negative experiences affected them as well and described experiences of dentists scolding them or the hygienist whining.(\u2026) Brushing your teeth is, as you say, in the bone marrow, I have always done it (\u2026) The informants also described facilitators and barriers in daily life and what motivated them to maintain their habits. The motivation for maintaining adequate oral hygiene habits was often the perceived importance of having good oral health, or the positive sensation within the mouth. This motivation provided oral hygiene habits that felt habitual or a natural part of their daily routine. Lack of motivation was not always the reason for not maintaining adequate habits as other factors acted as barriers. Experiences of feeling nauseous or being uncomfortable when using oral aids, were viewed as ongoing barriers which were hard to overcome. Other barriers could be managed, and the solution was often to make their oral hygiene habits a part of their everyday life, and they incorporated them in other daily activities, for example by performing tooth-brushing or interdental cleaning in other locations than the bathroom. But there were also those who were aware that they could do better but lacked the motivation to maintain or improve their oral hygiene habits, and who described themselves as careless, forgetful, or too comfortable.Yes, I have, it is probable that I have understood that there are some risks that when you\u2026 with the CPAP the oral hygiene could deteriorate. (\u2026) The informants were often willing to make changes to improve their, oral health habits when they experienced a negative change in their oral health. CPAP-treatment was described as having increased their awareness of their oral health, and the increased xerostomia made them actively change their oral hygiene or dietary habits to decrease the risk of oral disease or loss of teeth. Even if they experienced a need to make changes, some actively chose not to, while others were more passive by not thinking about their oral health. They also described their willingness to take responsibility for their oral health, which was described by claiming that they did their best or by justifying their choice to not make any changes. Sometimes they were willing to make changes, but due to their general health it was impossible. Being aware of potential negative side-effects of for example CPAP-treatment or noticing an improvement when making a change was described as increasing their willingness to maintain the changes they made.The informant\u2019s views on having access to oral healthcare included other aspects than availability by having a clinic nearby. They also described how for example control, trust in oral health care personnel, and financial or organisational aspects were viewed as elements of access to care.Otherwise, it is better to go to the dentist a bit more often, then you have more control, you know. (\u2026) It is important. Access to care was considered by the informants to be demonstrated by being offered and accepting the offer to visit an oral healthcare clinic, regularly or when needed. Their childhood experiences varied from lack of regular attendance due to lack of availability, to experiences of being a regular attender from an early age which could be related to their parents\u2019 education or profession. Being offered and accepting the offer of regular visits was experienced as facilitating preventive or promotive oral health care and acute visits. Due to lack of availability (e.g. moving around or no clinics in the area), some described previous difficulties facilitating regular visits as adults and only made acute visits. Confirming that one was a regular visitor or choosing to postpone visits were expressed as ways to have control of their oral health. Relying on the clinics\u2019 regular re-call system to send a reminder within pre-decided time intervals, and thereby handing over control of their oral health, was described. However, this could lead to negative feelings such as disappointment or loss of control if their expectations were not met. Negative experiences in the dental encounter could lead to rejecting an offered appointment or changing oral health care provider to take back control of their oral health. Becoming a regular visitor after years of non-attending was experienced as improving oral health as not being a regular visitor was associated with deteriorated oral health. However, some challenged this view, as being a regular attender per se does not provide any information regarding the quality of care.(\u2026) But I can also understand why some people don\u2019t go, it\u2019s too expensive. That\u2019s why I think it should be included in the regular health insurance system because it is equally as important as the rest of the body (\u2026) Postponing visits was experienced by the informants to lead to more expensive treatments, and regular visits were therefore viewed as more economical. However, no-one said that they had to postpone oral health care due to financial reasons but showed an understanding for those who had to. The cost for oral health care was viewed as inevitable; postponing visits only meant that you paid for treatments instead of preventive care. In relation to this, some of them also questioned the organisational and financial separation of oral and general healthcare. Financial aspects also included experiences of greedy dentists who cared more about making money, as well as organisational aspects regarding for example lack of dental hygienists or having to see a new dentist at every visit.A treatment like that as it was back then. It was forced treatment, because they tied one [me] to the dental chair to make me sit still. And then it was\u2026.and the drills (\u2026) they kept going until there was smoke, now it is water cooling so now it doesn\u2019t feel like that. The old drills were driven by big straps in the ceiling, it was a terrible appliance. Just to see the equipment made you, [I felt I] almost died. Having a trusting relationship with oral health personnel was viewed by the informants as important by many of the informants in this study. Experiences from childhood often included descriptions of terrifying visits, which could affect their trust in oral health care personnel for a while in adulthood.However, fearsome experiences as a child or adult, such as harsh dental professionals, and painful or uncomfortable treatments, did not impact their will to visit a clinic today as they had regained trust in their oral health care personnel. Most of them did not view themselves as afraid or anxious, and described themselves as regular visitors, which some related to their ability to adapt and thereby be able to overcome previous experiences. For some, the lack of trust in oral health care personnel made them consider or did change clinic. Meeting the same personnel was described as important for some of them, as it provided continuity and accountability. Some of them said that their trust was restored in adulthood as the oral health care personnel were experienced as thorough, better educated within their area of expertise, and more caring nowadays. Trust was also fostered by having the possibility to participate in the planning of their care, which was also related to having a trusting relationship.To the best of our knowledge, this study is the first to explore oral health determinants from the perspective of persons with long-term experience of CPAP-treated OSA by using the FDI\u2019s theoretical framework of oral health. The FDI\u2019s definition and theoretical framework was usefEarly life experiences, genetic predisposition, and saliva were viewed as influential oral health determinants which could have immediate consequences or affect the informant\u2019s oral health throughout life. Their views correspond to the consensus report on the interactions among lifestyle, behaviour, and systemic and oral diseases , where gThe community and the immediate family were described as influential throughout life on the informants\u2019 oral health, from childhood until today. The parental influence and communicative behaviour have previously been shown to affect children\u2019s understanding of oral health and their oral health behaviour , and in Besides the views on influential factors in the informants\u2019 childhood, changes in the local environment in adulthood during CPAP-treatment were viewed to have an impact on their oral health, such as the experience of xerostomia. They connected this with changes in air humidity and increased prevalence of having a blocked-up nose due to seasonal changes. Previously, seasonal changes (i.e. air humidity) have been reported to affect CPAP-adherence in a Japanese population where moMotivation and willingness to change affected the informant\u2019s behaviour, where support from oral healthcare personnel contributed either as a facilitator or barrier. CPAP-treatment was viewed to have increased their awareness of their oral health and therefore changed their oral hygiene habits to prevent adverse oral health outcomes. As CPAP-treatment is often life-long, it is important for CPAP-users to maintain positive changes in oral health behaviour for a long time. Previously, it has been reported that several factors can affect a person\u2019s ability to maintain adequate oral hygiene with increasing age . The infBased on the findings in this study some recommendations for clinical practice can be highlighted. The knowledge provided in this study could be useful in both identifying oral health needs as well as tailoring oral healthcare to this specific population. Person-reported information regarding oral health related barriers and facilitators for CPAP-treatment adherence is therefore significant. For oral health care personnel, it is important to create trustful relationships to promote beneficial oral health habits including regular visits over time, further implement shared-decision-making, and increase interprofessional collaboration with CPAP-practitioners. For CPAP-practitioners, identifying oral health-related side-effects of CPAP-treatment and providing adequate recommendations can be a challenge as studies on the patient perspective on oral health during CPAP-treatment are scarce. Therefore, it is suggested that future studies focus on the patient perspective on real-life oral health-related experiences before and during CPAP-treatment. However, until then, CPAP-practitioners could ask their patients regarding known side-effects such as xerostomia as a first step to include oral health within CPAP care.Finally, some methodological considerations should be noted. All persons eligible to participate in the interviews were invited to participate. Of the potential 42 informants, 18 persons chose to participate which was regarded as sufficient based on the predetermined aim of 15\u201320 informants. The informants had previously participated in research projects and were familiar with the research context. In addition, the informants had also met the interviewer in person before and/or been in contact with her per telephone which was clarified in the information letter. In our study, this enabled in-depth descriptions and had a positive effect on the informants\u2019 willingness to share their experiences. By the initial inductive approach, all views from the informants were considered for inclusion. The following deductive coding process allowed us to describe the determinants in a comprehensive way while still maintaining focus on the views described by the informants. Although face-to-face interviews, as\u00a0originally planned, would have been optimal, telephone interviews are common and accepted as a method for data collection . But eveThe qualitative data reflected the informants\u2019 views on oral health determinants, which were related to all five dimensions of the FDI\u00b4s driving determinants for oral health. Long-term CPAP-treatment can increase the awareness of one\u2019s oral health and improve oral hygiene habits, but can also induce xerostomia, and can therefore be considered an oral health determinant that interacts with other determinants related to the physical environment and health behaviour. The study points to a variety of individual oral health-related experiences worth considering when designing interventions to reduce xerostomia and prevent adverse oral health outcomes for persons with long-term CPAP-treatment."} +{"text": "The multidisciplinary and comprehensive nature of children`s oral health with mutual interactions among various determinants makes the area a window of more discussion among oral health policymakers, stakeholders, providers, and other interested parties. This commentary presents a triangle framework of the children`s oral health, including all the above groups, for new discussions in oral health policymaking.Families and community, determine the individual background, demographic, biological, genetic, and psychological factors, as well as community-based and social background, including cultural and socioeconomic factors. The second angle, Oral health providers, includes a variety of determinants from the provider`s perception toward oral health provision of services to availability of dental services, teledentistry and digital technology, surveillance, and monitoring systems for children`s oral health. And finally, as the third angle, Oral health policymakers affect the mechanism for funding dental care and supporting schemes, affordability of oral health services, regulations and standards and public education. Macro environmental policies related to the children`s ecosystem, community water fluoridation, and social marketing for promoting probiotics products` consumption are categorized in this category.Three leading influencers could be recognised in children`s oral health as a triangle despite the contextual differences among the countries. The first angle, The triangle framework of children`s oral health presents a big picture of the oral health concept at the multilevel. Although these determinant factors interact with each other, each can have a cumulative effect on children`s oral health; policymakers could try to consider them as a big picture with a systematic approach for better achievement of oral health among children considering the local and national contextual factors of the community.The online version contains supplementary material available at 10.1186/s13690-023-01073-8. Children`s oral health is a complex multidisciplinary concept affecting their general health and is determined by many interrelated factors. There is a close relationship between determinants of physical health and subjective perceptions of health. At the same time, oral and general health share potential causes and risk factors .Dental caries is considered as the major oral health problems affecting up to 60\u201390% of children and most adults worldwide . Dental The utilisation of dental services is affected by many other factors such as the socioeconomic status of the household, demographic variables, cultural and behavioural factors, level of satisfaction with dental services, and the financial and beneficial supports the same as dental schemes or insurance coverage .All this evidence emphasises the complex multidisciplinary nature of children`s oral health with mutual interactions among various determinants due to the complexity of the concept and multilateral interactions among the determinant factors that affect the children`s oral health at different levels. This commentary is presented a triangle framework of children`s oral health for new discussions in oral health policymaking.There are many recognized social determinants affecting the population`s oral health. Those significant determinants include health disparities, dental access, oral health literacy, oral health and general health, culture and oral health behaviours are notable . AccordiConsidering all the above and regarding the Bronfenbrenner ecological system theory , which sMany factors can be considered at this level; some of them have an individual background, such as demographic factors, biological and genetic factors, as well as psychological ones, while some others have the more community-based and social background, including cultural and socioeconomic factors , as well as religious factors. Two other categories of elements can be mentioned here as a mutual interaction between the children, family and the community regarding oral health behaviours and oral health literacy.In this category, we can mention the variety of determinants from the provider`s perception toward oral health provision of services to availability of dental services , teledentistry and digital technology as well as surveillance and monitoring systems for the children`s oral health.Public policies that can affect the affordability of oral health services, along with regulations and standards on food advertisement and labelling and public education, including the mechanism for funding dental care and supporting schemes such as dental insurance, are the first determinant factors in this category. In contrast, national policies such as political regimens and environmental policies related to the children`s ecosystem and community water fluoridation are considered as the second category of policymakers` perspective. Incorporating probiotics utilisation is also should be mentioned in the oral health policies and health promotion behaviours for moderating the community lifestyle.The present commentary discussed a new viewpoint in categorising the determinant factors affecting children`s oral health based on their ecological system, from the closes system to a child; microsystem to the farthest one; the chronosystem. At the same time, the commentary makes a link via the child levels of the ecological system and the triangle of the people, healthcare providers and policymakers as the main elements of health system governance. This new approach makes this commentary different from the available knowledge and existing models, the same Fisher-Owens (2007), which mainly focused on oral health outcomes from multilevel influences of individual, family, and community .Children`s oral health is related to their general health and is affected by a complex and multilateral interaction of factors at three levels family/community, oral health providers and policymakers. The triangle framework of children`s oral health presents a big picture of the oral health concept at the multilevel. The national and public policies, along with the oral health guidelines and policies, can be considered at both the chronosystem and macrosystem of a child regarding the level and significance of the policy. Then this determinant factor at the top of the triangle can affect the other determinants of family/community and oral health providers. The interactions among the children, their parents and families and the surrounding community, with an emphasis on the school environment and the oral health care providers, can be flowed in all three micro, meso and exosystem surrounding a child. Although these determinant factors have their interactions with each other and each can have a cumulative effect on children`s oral health, policymakers could try to consider them as a big picture with a systematic approach for better achievement of oral health among children considering the local and national contextual factors of the community.Below is the link to the electronic supplementary material.Supplementary Material 1"} +{"text": "SPOP (Speckle-type POZ protein) is an E3 ubiquitin ligase adaptor protein that mediates the ubiquitination of several substrates. Furthermore, SPOP is responsible for the regulation of both degradable and nondegradable polyubiquitination of a number of substrates with diverse biological functions. The recognition of SPOP and its physiological partners is mediated by two protein\u2013protein interaction domains. Among them, the MATH domain recognizes different substrates, and it is critical for orchestrating diverse cellular pathways, being mutated in several human diseases. Despite its importance, the mechanism by which the MATH domain recognizes its physiological partners has escaped a detailed experimental characterization. In this work, we present a characterization of the binding mechanism of the MATH domain of SPOP with three peptides mimicking the phosphatase Puc, the chromatin component MacroH2A, and the dual-specificity phosphatase PTEN. Furthermore, by taking advantage of site-directed mutagenesis, we address the role of some key residues of MATH in the binding process. Our findings are briefly discussed in the context of previously existing data on the MATH domain. The regulation of protein homeostasis is a critical biological process that relies on protein translation, chaperone-assisted folding, and protein degradation pathways ,2,3. In SPOP (Speckle-type POZ protein) is an E3 ubiquitin ligase adaptor protein that mediates the ubiquitination of several substrates and was originally discovered in 1997 by Nagai and coworkers , and celFrom a structural perspective, SPOP is characterized by a modular nature, comprising an N-terminal portion, containing a Meprin and TRAF-C Homology (MATH) domain, followed by a BTB domain, a 3-box, and an NLS disordered motif at its C-terminal part. Importantly, whilst the BTB domain facilitates the formation of the complex with CUL3, leading to the active form of the enzyme , the MATMATH domains typically consist of seven \u03b2-strands forming an antiparallel \u03b2-sandwich architecture . The thrPrevious structural work suggested that the interaction between the MATH domain and the SBC motif is primarily mediated by a cluster of residues, constituting its binding pocket . HoweverThe MATH domain of SPOP exerts its functions by specifically recognizing different substrates and, therefore, acting as an adaptor module between SPOP and its physiological ligands . In an eTo address the equilibrium binding affinity of SPOP MATH and its substrates, a fixed concentration of the protein (1 \u00b5M) was challenged with increasing peptide concentrations. In all cases, the fluorescence-monitored transition returned a hyperbolic behavior as described in the obsk at different peptide concentrations. We then plotted the obsk values over the concentration of dansylated peptide and fitted the data to a simple linear function equation:obsk is the observed rate constant; onk and offk are the microscopic association and dissociation rate constants, respectively in a stopped-flow apparatus at 298 K. The binding traces obtained by monitoring the time-resolved fluorescence were satisfactorily fitted to a single exponential equation, allowing calculating the observed rate constants ectively . The slokinetics . This fiPrevious structural characterization of the interaction between SPOP MATH and its partners suggested the binding site involved several charged groups . Hence, kon, whereas, on the whole, koff remained unchanged. On the basis of these observations, it may be concluded that electrostatic interactions play an important role in the formation of the complex by stabilizing the interactions in the transition state, with a very pronounced effect in the case of Puc. Interestingly, the net charge of both Puc and PTEN peptides is identical, both displaying two acidic groups, while showing a clearly different ionic strength dependence. This finding suggests that the distribution of charges around the SBC motif might be critical in dictating the electrostatic interactions stabilizing the transition state of binding. Inspection of the data reported in D calculated from equilibrium and kinetic experiments, in the case of both MacroH2A and PTEN, there is a detectable difference. Such observations reinforce our indication that the binding of these two peptides occurs via a more complex scenario. Finally, it is of interest to observe that\u2014analogous to what was noted above for the data recorded at a low ionic strength\u2014whilst in the case of Puc, the data are consistent with a two-state binding mechanism with a very good agreement between the KTo quantitatively address the role of specific residues in the binding mechanism of SPOP MATH, we resorted to taking advantage of the site-directed mutagenesis and generated ten site-directed mutants corresponding to the residues located in the binding pocket of the SPOP MATH domain. In particular, we produced a set of single mutants where residues R71, Y88, F103, Y124, F125, K130, D131, W132, F134, and K135 were substituted with alanine. Importantly, those residues were previously suggested to interact directly with the physiological binders of SPOP MATH, through the conserved consensus motif that consists of \u03c6-\u03c0-S-S/T-S/T. The dependence of the binding rate constant of the different mutant variants compared to the results obtained for the wildtype SPOP MATH is reported in Because of the low affinity, it was very difficult to quantitatively analyze the mutational work for the peptides mimicking PTEN and MacroH2A. On the other hand, in the case of Puc, it is of interest to analyze the structural distribution of the observed results. Understanding the mechanisms by which protein\u2013protein interaction modules interact with their partners is of critical importance to define their functions as well as to pave the way for the design of specific inhibitors. From this perspective, despite their critical importance and despite being present in hundreds of different eukaryotic proteins, MATH domains have, to date, eluded detailed experimental characterization. In this work, by taking advantage of a versatile MATH domain, recognizing at least three distinct physiological ligands, we resorted to analyzing in detail the equilibrium and kinetic properties of binding of a prototypical example of MATH domains, SPOP MATH.D extracted from equilibrium and kinetic experiments returned comparable values, for MacroH2A and PTEN, there were detectable differences. In fact, in these cases, the affinity of the complexes appeared higher when calculated from equilibrium experiments as compared to that observed from kinetics. On the basis of these observations, it is possible to conclude that, whilst the binding between SPOP MATH and Puc occurs in an all-or-none two-state fashion, in the case of the other partners, there is the presence of at least one intermediate. Notably, however, since we could neither observe any deviation from linearity in the dependence of the observed rate constant nor detect any additional kinetic phase, it is important to stress that, at this stage, the binding mechanism between MATH and Puc cannot be described in further detail.A common method to describe the properties of the binding mechanism between interacting molecules lies in comparing the thermodynamic parameters obtained from different experimental approaches . In the A binding mechanism processing through the accumulation of an intermediate is often linked to detectable conformational changes in the interacting partners ,23,24,25It is of interest to discuss the mutational data of SPOP MATH in light of the experiments carried out at different ionic strengths. In fact, for all three peptides, we observed a pronounced decrease in the calculated association rate constants with increasing ionic strength. This observation indicates that, despite the fact that the SPOP-binding motif does not directly imply the presence of charged residues , the transition state is primarily stabilized by the electrostatic interactions between SPOP MATH and its binding partners, indicating that the charges, as well as their physical distribution around the SPOP-binding motif, are critical for binding. From this perspective, it is of interest to note that positions Y88, K130, and D131, which are clustered in the center of the binding pocket, primarily affect the dissociation rate constants. On the other hand, positions Y124, F134, and K135 destabilize binding by affecting both the association and dissociation rate constants, thereby indicating these residues have a direct role in both transition state and ground state stabilization. Overall, our data support a mechanism whereby the early stages of binding are primarily dictated by electrostatic interactions between SPOP MATH and its binding partners, which is followed by a subsequent locking of the specific interactions located in the binding site of the domain. Future work with other members of the MATH domain family will clarify the general nature of these observations. Escherichia coli cells BL21 (DE3). Bacterial cells were grown in LB medium, containing 30 \u03bcg/mL of kanamycin, at 37 \u00b0C until OD600 = 0.7\u20130.8, and then protein expression was induced with 1mM IPTG. After induction, cells were grown at 25 \u00b0C overnight and then collected using centrifugation. To purify the His-tagged protein, the pellet was resuspended in buffer made of 50 mM Tris-HCl, 300 mM NaCl, and 10 mM Imidazole, with a pH of 7.5 and with the addition of antiprotease tablet , then sonicated and centrifuged. The soluble fraction from bacterial cell lysate was loaded onto a nickel-charged HisTrap Chelating HP column equilibrated with 50 mM Tris-HCl, 300 mM NaCl, 10 mM Imidazole, and a pH of 7.5. The protein was then eluted with a gradient from 0 to 1 M imidazole by using an \u00c4KTA-prime system. Fractions containing the protein were collected and the buffer was exchanged to 50 mM Tris-HCl and 300 mM NaCl, pH 7.5. The purity of the protein was analyzed through SDS-PAGE. Site-directed mutagenesis was performed using the NZYTech mutagenesis kit , according to the manufacturer\u2019s instructions. Peptides mimicking SPOP MATH substrates, Puc (sequence DEVTSTTSSS), MacroH2A (sequence KAASADSTTEGTPAD), and PTEN (sequence PSNPEASSSTSVTPD), with and without the dansyl N-terminal modification, were purchased from GenScript . A construct encoding the engineered MATH domain of SPOP protein (residues 1\u2013182), with cysteine residues substituted to serine, was subcloned in a pHTP1 plasmid vector and then transformed in Equilibrium binding experiments were performed on a Fluoromax-4 single-photon-counting spectrofluorometer . The SPOP MATH domain was excited at 280 nm and emission spectra were recorded between 300 and 400 nm, at increasing dansylated peptide concentrations. Experiments were performed with the engineered SPOP MATH wildtype at a constant concentration of 2 \u00b5M and titrated with increasing concentrations of dansylated peptides from 0 to 40 \u00b5M at 298 K, using a quartz cuvette with a path length of 1 cm. Data were fitted using the following hyperbolic function:Binding kinetic experiments were performed on an Applied Photophysics DX-17MV stopped-flow apparatus . Pseudo-first-order binding experiments were performed by mixing a constant concentration (2 \u03bcM) of MATH domain with increasing dansylated peptides concentrations, from 2 to 10 \u03bcM of Puc and MacroH2A, and 2 to 40 \u00b5M of PTEN. The samples were excited at 280 nm, and the emission fluorescence was recorded by using a bandpass 320 nm cutoff filter. The experiments were performed at 298 K. For ionic strength dependence with Puc and MacroH2A, the buffers used were 50 mM sodium-HEPES, pH 7.2, supplemented with 50 mM, 100 mM, 200 mM, or 500 mM of sodium chloride (NaCl). For the PTEN binding salt dependence experiments, lower concentrations of salt were added for the previous buffer list mentioned . For each acquisition, five traces were collected, averaged, and satisfactorily fitted to a single exponential equation.offk) were directly measured by performing displacement experiments on an Applied Photophysics DX-17MV stopped-flow apparatus (Applied Photophysics). A preincubated complex of MATH domain and dansylated peptides at a constant concentration (both 8 \u00b5M) was rapidly mixed with an excess of nondansylated peptides (50 \u03bcM). The samples were excited at 280 nm and fluorescence emission was collected by using a 330 \u00b1 30 nm bandpass filter. The experiments were performed at 298 K in the same buffer used for the binding experiments. The observed rate constants were calculated from the average of five single traces. The observed kinetics were consistent with a single exponential decay. A typical binding and displacement transition is reported in Microscopic dissociation rate constants ("} +{"text": "Pharmaceutical wastewater frequently contains high levels of toxic pollutants. If they are discharged untreated, they pose a threat to the environment. The traditional activated sludge process and the advanced oxidation process do not sufficiently remove toxic and conventional pollutants from pharmaceutical wastewater treatment plants (PWWTPs).We designed a pilot-scale reaction system to reduce toxic organic pollutants and conventional pollutants from pharmaceutical wastewater during the biochemical reaction stage. This system included a continuous stirred tank reactor (CSTR), microbial electrolysis cells (MECs), an expanded sludge bed reactor (EGSB), and a moving bed biofilm reactor (MBBR). We used this system to further investigate the benzothiazole degradation pathway.4+-N, TN). During the stable operation of the pilot-scale plant, the total removal rates of benzothiazole, indole, pyridine, and quinoline were 97.66, 94.13, 79.69, and 81.34%, respectively. The CSTR and MECs contributed the most to the removal of toxic pollutants, while the EGSB and MBBR contributed less to the removal of the four toxic pollutants. Benzothiazoles can be degraded via two pathways: the benzene ring-opening reaction and the heterocyclic ring-opening reaction. The heterocyclic ring-opening reaction was more important in degrading the benzothiazoles in this study.The system effectively degraded the toxic pollutants and the conventional chemicals (COD, NHThis study provides feasible design alternatives for PWWTPs to remove both toxic and conventional pollutants at the same time. GloballDifferent techniques have been used to reduce the toxicity of pharmaceutical wastewater and increase its biodegradability . The hydAnaerobic granular sludge and aerobic biofilm processes are more stable than the traditional activated sludge process for the biological removal of pharmaceutical wastewater because of the impact load and toxicity of pharmaceutical wastewater . The exp4+-N), and total nitrogen (TN). By pretreating the pharmaceutical wastewater with hydrolysis acidification and MECs, the biodegradability of the wastewater can be enhanced, and the toxic, harmful, and bio-inhibitory refractory wastewater can be more efficiently treated. Therefore, high efficiency reactors EGSB and MBBR can be used for subsequent treatment. The EGSB and MBBR biological treatment technologies can significantly remove carbon and nitrogen from the wastewater and further reduce organic pollutants. This study provides a new way to solve the problems of high energy consumption and low efficiency in the treatment of highly toxic pharmaceutical wastewater. It has important scientific significance and engineering application value and will exert a great influence on the design and production operations of pharmaceutical wastewater treatment systems.This study designed a pilot-scale wastewater treatment system consisting of a CSTR\u2009+\u2009MECs\u2009+\u2009EGSB\u2009+\u2009MBBR unit to investigate its effectiveness in treating pharmaceutical wastewater. According to the author\u2019s literature research, there is no case of combining the four systems and using them in a pilot-scale study of real pharmaceutical factory wastewater. We tested the removal of antibiotics and other refractory toxic and harmful substances and conventional indicators such as chemical oxygen demand (COD), ammonia nitrogen , and other refractory substances. The pharmaceutical factory wastewater also contained high salinity, including chloride ion concentrations of about 3,500\u2009mg/L and sulfate concentrations of about 1,098\u2009mg/L. In addition, the pharmaceutical wastewater also contained a few metal ions, among which the concentrations of Cr, Fe, Cu, and Zn are 0.042, 11.60, 5.37, and 0.23\u2009mg/L, respectively. The colored odorous wastewater foamed easily and had a lot of suspended matter. The biochemical treatment of pharmaceutical wastewater is typically challenging due to its biological toxicity. Based on gas chromatograph\u2013mass spectrometry (GC\u2013MS), the wastewater contains more than 100 distinct kinds of organic pollutants. The most prevalent pollutants included esters, phenols, anilines, indoles, and benzothiazoles.The pharmaceutical factory wastewater had a BOD2.2.via a pilot-scale plant that used the CSTR\u2009+\u2009MECs\u2009+\u2009EGSB\u2009+\u2009MBBR bacteria enhancement treatment method . The effective volume of the aerobic tank is about 300\u2009L with a length of 0.4\u2009m and a width of 0.8\u2009m and a height of 1\u2009m, and the effective volume of the micro-oxygen tank is about 600\u2009L with a length of 0.8\u2009m and a width of 0.8\u2009m and a height of 1\u2009m. The MBBR unit is filled with a multiphase flow dynamic membrane microbial carrier (see Patent CN104787874A). The concentration of dissolved oxygen in the aerobic portion of the MBBR was kept above 2\u2009mg/L. Heterotrophic bacteria in the MMBR further degraded the remaining organic matter in the water to create a favorable autotrophic environment for the removal of nitrogen. The dissolved oxygen was kept below 0.5\u2009mg/L by regulating the micro-oxygen pool through the aeration system. A dissolved oxygen gradient forms inside the biofilm attached to the patent filler, and simultaneous nitrification and denitrification occur inside the reactor. After the hydrolysis and acidification effect of the CSTR stabilized, the influent flow rate was about 1\u2009m3/d, and the residence times of the CSTR unit, the MEC unit, the EGSB unit, and the MBBR unit was 19.2, 12, 31.2, and 21.6\u2009h, respectively.Most of the pharmaceutical sludge and residue are removed from the high-concentration pharmaceutical wastewater in the regulating pool, after which the water temperature is adjusted by the heat exchange tank and the wastewater is lifted to the collection and distribution wells . The phaThe advantages of the pilot-scale plant in this study lie in its high processing efficiency and low processing cost. At the front end, the refractory organic matter in the wastewater is greatly reduced through the function of the CSTR and the MECs, as is the biological toxicity of the wastewater. At the back end, the efficient granular sludge unit and biofilm unit can be used to strengthen the treatment of organic matter and nitrogen in the wastewater so that the pilot-scale system can complete the efficient removal of the refractory pollutant and nitrogen. Although four reaction units are used in the pilot-scale system studied, they are all carried out by biological or related treating methods, so the cost of treatment is much lower than the operating costs of physical methods such as membrane processing and evaporation. Among them, the cost of the CSTR unit and the MBBR unit is very low due to having a simple operation mode and a mature and stable process, and the main operating costs of the system are to debug and run the MECs unit and the EGSB unit. In addition, in order to facilitate the application and popularization of this technology, we named the system \"Integrate Continuous stirred tank reactor, Microbial electroassisted catalysis, Expanded granular sludge blanket and Flow dynamic biofilm (ICMEF)\".3. Due to significant changes in the water quality, the inlet COD was measured every 12\u2009h. The volume load of the inlet was also calculated to adjust the inlet flow volume. After 6\u2009weeks of operation, the sludge in the system changed from black and odorless to gray and brown. The sludge had no obvious odor, and the effluent was moderately acidic. The COD removal rate remained steady at around 20\u201330% and was able to withstand significant COD influent fluctuations. At this point, the CSTR reactor has started up successfully. After the CSTR reactor was successfully started, the effluent that entered the MECs started to acclimate to its biofilm. A change in the electrode potential should be detected during the start-up process. The microbial electrical assistance system is considered to have successfully started when the anode potential value is stable. In addition, when the COD removal reaches more than 80% and the operation has been stable for about a week during the EGSB reactor\u2019s starting process, the reactor is successfully started. In the MBBR, the filling ratio of the filler was 40%, while the dissolved oxygen was controlled between 2 and 4\u2009mg/L. Based on the amount of sludge in the two-stage aerobic MBBR tank, the sludge in the sedimentation tank was returned either intermittently or continuously.For the initial start-up phase, anaerobic granular sludge purchased from the sewage plant was used in the EGSB reactor. However, the remaining sludge from the pharmaceutical factory\u2019s sewage treatment workshop\u2019s secondary sedimentation tank was used to seed the other reactors. The sludge inoculation ratio of each reactor was approximately 30%, and the reactors had an MLVSS/MLSS ratio of 0.75 and an MLVSS of 3,570\u2009mg/L. If the sludge were to be directly injected into the reactor, it would cause serious floating phenomena. Therefore, the sludge needed to be aerated in each reactor for 48\u2009h before sludge inoculation. To improve the bioavailability of wastewater and accelerate the speed of sludge domestication, industrial brown sugar was added to each reactor during aeration. The floating sludge, which was not bioactive in the initial start-up phase, was continuously discharged. After 2\u2009weeks, the sludge has adapted to the anaerobic environment. At this point, the effluent was almost free of any floating sludge. Due to the slow start-up of the anaerobic MECs and CSTR (Hydrolytic Acidification System) reactors and the poor biodegradability of the pharmaceutical wastewater, brown sugar solution was continuously added during the sludge culture stage of these two reactors. The organic load in the CSTR gradually rose from 1\u2009kg to 8\u2009kg\u2009COD/m2.3.4+-N and TN were measured using a spectrophotometer . The DO was measured using a DO meter , and the pH was measured with a pH meter . The volatile acid content was determined by gas chromatography. For the gas chromatograph , the carrier gases were nitrogen and hydrogen, at flow rates of 50 and 55\u2009mL/min, respectively. An FID hydrogen flame detector was used to analyze the samples. An injection volume of 10\u2009L was used at an injection port temperature of 250\u00b0C, a column temperature of 240\u00b0C, and a furnace temperature of 170\u00b0C, which was heated at a 20\u00b0C gradient and was maintained for 2\u2009min.Before analyzing the samples, the pooled samples were passed through a 0.45-\u03bcm filter. The potassium dichromate method was used to analyze the COD, while NH\u22121. For the benzothiazole detection, the UV light detection wavelength was 280\u2009nm, the injection volume was 10 \u03bcL, and the column temperature was 30\u00b0C. The mobile phase of the indole was acetonitrile water at a flow rate of 1\u2009mL/min\u22121. For the indole detection, the detection wavelength of the ultraviolet light was 254\u2009nm, the injection volume was 10 \u03bcL and the column temperature was 20\u00b0C. The mobile phase that was used to detect pyridine was methanol\u2013water at a flow rate of 1\u2009mL\u2009min\u22121. The detection wavelength of the ultraviolet light was 254\u2009nm, the injection volume was 10 \u03bcL and the column temperature was 20\u00b0C. The detection of quinoline was the same as that of pyridine, but at a UV detection wavelength of 275\u2009nm.High-performance liquid chromatography on a C18 column was used to detect benzothiazole, indole, pyridine, and quinoline. The mobile phase of the benzothiazole detection was methanol\u2013water at a flow rate of 1\u2009mL/min3.3.1.4+-N (80.58%), and TN (86.36%) after 13\u2009weeks of domestication , NHtication ; Table 2During the start-up stage of the MECs, the COD removal rate of the effluent of the MECs was between 12 and 28%. The startup of the cathode microorganisms in the MECs was quicker, and the electrode potential of the anode gradually stabilized. After the microbial electric assistance reactor was effectively activated, the electrode potential was stable. The anode potential was stabilized at about \u22120.35\u2009V, and the cathode potential was stabilized at about \u22120.9\u2009V. From about the 12th week, the COD effluent was not affected by the inflow fluctuations as much, and the COD removal rate ranged from 30 to 40%. This means that the MEC system removed CODs at a relatively stable rate. The microbial electro-assist system must enrich many electrochemically active bacteria on the electrode surface and ensure their activity. However, many small molecules, such as acetic acid, butyric acid, ethanol, and lactic acid, that are produced during hydrolysis and acidification, are more easily utilized in MECs. This helps the electrochemically active bacteria grow. A large number of microorganisms have survived on the electrode surface of the microbial electrical assistance system after the stable operation of the MECs. Over time, these microorganisms will adapt to the effluent of the CTSR and will catalyze refractory substances to accelerate electron transfer. These microorganisms could deliver tens of thousands of electrons to the cathode, which would continue to transfer to the refractory substances and greatly aid in the degradation of toxic pollutants.The start-up of EGSB is relatively slow due to the effect of refractory toxic substances such as antibiotics in pharmaceutical wastewater on granular sludge. During the early start-up period of EGSB, a large volume of sludge will flow out of the reactor, including sludge with poor settling performance, dead sludge, and granular sludge that disintegrated during inoculation, while sludge with good settling performance and activity will remain in the reactor. With the improvement of the degradation effect of the front-end CTSR\u2009+\u2009MECs unit on toxic substances such as antibiotics and the adaptation of the granular sludge in EGSB to antibiotics, the start-up of EGSB was completed after the 13th week. The concentration of COD in the effluent is high during this time. From the 13th week, when the reactor started up, the COD degradation rate dropped to 79.23%, after which it increased slightly during the stable operational stage. The final COD degradation rate maintained a level of about 80% during the stable operational stage of the subsequent reactor. This good degradation rate may be due to the good operating condition of the EGSB and the continuous formation of new antibiotic-resistant granular sludge. The COD removal rate of the biofilm system in the MBBR segment was low, mainly because most of the volatile acids have been consumed by carbon sources in the EGSB stage. The effluent COD fluctuated between 224 and 331\u2009mg/L after finally passing through the sedimentation tank, which indicates that the system still contained some organic matter that was difficult to biodegrade.4+-N and TN during the start-up and operational phases are shown in 4+-N in the influent ranged from 120 to 175\u2009mg/L, while the TN concentration in the influent ranged from 205.13 to 300.58\u2009mg/L. After the CSTR unit treatment, the NH4+-N concentration increased. This increase in NH4+-N concentration may be due to the decomposition of some organic nitrogen compounds in the influent water. However, the TN concentration decreased only a little, which may be due to the limited consumption of nitrogen by the proliferation of anaerobic bacteria during the cellular synthesis process. The NH4+-N and TN concentrations both decreased significantly after passing through the MECs. This may be because ammonia was oxidized to nitrite or nitrate in the air cathode, and then nitrite or nitrate was reduced to nitrogen in the anaerobic environment. The ammonia can be used as an electron donor and reactor between the cathode and anode to provide energy for microorganisms in the bio-electrochemical anammox process in MECs, while the high potential of the anode can further promote nitrification also gradually increased. The TN in the MBBR can be greatly degraded because the biofilm in the reactor allows the aerobic and anaerobic microenvironments to exist at the same time. This enables simultaneous nitrification and denitrification, which reduces the need for carbon sources in the MBBR and butyric acid are shown in Volatile acids are generally produced by hydrolytic acidification. The system pH mainly affects the type and acidification rate of the hydrolytic acidification products. Many organic acids and alcohols are produced during hydrolytic acidification in the CSTR. The accumulation of these substances decreases the pH of the system. The pH of the anaerobic system will have a greater impact on the entire biochemical reaction process compared with an aerobic system. The pH of the inlet and outlet water of the hydrolysis and acidification reactor must therefore be continuously monitored. By measuring the pH change between the inlet and outlet water of the system, the degree of hydrolytic acidification can be determined. 4+-N produced by the anaerobic reaction also increases the pH of the EGSB effluent. The pH of MBBR effluent fluctuated within a certain range. The simultaneous removal of carbon and nitrogen by simultaneous nitrification and denitrification processes in MBBR caused this fluctuation.MECs have a small effect on the pH of the system . The pH 3.3.Four typical toxic pollutants were selected from pharmaceutical wastewater, and the removal of the four substances was monitored in the start-up and stable operational stages . During The removal of pharmaceuticals in wastewater treatment plants was mainly achieved through biodegradation, adsorption, chemical oxidation by oxygen, and hydrolysis, of which biodegradation is the most important way . As show3.4.In our study, ion chromatography was used to analyze the inorganic products in the effluent, while a gas chromatograph-mass spectrometer (GC\u2013MS) was used to test mixed samples of effluent that had been aerated for two, four, and 7\u2009h. The parameters, such as retention time and the relative molecular weight of the main intermediates of the benzothiazole degradation process, were obtained from the GC\u2013MS analysis .The degradation pathway of benzothiazole was inferred from the results of the intermediate products and the ion chromatography. The degradation of benzothiazole typically begins with the hydroxylation of the heterocycle and proceeds with the cleavage of the ring since it is a heterocyclic compound. The five-membered ring adjacent to the six-membered ring usually was oxidized first to form 2-hydroxybenzothiazole (OBT) during benzothiazole degradation. The mass concentrations of benzothiazole and OBT in the MEC reactor were determined by high-performance liquid chromatography. It was found that OBT concentration increased with the decrease in benzothiazole concentration. When benzothiazole concentrations were low, OBT concentrations reached a peak and then began to decline. Therefore, OBT was considered to be the intermediate product of benzothiazole. According to the literature research, we could find that OBT may have two biodegradation pathways . FurtherPathway A: OBT (1) undergoes hydroxylation and further degrades to form 2,6-dihydroxybenzothiazole (2) under the action of microorganisms. Benzothiazole-2,4,5-triol (3) was mainly formed during the further hydroxylation of 2,6-dihydroxybenzothiazole (2), due to the different activities at the different benzene ring substitution sites. Under the continuous action of the microorganism\u2019s dioxygenase, the final benzene ring underwent a ring-opening reaction, which caused the formation of the acidic substance (4).32\u2212, NO3\u2212, and SO42\u2212.Pathway B: OBT first generates 2-sulfobenzothiazole (5), and S and N heterocycles are cleaved by microorganisms. A desulfonate group reaction takes place during this process, and part of the organic sulfur is converted to inorganic sulfate to form 2-(methylthio)aniline (6). This desulfonic acid group reaction is very important for removing the toxic and irritating odor of benzothiazoles and their derivatives. Further oxidation converts the 2-(methylthio)aniline (6) to 2-(methylsulfinyl)aniline (7) and 2-(methylsulfonyl)aniline (8). The continuous action of the microorganisms further converted the resulting 2-(methylsulfinyl)aniline (7) to 2-(methylsulfonyl)aniline (8). Microorganism-produced enzymes further decompose or hydrolyze the 2-(methylsulfonyl)aniline (8), resulting in dealkylation and the formation of the 2-aminobenzenesulfinate (9). The 2-aminobenzenesulfinate (9) was further desulfated and deaminated, and it was finally mineralized to COWhen comparing and analyzing the degradation pathways of these two benzothiazoles, it can be seen that the main intermediate metabolite of pathway A is an acidic substance (4). Pathway A is the ring opening reaction of the benzene ring. However, the main metabolites of pathway B are 2-(methylsulfinyl)aniline (7) and 2-(methylsulfonyl)aniline (8). Pathway B is ring-opening reactions of heterocycles. The complete mineralization of benzothiazole requires the cleavage of the heterocycles.42\u2212 was determined to be 15\u2009mg/L. Nessler\u2019s reagent spectrophotometry was used to detect the NH4+-N concentration in the inlet and outlet water. In the inlet water, the NH4+-N concentration was not detected or was below the detection limit, while the NH4+-N concentration in the outlet water was 11.533\u2009mg/L. This means that the NH4+ and SO42\u2212 were produced during the reaction. Pathway B is therefore considered to be the main pathway for the degradation of benzothiazole. Acidic substance (4) was also detected by GC\u2013MS, but at low concentrations, and therefore path A is the auxiliary path of the benzothiazole degradation.Ion chromatography was used to detect the inorganic products of the effluent. The concentration of SO4.This study used the combined CSTR\u2009+\u2009MECs\u2009+\u2009EGSB\u2009+\u2009MBBR to treat actual pharmaceutical wastewater. This system proved to be effective in removing pollutants from pharmaceutical wastewater. We also analyzed the degradation of the main characteristic pollutants in pharmaceutical wastewater in detail. The hydrolytic acidification unit, when used as a pre-treatment unit, can adapt well to the refractory substances contained in the wastewater and has a certain tolerance for toxic substances. Compared with the hydrolytic acidification unit, the bioelectrochemical system used in this study provides a greater contribution to the removal of organic pollutants and can also partially remove conventional pollutants. The EGSB\u2009+\u2009MBBR unit, which is the main biochemical process at the back end, removed most of the nutrients and further reduced characteristic pollutants. This study provides experimental data and feasible suggestions for the design and modification of a full-size PWWTP.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.WD: conceptualization, methodology, supervision, writing original draft. J-WP, JD, Y-QW, L-YZ, N-QR, and S-SY: data collection and analysis, conceptualization, supervision, validation, writing and editing. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (Grant No. 52170073); the National Engineering Research Center for Bioenergy , and the Open Project of State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology) (Grant No. 2022TS35).J-WP was employed by China Energy Conservation and Environmental Protection Group, CECEP Talroad Technology Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "In general, urinary and faecal incontinence is a multifaceted problem that is associated with increasing burdens for those affected, a massive impairment of quality of life and economic consequences. Incontinence is associated with a high level of shame, which in particular reduces the self-esteem of those being incontinent and leads to additional vulnerability. Those affected by incontinence often perceive incontinence and the care they receiveas humiliating, hence they can no longer control their own urination; nursing care and cleansing support then lead to additional dependency. People with incontinence in need of care not uncommonly experience a poor communication and many taboos surrounding the issue as well as the use of force when incontinence products are changed.n\u2009=\u200980). One intervention group will be equipped with a sensor-based digital assistance system, which will transmit care-related information to the nursing staff (via smartphone). The collected data will be compared to the data of the control group. Primary endpoints are falls occurring; secondary endpoints are quality of life and sleep, sleep disturbances and material consumption. In addition, nursing staff (n\u2009=\u200915\u201320) will be interviewed regarding the effects, experience, acceptance and satisfaction.This RCT aims to validate the benefits of using a digital assistance system to optimise incontinence care and to enable statements concerning the effect of the assistance technology on nursing and social structures and processes as well as on the quality of life from the perspective of the person in need of care. In a two-arm, stratified, randomised, controlled interventional study, primarily incontinence-affected residents of four inpatient nursing facilities will be examined . This RCT is registered at the German Clinical Trials Register on July 8 Incontinence refers to the body\u2019s inability to retain and release urine (urinary incontinence) or faeces in a controlled manner, resulting in an involuntary outflow. Experts estimate that between six and eight million people in Germany are affected by urinary incontinence and about four million are affected by faecal incontinence\u20133. The eRoutine visual inspections of care recipients' incontinence products are currently performed as standard practice in both outpatient and inpatient settings. This procedure significantly compromises a variety of aspects such as hygiene, ethics, the structure of daily life and work efficiency. A digital, sensor-based assistance system could replace visual checks in both outpatient and inpatient settings and improve the quality of incontinence care for both those in need of care as well as for nursing staff. Although the benefits of digital assistance systems in the field of incontinence care are promising, primarily the users need to be convinced. Technical aids and digital assistance systems often lead to scepticism among nursing staff, this manifests itself in misunderstandings, worries and fears, a lack of information and insecurities concerning data usage . This skWhat are the benefits of planned assistance technologies for incontinent care recipients?Can assistance technologies reduce falls and improve quality of life as well as quality of sleep of incontinent individuals?What effects does the assistance technology have on existing structures and care processes in long-term care?How can implementation in nursing practice be organized and what kind of logistical measures are required for usage in long-term care ? How can the technology be incorporated into routine processes in long-term care?In terms of economic perspectives, what results does a measurement or prognosis of success provide to potential users of assistance technologies?How can a sustainable utilisation of the technologies be assured?This RCT aims to validate the benefits of using a digital assistance system to optimise incontinence care and to enable statements concerning the effect of assistance technology on nursing and social structures and processes as well as on the quality of life from the perspective of individuals in need of care. Answers are to be found in particular to the following questions:In the long term this project aims to contribute to new quality-assured modes of care in nursing practice.This RCT is based on a mixed-methods approach and consists of a quantitative and a qualitative method strand, which are to be arranged in a parallel design to each other, allowing an integrative presentation of the results. The focus is on individuals requiring care in inpatient nursing facilities (primary target group) and on nursing staff (secondary target group). The approach selected intends to generate a comprehensive understanding concerning the use of digital assistance systems for incontinence care, which represents the different perspectives of the target groups and captures the complexity of the research subject as a whole.This study protocol is guided by the SPIRIT Statement .primary target group of care recipients, a two-arm, stratified and randomised, controlled interventional study is selected for the study design (RCT). Participants will be assigned to the intervention group and control group study arms, and the assignment of study participants to arms will be randomized. To ensure a homogeneous distribution regarding the prognostic factor of impaired abilities to the IG and CG, a simple disproportionate stratification will be performed prior to the randomisation. This will ensure that a valid interpretation on the efficacy of the intervention is possible. Other advantages of this approach refer to a reduced probability of an \u03b1 and/or \u03b2 error when interpreting the results, furthermore the statistical validity of the results with a comparably low participant number is increased, and interim analyses are simplified [Concerning the mplified \u201333. The mplified , 35. Howmplified . Particisecondary target group of nursing staff, a single-armed observational study design is selected. All participants will be surveyed both quantitatively and qualitatively at four time points.Concerning the When randomizing both groups, additional attention will be paid to the individual framework conditions of the participating inpatient care facilities and they will be appropriately responded to.Within the framework of the RCT, care recipients affected by incontinence and their responsible nursing staff will be monitored in four selected inpatient care facilities in Berlin and Brandenburg over a system test period of four months.have a moderate to severe incontinence andhave given their written informed consent to participate in the RCT after receiving information and clarification. If individuals are not able to consent to study participation by themself due to cognitive impairments, their family caregivers or legal guardians may provide consent.To consider the different comorbidities of the primary target group and to be able to realistically assess the benefits of the digital assistance system, the inclusion criteria were defined relatively broadly. Individuals included in the RCT:The severity of incontinence is determined by the consumption of incontinence material per person in need of care as recorded in the care documentation.Due to the multifaceted occurrence of incontinence, other factors such as cognitive ability, mobility as well as the degree of care of the person affected do not play a role as criteria for study participation. Individuals who are cognitively impaired as well as not impaired; who are mobile as well as immobile; and who have a high or low level of care will be included in the RCT.Since nursing staff is to incorporate and evaluate the digital assistance system as part of their professional activities, only the nursing staff responsible for incontinence care is included in the study. By doing so, differentiated statements about the system can be voiced by the nursing staff. Ideally, the included nursing staff is also responsible for participants in the control group, enabling them to identify and describe any changes concerning nursing care with an even greater nuance. Furthermore, a written consent to participate in the study after being informed and educated is necessary. In addition, nursing staff with similar as well as different characteristics such as professional experience, age and gender is included. Thereby, the individuals should differ as minimally and maximally as possible. This contrastive sampling procedure intendeds to reflect the heterogeneity of the research subject through the selection of individuals to be interviewed .In order to include as many individuals as possible in the RCT, the exclusion criteria will be defined relatively narrow.they or their responsible nursing staff decline to participate in the study and therefore do not sign the written declaration of informed consent, and ifthey only have a mild form of incontinence .Individuals will be excluded from the RCT if:Nursing staff will only be excluded from the study if they do not agree in writing to participate and/or if they are not responsible for the incontinence care of the primary target group.Individuals may withdraw from their study participation at any time. They or their responsible nursing staff can revoke their consent to participate in writing. Likewise, they are automatically excluded from the RCT if they die during the intervention.A deterioration in their health does not necessarily lead to exclusion from the RCT. Those affected are only excluded from further study events if the deterioration is due to compelling medical and/or nursing reasons requiring a termination of their participation in the study.Nursing staff may withdraw their consent to participate at any time. They can do this by revoking their consent to participate in writing.In addition, nursing staff can be excluded from the study if their poor technical affinity or acceptance leads to massive restrictions concerning the correct implementation and use of the digital assistance system in the context of incontinence care. In order to ensure a correct application of the technology, training sessions for the nursing staff will be held over several days at the beginning of the measurement period. During this training nursing staff will be introduced in depth to the digital assistance system. This is followed by a test phase of one to two weeks in the inpatient care facility, during which the nursing staff independently tests the system in regular care. If, during this period, a professional is found to lack technical user skills or feedback is received from the person concerned, this person will not continue to participate in the study.For both the primary and secondary target groups, further progression of the Covid pandemic may result in exclusion or withdrawal from the study. If, in an inpatient care facility, a Covid outbreak is deemed by management to have a lasting impact on normal business operations, the facility may decide to not continue its participation in the measurements.The RCT addresses the use of digital assistance technologies in the field of incontinence care for care recipients. The RCT plans to equip incontinence products of any type (urine and stool) with a sensor that transmits care-related information to connected systems (such as smartphones and PCs). A local tracking mechanism allows the incontinence product to be located, but this function will not be used within this RCT.The sensor technology can be used within the facilities even without a dedicated W-LAN, as it is used via a Bluetooth Low Energy (BLE) as well as via LTE network; set up by AssistMe GmbH (practice partner). AssistMe provides the complete sensor, hardware and network technology. This also includes the smartphones provided to the nursing staff.At this stage multiple utilization of the sensors with incontinence material is not possible for the entire course of this study. However, in some cases, particularly at the start of the RCT, sensors will undergo a re-use process to check and test if and to what extent multiple utilization is possible. In view of the above, a multiple utilization of the sensors will be aimed at for test purposes, in order to identify opportunities for cost-effective mass production while also ensuring a sustainable use of the product. The project in applied practice thus enables further investigations which can provide recommendations for mass production of incontinence products equipped with (printed) sensors. The experience of the practical partner AssistMe GmbH in the field of cleaning and disinfecting sensor technology will also be used for quality assurance and development of logistical recommendations.The digital assistance system from AssistMe GmbH provides the responsible nursing staff within the care facilities with an automated message concerning the status of the incontinence material (ICM) of the home residents within the IG. The status report does not give any indication about whether the ICM needs to be changed but only describes the status of the ICM itself . The decision to change the ICM is made by the responsible nursing staff and not by the digital assistance system from AssistMe GmbH. In addition, it is possible to store individual incontinence profiles of the home residents in order to obtain an additional source of information concerning the decision among the nursing staff as to when an optimal change of the ICM should take place.The digital assistance system from AssistMe GmbH is to be understood as a supporting, digital measure in the context of incontinence management.Preparation and implementation phaseTraining and testing phaseRealisation phaseThe technical procedure of the intervention is divided into three phases:(1) Preparation and implementation phaseThis thus implies, on the one hand, the implementation of an IT infrastructure within the care facility and the associated assurance of a complete connectivity of the wearables equipped to the ICM with the network. On the other hand, the logistical process of the ICM requirements for the home residents will be jointly coordinated in detail with the respective care facilities and service providers (TZMO and geria\u2009+\u2009med) so that a) no supply bottlenecks arise and b) the study process can be realized as smoothly as possible.(2) Training and testing phaseThe training and testing phase is used to familiarise the participating nursing staff with the digital assistance system from AssistMe GmbH. This means both a multi-hour training session for nursing staff and a 1\u20132\u00a0week trial period to \"get to know\" and \"better understand\" the system will be conducted, in order to reduce any technical challenges during the course of the study and to address any questions that arise from the nursing staff in advance.(3) Realisation phaseThe realisation phase represents the actual RCT and intervention. In this phase the digital assistance system is connected within the nursing facility, the nursing staff has been trained and the study project begins see Fig.\u00a0.Fig. 2OvThe ICM equipped with a sensor is worn with a so called wearable. The wearable records the sensor data and transmits the data via BLE to the corresponding charger or repeater nodes.The charger and repeater nodes act as network within the nursing facility ensuring a permanent connectivity between wearable and IT infrastructure. The charger and repeater nodes record the signal transmitted via BLE and then forward it via WLAN to defined access points within the nursing facility. These access points send the relevant data to an edge computer in the corresponding nursing facility and then to a LTE router which sends the data to the AWS cloud service, which complies with data protection requirements. The data is processed in the cloud and then forwarded to the mobile application of the responsible nursing staff.Two wearables will be individually assigned to each resident of the IG and will be accordingly available to the nursing staff. Hence one wearable can be worn with the ICM while the other wearable is charging on the station (charger node) ensuring a seamless change of the wearable when the battery is low.The aim is to implement a fully functioning end-to-end system in the nursing facility so that the ICM status of each facility resident can be monitored in real time on the smartphone of the responsible nursing staff.Occurring falls represent the primary endpoint of the RCT. Secondary endpoints include cognitive ability, quality of life, sleep quality and disruptions of sleep, incontinence material consumption and general health.The secondary target group will be surveyed by means of interviews regarding the acceptance, experience and perceived effects of the digital assistance systems and the resulting changes to their work processes (secondary endpoints). This qualitative data collection captures the important perspectives of nursing staff and nursing management.In addition, the nursing staff will also be surveyed quantitatively about their subjective attitude toward the use of technology in their professional context and concnerning the use of the digital assistance system in particular. The subscales of the questionnaire to measure the affinity for technology scale (TA-EG) are used as guidance. The TA-EG consists of 19 items and four scales and has been adequately reviewed in terms of its validity and reliability. Due to its scope, it will be used in a shortened form in the study and adapted to the specific core of the intervention. The modified questionnaire was tested in pretests with regard to comprehensibility, practicability and temporal scope. Subsequently, editorial changes were incorporated and one at the end (t6) of the four-month study period. Between these two points (t0 and t6), the quantitative surveys will be conducted every three weeks . If necessary, when taking the measurements, the study staff of the Neubrandenburg University of Applied Sciences may be assisted by the nursing staff of the relevant inpatient nursing facility (e.g. if a participant has a greater cognitive impairment).Four measurement points are planned for the individuals of the secondary target group. One measurement will take place at the beginning (t0) and one at the end t3) of the four-month study period. In the meantime, the qualitative and quantitative surveys will take place every six weeks t1, t2) The study staff of the Neubrandenburg University of Applied Sciences and those responsible from AssistMe will visit the participating inpatient nursing facilities. In an open event all residents of the facility who are interested will be informed about the RCT . This is meant as opportunity to ask questions to the persons responsible for the RCT. This shall emphasise the absolute voluntary nature of study participation. 2) If the residents or their family caregivers/legal guardians indicate interest in participating in the study, they inform the responsible nursing staff in the facility. The nursing staff consolidate the interested parties and forward their names to the study staff at the Neubrandenburg University of Applied Sciences. The study staff then visits the interested parties again. In a conversation, they will be again informed in detail about the RCT as well as the intervention. In addition, the inclusion and exclusion criteria will be checked. Residents who meet the eligibility criteria and who would like to participate in the RCT are then examined regarding their cognitive performance using the Mini Mental Status Examination (MMSE). Depending on their score, they are assigned to the respective strata \"cognitively impaired\" or \"not cognitively impaired.\" Subsequently, both subgroups are randomized individually into the intervention and control group.Should individuals in the primary target group drop out of the RCT prematurely , new particpipants will be recruited and included if they meet the inclusion criteria. This post-recruitment is foreseen up to a maximum of two months after the start of the system test period.Only nursing staff responsible for incontinence care of participants in the primary target group within the respective inpatient nursing facility will be recruited. In addition, they must have attended the training sessions concerning the use of the digital assistance system and must have consented to participate in the study.The aim is to ensure that IG and CG do not differ beyond the intervention with regard to the prognostic factor of the individuals' cognitive performance. Especially with a small sample size, a different approach could promote an unbalanced relationship between the two groups . An indeThe study information for participants includes information about the assigned arms and the randomised design of the study. An independent researcher not involved in the study performs the allocation using Excel. Participants, nursing staff, the provider of the digital assistance system and research study staff will not be blinded to the group assignment.Data management is divided into two strands. First, data from all survey points, all measurement times and assessment tools will be documented using electronic case report forms (eCRF). If possible, a plausibility check is performed when the data is first entered into the system. Only the anonymised particpant IDs are used for the eCRFs. Identifying data that enables the allocation of a participant ID to an individual are stored separately. Only the project management has access to this data after random allocation and assignment of the participants IDs. The data is stored on a central network system based on the data management system of the Neubrandenburg University of Applied Sciences in line with current standards for data security. Access to this network system is only granted to study staff at the Neubrandenburg University of Applied Sciences. Access to the system can only be granted by the technical administrator of the Faculty of Health, Nursing, Management after prior agreement. After completion of the study, the identifying data will be stored physically separate in the Neubrandenburg University of Applied Sciences. All study information in paper format, including the written declarations of informed consent and the questionnaires, will be stored in sealed filing cabinets in areas with restricted access. The individuals included in the study provide a written declaration of informed consent based on comprehensive verbal and written information provided about the study. All consent forms will be stored in the Neubrandenburg University of Applied Sciences. If a study participant limits his or her consent, the limitation is documented and the subsequent data analysis is performed in accordance with the imposed limitations. If a person withdraws his or her consent, all data will be removed, with the exception of data that was published prior to the withdrawal of the consent. All data will be analysed only in an anonymised format. For publications, only aggregate data that does not permit personal identification will be used. Data will only be shared without identification of the participants. Data management and the documentation process will be monitored by the study management.The second part of the data management covers data collected during the study intervention. The data transfer between the individual components of the digital assistance system is end-to-end encrypted using a high security standard. End-to-end encryption means that no plain text is transmitted at any point during data transmission and the transferred data can only be decrypted by the intended senders and recipients. The sensor data will be transferred in anonymised format by allocating it to the ID of the wearable. Personal data is also stored in a particular part of the cloud backend, the Business Logic Server, and the sensor data will be allocated to the participant\u2019s personal data using the wearable ID. This allocation is essential because otherwise a detected incontinence event cannot be matched to a resident and the responsible nursing staff cannot be informed via digital end device. The merging of sensor data and personal data in the cloud backend meets high security standards and thus ensures that the system offers as few attack points as possible for an unauthorized extraction, manipulation or use of personal data, e.g. by hackers.After completion of the project, the records, transcripts and questionnaires will be sealed, stored for ten years and then destroyed. The contact data necessary to carry out the study is stored separately from the records, transcripts, questionnaires and description of the results. Both data sets will be kept locked. The mandatory data protection information required by law is complied with (2016/679/EU and State Data Protection Act for the State of Mecklenburg-Vorpommern of 22.05.2018).A Data Monitoring Committee (DMC) is not established as there is no blinding and adverse events can be reported directly to the study staff. All aspects of the intervention are part of the conventional nursing care of incontinence. There are no interim analyses planned and all results will be analysed after the data collection is completed.The data material will be analysed using SPSS. The focus of the evaluation will be on descriptive analyses and correlation analyses. Conclusively, the results will be evaluated and presented in a stratified manner. The following methods will be used to answer the research questions: Regression analyses, MANOVA with measurement repetition, correlation analysis, multilevel analysis and chi-squared tests. The results of the qualitative interviews will first be transcribed and then analysed using qualitative contents analysis and MAXQIn the context of the RCT, in addition to the technical and methodological points, the underlying logistical process must also be comprehensively defined and coordinated with the project partners involved so that regular nursing care is not restricted at any time. Therefore, after randomization of the sample into intervention and control group, detailed micturition profiles of the participating residents are additionally established by the incontinence care provider responsible for the respective nursing facility. The ICM requirements will be reported to the ICM manufacturer. Concerning the intervention group, AssistMe obtains the personal ICM requirements for a usage time period of four to six weeks as well as the ICM from the ICM manufacturer. AssistMe then attaches the digital incontinence sensors to the ICM and sends the material to the according nursing facility. In parallel, the ICM manufacturer delivers the original ICM requirement to the nursing facilities to ensure that the supply is not limited at any point. This will be particularly effective if the RCT has to be paused or ended due to dropout criteria. This will ensure that the standard care can be resumed at any time, independent of the digital incontinence management see Fig.\u00a0.Fig. 4LoThe project partner AssistMe GmbH has already coducted numerous tests of the digital assistance system for incontinence care in the past. Harm due to heat through the sensor or other personally hazardous technical malfunctions can be ruled out.To ensure that the system and the app function without interference, technical devices with electromagnetic emissions in microwave range are installed within the inpatient care facility . Damage or incidents due to electromagnetic exposure or compatibility problems are not to be expected, as all necessary electromagnetic safety checks (electromagnetic compatibility) have been carried out and the specifications are fully met by AssistMe GmbH. The tests also showed no restrictions for the participants with regard to freedom of movement or wearing comfort of the ICM. Further comfort tests are currently being carried out to determine whether patients experience unpleasant pressure sensations from the sensor attached to the incontinence material when lying in certain positions . However, significant problems are not expected due to the small size and height of the sensor. Regarding the quality of the care, no deterioration is expected since the digital assistance system will only complement traditional nursing measures as part of incontinence care but shall by no means replace them. Standard care is assured for all participants. If problems with the system should develop, those affected in the intervention group can immediately switch to standard care.In rare cases, participants in both target groups may experience psychological discomfort. On the one hand, this may be due to the fundamental topic of incontinence and incontinence care, which is often still a taboo subject in society. On the other hand, the sensors on the incontinence material record data continuously (24/7), which may result in an unpleasant feeling of being monitored. Particularly in the case of nursing staff, the impression could arise that they may be checked for possible nursing fails. These potential negative perceptions should be countered with transparent information at the beginning of the study as well as with close-meshed discussions with the persons concerned if necessary.In addition, the secondary target group may also experience a time burden and technical overload, as they are expected to use and apply a new care system in parallel to the standard care of incontinence. Especially at the beginning of the study, when there is no experience in using the assistance system yet, or in already stressful situations in the daily work of the nursing staff, the use of the system could rather be perceived as a burden. These expected impairments are to be countered on the one hand with an intensive explanation of the handling and use of the system, and on the other hand with close-meshed conversations in the case of perceived challenges, especially in the first weeks of the study. Basically, all problems and wishes expressed by both target groups are taken very seriously by the study staff, and they are always responded to promptly.A further risk could be the occurrence of COVID cases in the care facilities, whereby the facilities or the care personnel may wish to withdraw from participation due to the time required for the study project, or further participation is only possible under more difficult conditions.Furthermore, there may be unwanted consequences for the study project from an environmental perspective due to the single use of the sensors. As part of the study project, resource-efficient production as well as the possibility of multiple use will be tested. In this regard, the study project aims to find ways to enable a multiple use or sustainable mass production of the incontinence sensors. As part of the project, indications for a more favorable mass production and/or multiple use of the sensor will be developed.All funds and invoices will be managed by study-independent employees in the administration of the University of Applied Sciences Neubrandenburg.The study results will be made available to the scientific public through conferences and international journals, and to the general public through presentations in healthcare organizations and national congresses. In the long term, the project is intended to contribute to the further development of new quality-assured forms of care and action in nursing practice.The results of the RCT are expected to provide reliable data regarding improved incontinence care. The focus is primarily on the possible improvement of the quality of life and dignity of those affected, as they are severely impacted by the taboo that often surrounds the issue and by additional health problems associated with incontinence . The stuconceptual elaboration concerning nursing facilities with recommendations for data protectioninformation of those in need of caretechnical implementation, use and maintenance of the digital assistance systemcleaning and disposal of the sensor technologynursing staff trainingIn addition, data will be determined as to whether and how processes in inpatient care settings can be improved using a digital assistance system. In the long term, the use of digital assistance systems for incontinence care may lead to new nursing and care concepts that support inpatient long-term care. The results of the study are to be processed in form of recommendations for action and to provide information on possible transferability to standard care. Focal points are:Another focus is on economic feasibility studies, which consider implementation and transfer to care settings in inpatient facilities even after the end of the funding period. For outpatient services, the study also provides information on the application and implementation of the digital assistance system." \ No newline at end of file