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+{"text": "Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified.SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis.The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an \"orthodox\" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the \u03b1-class (EcoRI-like), and the \u03b2-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses. The enzymes that do not fit this definition or exhibit certain structural and functional peculiarities, have been classified into several subtypes (review: [kcat/Km for DNA cleavage by a factor \u2265106 [Type II restriction endonucleases (REases) comprise one of the major families of endonucleases and one of the largest groups of experimentally characterized enzymes (comprehensively reviewed in: ). The \"otor \u2265106 .D-Xn-(D/E)-X-K (where X is any amino acid), together with a number of non-specific and structure-specific nucleases, suggesting that these proteins are evolutionarily related despite the absence of overall sequence similarity : BfiI iStreptomyces fimbriatus. It recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3', where N denotes any base, and cleaves it as indicated by \"^\", leaving 3' extensions 3 nt long [SfiI is a REase isolated from  nt long . SfiI is nt long . However nt long -25, but In the absence of experimentally determined protein structures, homology-based models may serve as working models for the investigation of sequence-structure-function relationships between diverged enzymes . HomologThe sequence of SfiI showed no significant similarity to any other protein sequences. Also among the proteins reported by BLAST with sub-optimal scores, there were no proteins of known structure and no nucleases (data not shown) that could hint at potential relationships of SfiI to any previously characterized protein superfamily. Thus, in order to identify a template structure for modeling of SfI we used the threading approach, which allows to assess the compatibility of the target sequence with the available protein folds based not only on the sequence similarity but also on the structural considerations  . TheA homology model of SfiI was constructed based on the alignments reported by threading methods, using the \"FRankenstein's Monster\" approach  , followed by local geometry optimization. One of the adenine residues in the mismatched A/A base pair in the middle of the DNA molecule was \"mutated\" to T/A. The curvature of the DNA remained unchanged. Essentially, the global structure of the protein-DNA complex for SfiI remains exactly as in the BglI structure, as reliable modeling of macromolecular interactions remains beyond the capabilities of the existing methods. The model of SfiI dimer is shown in Figure .BglI belongs to the \"EcoRV-like\" \u03b2-class of PD-DxK nucleases. The most typical features of REases from this class are: antiparallel orientation of the 5elements ,9. Most elements . Brieflyelements  and \"mutIn the proposed model of SfiI, the spatial configuration of the catalytic residues is typical for an active site architecture conserved among PD-DxK nucleases. We predict that the active site of SfiI comprises residues: E55, D79, D100 and K102, which superimpose well on the catalytic residues of BglI: E87, D116, D142 and K144, respectively Figure , backbonInterestingly, our model suggests that SfiI lacks the counterpart of a loop corresponding to aa 63\u201380 in BglI used by this enzyme to interact with the target site from the minor groove side. Thus, SfiI appears to recognize its target solely from the major groove side and to use fewer specific contacts than BglI to recognize its cognate site. This suggests that SfiI may be an easier target for the engineering of REases with new sequence specificities.The dimerization interface of SfiI is comparable to that of BglI. We predict that the following residues may be important for monomer-monomer interactions: Q59, Y60, E63, E66, R73, F74, G76 and that mutating them to change the volume of the side chain (for instance G76R) or introducing (or reversing) the charge  could disrupt the formation of the SfiI dimer and destroy the REase activity.SfiI is a Type IIF enzyme, i.e. a tetramer that binds simultaneously to two recognition sites and cleaves both sites concertedly , while BComparative analysis of nucleases from the PD-DxK superfamily suggests that they can be classified into two remotely related lineages: \u03b1 (EcoRI-like) and \u03b2 (EcoRV-like) -9. It waOur results strongly suggest that the archetypal Type IIF enzyme SfiI is closely related to a \u03b2-lineage member, an \"orthodox\" Type IIP REase BglI. Another well-characterized group of Type IIF REases comprises \u03b1-lineage members for which crystal structures were solved  -41. ThusSequence searches of the non-redundant (nr) database and of the putative translations from finished and unfinished microbial genomes were carried out at the NCBI using PSI-BLAST . SecondaThe alignments between the sequence of SfiI and the structures of selected templates (members of the fold identified by Pcons) were used as a starting point for modeling of the SfiI tertiary structure using the \"FRankenstein's Monster\" approach , comprisaa, amino acid(s); bp, base pair(s); nt, nucleotide; e, expectation; REase, restriction endonuclease; MTase, methyltransferase; ORF, product of an open reading frame, RM, restriction-modification;JMB carried out the fold-recognition analysis for SfiI, built the preliminary models, drafted the manuscript and coordinated the whole study. AC built the final models and identified functionally important residues. KS participated in interpretation of the data and writing the manuscript. All authors have read and accepted the final version of the manuscript."}
+{"text": "Acinetobacter calcoaceticus has a great potential for application as a glucose sensor constituent. Because this enzyme shows no activity in its monomeric form, correct quaternary structure is essential for the formation of active enzyme. We have previously reported on the increasing of the stability of PQQGDH-B by preventing the subunit dissociation. Previous studies were based on decreasing the entropy of quaternary structure dissociation but not on increasing the interaction between the two subunits. We therefore attempted to introduce a hydrophobic interaction in the dimeric interface to increase the stability of PQQGDH-B.Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from Amino acid residues Asn340 and Tyr418 face each other at the dimer interface of PQQGDH-B, however no interaction exists between their side chains. We simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile, to create the two mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile. Furthermore, residues Leu280, Val282 and Val342 form a hydrophobic region that faces, on the other subunit, residues Thr416 and Thr417, again without any specific interaction. We simultaneously substituted Thr416 and Thr417 to Val, to create the mutant Thr416Val/Thr417Val. The temperatures resulting in lose of half of the initial activity of the constructed mutants were increased by 3\u20134\u00b0C higher over wild type. All mutants showed 2-fold higher thermal stability at 55\u00b0C than the wild-type enzyme, without decreasing their catalytic activities. From the 3D models of all the mutant enzymes, the predicted binding energies were found to be significantly greater that in the wild-type enzyme, consistent with the increases in thermal stabilities.We have achieved via site-directed mutagenesis the improvement of the thermal stability of PQQGDH-B by increasing the dimer interface interaction. Through rational design based on the quaternary structure of the enzyme, we selected residues located at the dimer interface that do not contribute to the intersubunit interaction. By substituting these residues to hydrophobic ones, the thermal stability of PQQGDH-B was increased without decreasing its catalytic activity. Acinetobacter calcoaceticus has great potential for application as a constituent of an electron mediator-type glucose sensor. The conventionally utilized enzyme for glucose measurement, glucose oxidase (GOD), inherently utilizes oxygen as its electron acceptor during the oxidation of glucose. In contrast, PQQGDH-B is completely independent of oxygen, resulting in improved accuracy and rapidity during glucose measurement. However, because the substrate specificity and stability of PQQGDH-B remain somewhat inferior to those of GOD, we have been engaged in the improvement of these enzymatic properties through protein engineering to further increase the application of PQQGDH-B in glucose monitoring systems [Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from  systems -17.The subunit structure of PQQGDH-B was determined to be homodimeric with no activity observed in its monomeric form . CorrectIn this paper, we report on our rational designing of hydrophobic interaction in the dimer interface to increase the stability of PQQGDH-B. We identified protein regions at the dimer interface where potential novel hydrophobic interactions could be introduced by amino acid substitution, thereby increasing the stability.Among 19 amino acid residues located at the dimer interface, 8 residues were predicted not to be involved in the formation of hydrogen bonds, electrostatic interactions, or hydrophobic interactions Fig. , no inteThe activity and stability of each of the three constructed double mutant enzymes were then analyzed. All three mutant enzymes showed slightly higher thermal stability than wild-type PQQGDH-B upon incubation for 10 min at various temperatures Fig. . The temInvestigation of the kinetic parameters of the wild-type and mutant enzymes shows that the mutations did not significantly affect the overall kinetic properties of PQQGDH-B Table . The spePQQGDH-B is a 6-blade \u03b2-propeller protein, with each blade consisting of a 4-stranded anti-parallel \u03b2-sheet (W-motif) . The strBased on the predicted structure of the mutant enzymes shown in Figure The addition of hydrophobic interactions in other enzymes has been reported to result in thermostability increases of 2 to 10\u00b0C -24, compWe improved PQQGDH-B's thermal stability by increasing the dimer interface interaction through rational design based on its quaternary structure. We demonstrated that this can be achieved by selecting residues located at the dimer interface that do not contribute to the intersubunit interaction and substituting them to hydrophobic ones via site-directed mutagenesis. In each case tested, the enzyme's thermal stability was increased without decreasing its catalytic activity. This rational design approach will provide relevant information for future designs by combining with other mutant PQQGDH-Bs with narrowed substrate specificity and improved catalytic efficiency.KpnI was obtained from TOYOBO  and HindIII from New England BioLabs .Glucose, phenazine methosulfate (PMS), 2,6-dichlorophenolindophenol (DCIP), and magnesium chloride were obtained from Kanto Kagaku , 3-(N-morpholino) propane sulfonate (MOPS) from Dojin , and pyrroloquinoline quinone from Mitsubishi Gas Chemical Company . All other regents were of analytical grade. KpnI-HindIII fragment containing the PQQGDH-B gene was transferred from pGB to pKF18k and mutagenesis was carried out with the Mutan-Express Km kit (Takara) according to the manufacturer's instructions with the oligonucleotides Asn340Phe (5'-GGTGGGACAAAGAATTTACCAGTCC-3'), Tyr418Phe (5'-CGGTACAGCGTCATCAAAAGTAGTGC-3'), Tyr418Ile (5'-CGGTACAGCGTCATCAATAGTAGTGC-3'), and Thr416Val/Thr417Val (5'-CAGCGTCATCATAAACAACGCTATAAGTTGGATC-3'). The mutations (underlined) were confirmed by automated DNA sequencing . The mutated genes were digested with KpnI and HindIII and were replaced into pGB to construct expression vectors containing mutated PQQGDH-B. Numbering of the amino acid positions starts from the first residue of the signal peptide (24 residues).The structural gene for wild-type PQQGDH-B was previously amplified by polymerase chain reaction (PCR) and inserted into the expression vector pTrc99A (Pharmacia) to create pGB . A 1.2-kEscherichia coli and the enzymes purified as previously reported [2 at room temperature (25\u00b0C) to produce the holoenzyme, GDH activity was measured by using 0.6 mM PMS and 0.06 mM DCIP. The enzyme activity was determined by measuring the decrease in absorbance of DCIP at 600 nm.The PQQGDH-B genes were expressed in reported ,16. FollThe thermal stability of wild-type and mutant PQQGDH-B was determined with 0.075 \u03bcg/mL protein, as previously reported . ThermalThree-dimensional and quaternary structures were predicted using Molecular Operating Environment (MOE) . By using the available PDB data of the wild-type PQQGDH-B, 1QBI , we madeST carried out the site-directed mutagenesis of PQQGDH-B as well as the preparation and characterization of the resulting proteins. SI carried out the 3D modeling and participated in the design of the study. SF participated in interpretation of the results and in drafting the manuscript. KS conceived of the study, participated in its design and coordination, as well as in drafting the manuscript. All authors read and approved the final manuscript."}
+{"text": "The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally.Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues  and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme.Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily. REases that do not fit this definition or exhibit certain structural and functional peculiarities have been classified into several subtypes  ? with plasmid p29Cm (Cmr) bearing eco29kIM gene  ATP using T4 polynucleotide kinase (Fermentas).The double-stranded (ds) DNA cleavage activity and nicking activities of wt R.Eco29kI and the mutants were determined. The ds DNA cleavage activity was assayed using the bacteriophage \u03c680vir DNA  and 200 2, 50 mM NaCl) containing 1 \u03bcg of phage \u03c680vir DNA or 0.01 \u03bcM of DNA fragment. Mutant proteins and serially diluted wild type were incubated with DNA substrate at 37\u00b0C. The aliquots were removed after fixed time intervals until the reaction rate plateaued. The cleavage reaction products of \u03c680vir DNA were analyzed by 0.8% agarose gel electrophoresis. The cleavage reaction products of 200 bp DNA fragment were applied onto 5% polyacrylamide gel under non-denaturing conditions. Then the gel was autoradiographed.The reactions were carried out in 10 \u03bcl reaction buffer RB . EMI and MVZ carried out the molecular genetic studies, participated in analysis of DNA-protein interactions and drafted the manuscript. MVZ collected the data and created the analysis procedures. MON and MMD contributed in design approaches of the protein purification. BSM and VBB analyzed the substrate-enzyme interactions and enzyme specificity. VBB performed the statistical analysis. ESB helped to perform the mutagenesis experiments. ASS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript."}
+{"text": "O6-alkylating agents, which comprise methylating and chloroethylating agents, is the ability of cells to repair alkylation damage at the O6-position of guanine in DNA. This is achieved by a specific DNA repair enzyme O6-alkylguanine DNA-alkyltransferase. In this study O6-alkylguanine DNA-alkyltransferase expression was measured in human breast tumours using both biochemical and immunohistochemical techniques. O6-alkylguanine DNA-alkyltransferase activity was then compared with known clinical prognostic indices to assess the potential role of O6-alkylguanine DNA-alkyltransferase in predicting the behaviour of this common malignancy. The application of both biochemical and immunohistochemical techniques was feasible and practical. Most breast tumours expressed high levels of O6-alkylguanine DNA-alkyltransferase. Immunohistochemical analysis showed marked variation in expression not only between individuals but also within individual tumours, and in the same patient, between metastases and between primary tumour and metastatic site. O6-alkylguanine DNA-alkyltransferase activity in tissue extracts significantly correlated not only with immunohistochemical staining intensity determined by subjective quantitation, but also with measures of protein levels using a computerised image analysis system including mean grey (P<0.001), percentage of cells positive for O6-alkylguanine DNA-alkyltransferase (P<0.001), and integrated optical density (P<0.001). O6-alkylguanine DNA-alkyltransferase expression did not correlate with any of the established clinical prognostic indicators for current treatment regimens. However, immunohistochemical offers a rapid and convenient method for assessing potential utility of O6-alkylating agents or O6-alkylguanine DNA-alkyltransferase inactivating agents in future studies of breast cancer treatment.An important determinant of cellular resistance to chemotherapeutic British Journal of Cancer (2002) 86, 1797\u20131802. doi:10.1038/sj.bjc.6600324www.bjcancer.com\u00a9 2002 Cancer Research UK Standard first-line chemotherapy for advanced breast cancer results in disease regression in the majority of patients but these responses are rarely complete or sustained , through the covalent transfer of the alkyl group from the O6 position of guanine to a cysteine residue within the active site of the ATase protein, a reaction that is stoichiometric and autoinactivating  were used for routine pathological diagnosis while other sections were stained for ATase (see below).Samples for resected tumours obtained at the time of primary surgery at the Christie Hospital were snap-frozen in liquid nitrogen and stored until subsequent assay. Samples were rapidly defrosted in batches of 10 and each tumour divided into two parts. Half was immediately assayed for ATase activity and the other half fixed in 70% alcohol for subsequent IHC. These sections were assessed for ATase using a conventional IHC scoring method and an image analysis system. Clinical, staging and prognostic information was available for all these patients.The preparation of tissue extracts and quantitation of ATase activity was performed as previously described .M citric acid pH\u20096.0 at this stage to retrieve antigenic sites. After washing in 50\u2009mM TBS (pH\u20097.6) endogenous peroxidase activity was blocked with 3% H2O2 in Tris-buffered saline (TBS) for 15\u2009min at room temperature and washing in running water followed by TBS. The sections were incubated with 5% normal swine serum in TBS for 20\u2009min at room temperature to block nonspecific binding of IgG, and were then incubated with polyclonal rabbit anti-human ATase antiserum (1\u200a:\u200a1500) in TBS overnight at 4\u00b0C. Control sections were incubated with pre-adsorbed serum at the same dilution. After washing in TBS the slides were incubated with biotinylated swine anti-rabbit serum at 1\u200a:\u200a400 dilution for 30\u2009min at room temperature, washed and then incubated with an avidin-biotin-horseradish peroxidase complex  for 45\u2009min. Sections were finally developed with 3,3-diaminobenzidine (DAB/H2O2) for 5\u2009min, washed well in TBS and counterstained with the red fluorescent DNA dye propidium iodide (5\u2009\u03bcg\u2009ml\u22121 in PBS for 30\u2009min at room temperature).Sections (3\u2009\u03bcm) were prepared, mounted onto 3-aminopropyltriethoxysilane  coated slides, dried overnight, dewaxed in xylene and rehydrated through graded alcohols . Sections of formalin fixed material were microwaved for 25\u2009min in 1\u2009l of 0.1\u2009Each specimen was assessed by a consultant histopathologist  and for each biopsy, 100\u2013400 tumour cells were counted and the intensity of staining of each cell determined visually and classified semi-quantitatively: negative (0)=no staining; 1+=weak staining; 2+=medium staining; 3+=strong staining, 4+=very strong staining. The fraction of negative, 1+, 2+, 3+ and 4+ cells was calculated for each tumour and staining patterns discriminated as nuclear, cytoplasmic or both.The intensity of ATase staining was measured using a Lucia Image Analyser . Duplicate sections were prepared for each tumour, one section was stained with primary antibody, and the other with antigen adsorbed serum (control). Threshold levels were set using the staining of the control section as a cut off point for each patient. This did not vary by greater or less than three grey levels within the 255 levels of the dynamic range of the system. Any staining above this threshold was counted as positive staining. Ten fields were counted using the \u00d720 objective lens and for each field several parameters were analysed (see below), the total area of the DAB stained cells measured and the binary image recorded. Fluorescent green light  was used to highlight the propidium iodide present in ATase negative cell nuclei in the field and these were thresholded. The ATase positive binary image was added to the propidium iodide positive binary image and the total nuclear area recorded. This allowed expression of all image analysis parameters as a function of total nuclear area in the subsequent quantitative assessment. The intensity of the labelling was determined by the computer programme and gave a grey value from 0 (black) to 255 (white). After capturing the image on the computer screen, stained areas were identified as those regions with a grey value for each pixel below the threshold, which was set at 230.\u22121 of protein) measured by biochemical assay of tissue extracts. (5) The percentage of cells displaying a grey value of <100. This gives an estimate of the distribution of highly stained cells in a sample: the grey value was arbitrarily selected.The parameters assessed in the detected areas were: (1) Mean grey: the sum of all the grey levels in the tumour cells divided by the number of positively staining cells. This was averaged over all the fields viewed. (2) Per cent positivity: the number of cells positively staining for ATase (using the pre-adsorbed control field as the threshold) as a percentage of the total number of cells. (3) Integrated optical density (IOD): the sum of grey value for each pixel in the detected area, indicating the total amount of light-absorbing material in the defined region. (4) Integrated optical density per area: IOD divided by the total area covered by breast cancer cells. This value was compared with the level of ATase , IHC semi-quantitative scoring by a histopathologist and IHC followed by computerised image analysis. Each of these results were compared with a series of established prognostic variables available for that particular patient. This prognostic information included tumour size, grade, histological type, Manchester score , menopausal status, tumour grade and whether or not axillary lymph nodes are clinically positive), axillary lymph node status as well as tumour oestrogen receptor (ER), progesterone receptor (PR), Ki 67 and epidermal growth factor receptor (EGFR) expression.U-test was used. For the comparisons with the subjective assessment of ATase expression, rank correlations were again used as the score  is naturally ordered.Due to the large number of variables being assessed, simple correlations were used. Spearman's rank pair correlation was used to compare the individual data points in ordered rank pairs. The Kruskal\u2013Wallis one-way ANOVA test was used to compare differences in the median score between particular groups. For those sets of data where only two groupings existed (e.g. EGFR: absent or present) the Mann\u2013Whitney Sixty-one primary breast tumours were analysed. Fifty-five of the tumours were ductal carcinomas, six lobular carcinomas, one spindle cell and one medullary carcinoma. Of the ductal carcinomas 12 were histopathological Grade 1, 24 were Grade 2 and 17 were Grade 3   was taken, where x is the in vitro assay result used in the correlation. Patient information for all of the variables was not always available hence, the number of data points available for each variable was not always the same.Known prognostic variables were compared with the various measures of ATase activity using the Kruskal\u2013Wallis one-way ANOVA. Table 4Data for 63 patients was available and of these, 54 were alive with no evidence of disease recurrence at the time of data analysis, three had relapsed and six had died. Insufficient actual survival data was therefore available at this stage to assess whether or not any of the different methods of measuring ATase activity were correlated with the patient's ultimate prognosis. No measure of activity was correlated with lymph node status, tumour grade and Manchester Score.P=0.002) and DNA (P=0.008) but also with mean grey (P<0.001), percentage of cells positive for ATase (P<0.001), IOD (P<0.001), IOD/area (P<0.001) and number (%) of cells <100 grey (P<0.001).  only a relatively small number of samples with non-ductal tumours were analysed. The subjective IHC staining intensity score was significantly correlated not only with the ATase activity by protein (<0.001). P<0.001).As most studies cite ATase expression in human tumours with respect to either DNA or protein content, these two methods of ATase assessment were compared with each other and were found to be significantly correlated with each other .\u22121 protein and 6.7\u2009fmol\u2009\u03bcg\u22121 DNA which is consistent with previously reported series . However, the Western blots were quantitated on the basis of control lanes loaded according to ATase activity and not total ATase protein and this might not be ideal. Two parameters of quantitative IHC, integrated grey and mean grey, were also correlated with each other (P<0.002) and with biochemical and Western blot estimates (P=0.004\u20130.04).There have been relatively few similar studies on which to base an assessment of whether biochemical assay or IHC is the clinically relevant score. O6-alkylating agents such as DTIC (O6-alkylating agents than those with tumours expressing higher levels. However, given the heterogeneity of ATase expression, individual, or even multiple, biopsies and IHC assessment or in vitro assay is unlikely to be a consistent predictive parameter of response.The present study indicates that there is very extensive heterogeneity of ATase staining and that separate biopsies of the same primary tumour (Figure 4"}
+{"text": "Any weak (positive or negative) electric disturbances of their resting potential are quickly corrected by chloride shifts.Proteins in any solution with a pH value that differs from their isoelectric point exert both an electric Donnan effect (DE) and colloid osmotic pressure. While the former alters the distribution of ions, the latter forces water diffusion. In cells with highly Cl-.In many excitable cells, the spreading of action potentials is mediated through fast, voltage-gated sodium channels. Tissue cells share similar concentrations of cytoplasmic proteins and almost the same exposure to the interstitial fluid (IF) chloride concentration. The consequence is that similar intra- and extra-cellular chloride concentrations make these cells share the same Nernst value for Cl- ions needs to be >20 times higher outside the cell.Further extrapolation indicates that cells with the same chloride Nernst value and high chloride permeability should have similar resting membrane potentials, more negative than -80 mV. Fast sodium channels require potassium levels >20 times higher inside the cell than around it, while the concentration of ClWhen osmotic forces, electroneutrality and other ions are all taken into account, the overall osmolarity needs to be near 280 to 300 mosm/L to reach the required resting potential in excitable cells. High plasma protein concentrations keep the IF chloride concentration stable, which is important in keeping the resting membrane potential similar in all chloride-permeable cells. Probable consequences of this concept for neuron excitability, erythrocyte membrane permeability and several features of circulation design are briefly discussed. This theoretical paper seeks to interpret similarities in pH, electrolyte and protein compositions of body fluids among diverse animals as requirements imposed by their excitable tissues, particularly neurons and muscle cells.+ concentration is determined by the average osmotic burden on animal tissue cells.The logic that follows is based on a previously published argument that similar body fluid osmolarity in various animals is dictated by the opposed Donnan effects of cell proteins and of sodium ions sequestered in the extracellular fluid (ECF) . The conThe presence of proteins in any solution exerts two effects on the traffic of ions and water. The first is the electric Donnan effect (DE), which alters the distribution of ions, and the second is the colloid osmotic pressure, which forces water diffusion. Both phenomena are measurable when a protein-rich fluid is in contact with a protein-free fluid through a semipermeable membrane that does not allow protein molecules to diffuse .+/K+ pumps. They expel 3 Na+ and import 2 K+ in every cycle, so some water also leaves the cell. If the membrane permeability for Na+ ions is low, sodium ions are virtually sequestered in the extracellular fluid (ECF), so although sodium ions are not proteins, their sequestered positive charges alter the ion distribution across the cell membrane. This action is analogous to the Donnan effect: ECF sodium ions pull more anions out of the cell. The only way for the cell to reach osmotic equilibrium is to alter its volume until the concentration of nondiffusible intracellular ions (mainly charges on intracellular proteins) is equal to the concentration of ECF-restricted ions (mainly Na+) [If protein-bound charges on one side of a semipermeable membrane cannot diffuse through the membrane, the distribution of other ions to which the membrane is permeable is altered . These pOwing to the balance of these two opposed Donnan effects, water diffusion is reduced and the osmotic burden on tissue cells is diminished .When the three main body fluid compartments - plasma, interstitial (IF) and cellular fluid - are considered, differences in chloride distribution across cell membranes and capillary walls result not from chloride pumping, but from the Donnan effect of cytoplasmic and plasma proteins . Both ce--permeable is more dependent on the cytoplasmic pH value that alters the Donnan effect on chloride ions than on the momentary pumping of sodium and potassium ions. The skeletal muscle resting potential is very close to the chloride Nernst potential since the membrane is highly permeable to chloride ions . A possThe presence of proteins in solution forces water to diffuse from the protein-free compartment and dilute the protein-containing fluid. This pressure is pH-independent and is mainly related to the number of protein molecules acting as particles in solution.Proteins in any solution with a pH value that differs from their isoelectric point always exert both a DE and colloid osmotic pressure. Since the normal pH of our body fluids ranges from 6.8 (cell fluid) to 7.35 , normal body fluid pH values are much higher than the average amino acid isoelectric point. This makes all proteins in our body fluids negatively charged . So, eve- ions act as stabilizers of their resting membrane potential because of high chloride permeability [--permeable cells. The consequence is that similar intra- and extra-cellular chloride concentrations make these cells share the same Nernst value for Cl-. A further extrapolation is that these cells, with the same chloride Nernst value and high chloride permeability, should have similar resting membrane potentials, more negative than -80 mV.The model presented here is focused on excitable cells in which Cleability ,5. VarioIf cells are surrounded with protein-containing fluid, the Donnan effect of the cytoplasmic proteins is opposed by the same effect of the extracellular proteins. Two opposed Donnan effects result in higher intracellular chloride concentrations, as has been reported in blood cells that normally float in protein-rich plasma . In thes- ions described above (from protein-rich to protein-poor fluid), the highest chloride concentrations are reported in cerebrospinal fluid (CSF), interstitial tissue fluid and lymph [- ions, so interstitial chloride ions are forced to remain in the tissue to be returned to the blood only via lymph drainage, helped by skeletal muscle work. In other words, high plasma protein levels maintain the stability of the IF chloride concentration through their Donnan effect, important in keeping the resting membrane potential similar in all chloride-permeable cells.Because of the DE-mediated shift of Clnd lymph ,7,8. ThuThe model presented here is shown in Fiure 1, which is organized in two sections with four paths. Each path is coded by a different letter and color. The pivotal fields that link cell physiology with body systems have been intentionally left without color code in the center of Figure A1 to A9: from the spreading of action potentials to cellular control of pH and isotonicity in all body fluid compartments and the plasma protein content.The following discussion refers to the left hand side of Figure Optimal function of neurons, skeletal and heart muscle cells is a prerequisite for animal mobility and survival. Cellular reactions depend on prompt and well-defined occurrence and spreading of action potentials. Action potential spreading in the aforementioned cell types is mediated through fast, voltage-gated sodium channels. These channels initiate rapid depolarization of the surrounding cell membrane, when the local membrane potential is altered from the resting <-80 to -55 mV or less negative.Several features are important for the function of fast sodium channels:Membrane permeabilities to sodium, potassium and chloride ions define the resting membrane potential, as defined by the Goldman equation Table .A1) can be achieved only if the membranes are almost impermeable to Na+ (field A2), since any substantial sodium current would make the resting membrane potential less negative than -80 mV, and this would leave sodium channels inactive after repolarization.A sufficiently low resting potential that allows the sodium channels to function Figure , A1 can A3) and chloride (field A4) moves the resting membrane potential to the required range, more negative than -80 mV, if the concentration gradients for these two ions are sufficient:Combined high permeability for potassium , which requires normal cytoplasmic pH.- Cl+ Nernst potential more negative than -80 mV. This means that the K+ concentration needs to be >20 times higher inside the cell than outside. The situation is similar to that of Cl- ions: >20 times higher concentration is required outside the cell to make the Nernst potential for chloride more negative than -80 mV. This means that a high concentration of chloride ions is needed in the IF and of potassium ions in the cytoplasm. Without these gradients, the resting membrane potential would be less negative than -80mV and this would compromise the spreading of action potentials through the activation of fast sodium channels. When osmotic forces, electroneutrality and other ions are taken into account with >20 times ionic gradients, the overall osmolarity needs to be near 280 to 300 mosm/L, or isotonic (field A9). In short, the necessity for a stable resting potential more negative than -80 mV in our excitable tissues determines body fluid osmolarity.Sufficient potassium gradients are associated with a KA7). A similar action is exerted if the extracellular protein concentration is excessive (field A6). A possible extrapolation is that the requirement for a stable resting potential in our excitable tissues might have determined the low concentration of IF proteins, making animal capillaries less protein-permeable. The same argument stands for the fine control of cellular and extracellular pH values (field A9).The Donnan effect of cytoplasmic proteins depends on the intracellular pH. The electric fields surrounding cell proteins are less strong if there is intracellular acidosis. This diminishes the effect of proteins on the ion distribution within the cell and in its vicinity . This setting allows higher Cl- concentrations to be established in the cerebrospinal fluid and interstitial fluid than in the plasma. The consequence is that similar chloride gradients are expected in all neurons with normal cytoplasmic pH, making their resting potentials similar and stable (field B3). This stability can be compromised under the following conditions:The high permeability of neurons to chloride ions  position- ions across their membranes (reduced IF chloride and increased cellular chloride concentrations).Diseases that accumulate extracellular proteins behind the blood brain barrier can alter neuron function through an altered gradient of ClIn edematous neurons, more cellular water dilutes the cell proteins, so the charges are less dense, and this allows more chloride to remain inside. This makes the resting membrane potential less negative.C1 to C3: the transport of gases, the chloride shift, and the membrane permeability of red blood cells.The following discussion relates to the right hand side of Figure The path describes the effect of RBC membrane permeability on the chloride shift and transport of gases:- ions enter or leave erythrocytes without difficulty; evolution developed RBCs with membranes permeable to sodium (field C1). The consequence is that the normal RBC membrane potential is close to the Nernst potential of these cells for chloride ions [- traffic and this enhances the capacity of RBCs for gas transport (fields C2 and C3).Gas transport in RBCs is helped by the chloride shift through the erythrocyte membrane every time an RBC enters pulmonary or peripheral capillaries . Since tide ions . In thisD1 to D3: plasma colloid osmotic pressure, capillary permeability and the design of the circulation.The following discussion relates to the right hand side of Figure This path describes the effect of plasma colloid osmotic pressure on circulatory design:In some fish, the capillaries are so permeable to proteins  that theD1). The consequence for mammals is that lymphatics are less dense and carry only some 10% of the tissue fluid back to the circulation, while the rest is reabsorbed by the peripheral capillaries.The reported higher arterial pressures in terrestrial amphibians  can be aD4). More proteins require even higher arterial pressures to ensure the expected 20% filtration fraction in the kidneys. The compromise plasma protein content, shared by humans and various other animals, is near 70 g/L, combined with a mean arterial pressure near 90 mmHg. This setting seems to provide skeletal muscle perfusion pressures that should suffice to survive the expected challenges before procreation.Although it might seem that higher concentrations of plasma proteins mean that even greater hydrostatic pressure and higher perfusion rates can be applied in narrow capillaries without risking tissue edema, a ceiling on plasma protein concentration is imposed by the kidneys (field D2 and D3).This optimal level of plasma proteins is matched by an adequately low capillary wall permeability for proteins, allowing the perfusion rates in pulmonary and peripheral capillaries to be high without developing pulmonary or peripheral tissue edema (fields The model presented here is focused on interactions that link seemingly unrelated aspects of our circulation in concordance with the reported densities of sodium pumps: a small number on red blood cells and a high density on neurons and muscle cells . The dif- Nernst potential value, which allows chloride shifts to occur easily in both directions. This feature of cell membranes reduces the chances of accidental action potential propagation. A possible important exception might be the heart pacemaker cells. Their unstable resting membrane potential depends on high sodium permeability [+/Ca++ exchanger. For pacemaker cell function, Cl- permeability seems less important than Na+/K+ pumping, making these cells more sensitive to ouabain exposure than to internal acidosis.Most excitable cells depend on sodium channels for action potential spreading so their resting potentials are anchored by the Cleability ,14 and iIf the plasma protein level allows higher pressures and perfusion rates to develop in skeletal muscle capillary beds, a relationship is expected between hemoglobin and albumin levels in different animals. Normal blood value data can be found for mammals , but unfFigure"}
+{"text": "In vivo self-endothelialization by endothelial cell adhesion on cardiovascular implants is highly desirable. DNA-oligonucleotides are an intriguing coating material with nonimmunogenic characteristics and the feasibility of easy and rapid chemical fabrication. The objective of this study was the creation of cell adhesive DNA-oligonucleotide coatings on vascular implant surfaces. DNA-oligonucleotides immobilized by adsorption on parylene (poly(monoaminomethyl-para-xylene)) coated polystyrene and ePTFE were resistant to high shear stress (9.5\u2009N/m2) and human blood serum for up to 96\u2009h. Adhesion of murine endothelial progenitor cells, HUVECs and endothelial cells from human adult saphenous veins as well as viability over a period of 14 days of HUVECs on oligonucleotide coated samples under dynamic culture conditions was significantly enhanced (P < 0.05). Oligonucleotide-coated surfaces revealed low thrombogenicity and excellent hemocompatibility after incubation with human blood. These properties suggest the suitability of immobilization of DNA-oligonucleotides for biofunctionalization of blood vessel substitutes for improved in vivo endothelialization. Current blood vessel replacement concepts, which use either prosthetic or biological grafts, achieve excellent mid-term results. The clinical application, however, is accompanied by a variety of limitations. Biological conduits  have better hemodynamic characteristics and avoid long-term anticoagulation but are limited in availability. On the long term, however, they fail due to intimal hyperplasia and fibrosis. Allograft transplants such as superficial femoral arteries have optimal hemodynamic properties, avoid any anticoagulation, and are resistant to infections to a certain extent. However, due to tissue scarcity, their availability is limited. In general, 5\u201330% of patients have no suitable autologous grafts available due to previous use or concomitant disease. In these cases, expanded polytetrafluoroethylene (ePTFE) grafts are used. However, despite their prevalence, these grafts have a lower patency rate due to noncompliance, thrombogenic surface, and the tendency to form intimal hyperplasia in particular in anastomotic areas . None ofin vitro established autologous endothelial cell layer on the implant surface prior to implantation provides an excellent barrier between the synthetic material and the blood flow and has been reported to reduce thrombosis and intimal hyperplasia [in vitro culture (which takes up to 6 weeks from cell harvest to implantation of the engineered product), the process requires a cost-intensive infrastructure.The multidisciplinary approach of tissue engineering might offer an attractive pathway to overcome these shortcomings and develop blood vessel substitutes identical or similar to native human arteries. In vascular grafts, an erplasia \u20134. Overcerplasia \u20135. Indeeerplasia . Howeverin vivo endothelialization of vascular implants [in vivo. One is the capture, immobilization and adhesion of circulating endothelial (EC) or endothelial progenitor cells (EPC) from the blood stream. An alternative is promotion of transanastomotic ingrowth of endothelial cells from the native vessel on and into the graft. Both require a suitable graft surface for cell adhesion and proliferation. However, it is reported that EC do not adhere to currently available vascular graft materials like ePTFE and polyethylene terephtalate (Dacron) [in situ endothelialization of cardiovascular implants  would be highly desirable. Different approaches exist, mostly using extracellular-matrix- (ECM-) derived proteins or peptides with functional domains of ECM proteins [Accordingly, ongoing research focuses on the development of facilitated spontaneous implants \u201311. Seve(Dacron) , 13. Fur(Dacron) . Hence, proteins . Another intriguing coating material for implant surfaces is deoxyribonucleic acid (DNA). DNA is a naturally occurring material with a homogenous molecular structure in all vertebrate species , 16. DNAin vivo adherence to the implant surface. These properties are known to be supplied by poly (p-xylylene) (PPX) and its derivates, known by their commercial trade name as parylenes [Apart from controlled cell adhesion, the coating matrix needs to possess excellent mechanical properties in terms of flexibility and long-lasting arylenes , 25. Pararylenes , 27.Here, we report a concept of a stable DNA coating and EC and EPC adhesion on DNA coatings using continuous shear stress application.40-GATGGACGAATATCGTCTCCC-3\u2032. For detection, oligonucleotides were 5\u2032-labeled with 6-carboxyfluorescein (6FAM). For immobilization, a 5\u2032-C12-NH2 modification was used.Oligonucleotides were synthesized by TIB Molbiol . As single-sequence 5\u2032-GGGAGCTCAGAATAAACGCTCAACAACCCGTCAACGAACCGGAGTGTGGCAGGTTCGACATGAGGCCCGGATC-3\u2032 was used. The DNA oligonucleotide library contained a 40-base central random sequence: 5\u2032- GAATTCAGTCGGACAGCG-N\u03b2-mercaptoethanol , 1\u2009mmol/l nonessential amino acids , 100\u2009U/mL penicillin, 100\u2009\u03bcg/mL streptomycin , and 2\u2009mmol/l L-glutamine .Murine embryonic EPC (eEPC) line T17b cells  were culHuman umbilical vein endothelial cells (HUVECs) were cultured in VascuLife VEGF Culture Medium containing 2% FCS .2. The culture medium consisted of DMEM  supplemented with recombinant basic fibroblast growth factor , 100\u2009U/mL penicillin, 100\u2009\u03bcg/mL streptomycin , and 10% FCS.Human ECs were harvested from human saphenous veins. 4 to 5\u2009cm long remnants of saphenous veins from patients undergoing aortocoronary bypass procedures were used as a cell source. Tissue procurement and use were approved by our local ethics committee and informed consent was obtained. The veins were cannulated, flushed with DMEM , and filled with 0.2% collagenase A . Cells were harvested after 20\u2009min of collagenase incubation at 37\u00b0C, 5% CO\u03bcm) diX AM coating was deposited onto a thicker (ca. 2\u2009\u03bcm) intermediate layer of inert Parylene C according to recommendations from the diX AM manufacturer. After deposition, the samples were stored in the dark using argon atmosphere to avoid amine's oxidation.Standard ePTFE vascular prostheses were obtained from Jotec GmbH, Hechingen, Germany (FlowLine Bipore and FlowLine Bipore Heparin), and ePTFE patch material from W.L. Gore & Associates, Inc., Flagstaff, Arizona, USA (Gore-Tex Cardiovascular Patch). ePTFE was activated using microwave plasma  prior to coating with parylenes. Poly (monochloro-para-xylene)  and poly (monoaminomethyl-para-xylene)  were coated on polystyrene dishes, polypropylene sheets, and activated ePTFE using a chemical vapour deposition (CVD) process in a PDS 2010 coater . A general description of parylene CVD processes is given by Lahann . This teThe thickness of the intermediate Parylene C layer was determined by both gravimetry and ellipsometry on reference samples. In the first case, the mass increase of an aluminum foil which was parylene coated in the same deposition run was determined. For ellipsometry, single crystalline Si substrates were used. The thickness of diX AM parylene top coatings was determined by ellipsometry only. The ellipsometric measurements were performed on a SE 801 spectroscopic ellipsometer .\u03b1 X-Ray source. For all samples, overview spectra and detailed spectra of all elements found in the overview were recorded.The samples were stored in dark under argon and transferred into the XPS apparatus within 24\u2009h of deposition. The surface elemental composition was verified by X-ray photoelectron spectroscopy (XPS) analysis using an Axis Nova system  with monochromatic Al KFor determination of the number of chemically available amino groups on the diX AM surface electron spin resonance spectroscopy (ESR) in combination with chemical derivatization was applied . An air bubble was brought in contact with the solid sample from below. After a few seconds, the static contact angle near the three-phase line was measured. For each sample, at least six contact angles were measured.\u03bcM DNA-oligonucleotides were applied to the diX AM-coated surface in HEPES buffer  at room temperature. After 2\u2009h, Tris-HCl was added to a concentration of 50\u2009mM and incubated for 15\u2009min. For experiments with cell incubation, the surfaces were washed with 0.2% BSA. Then, a triple washing step with PBS followed. For cell experiments, an incubation with a PBS-antibiotic solution  for 24\u2009h at 4\u00b0C was performed. If not stated differently, 1.7\u2009\u03bcM) were exposed to flow-induced shear stress for 1\u2009h at 37\u00b0C in a closed circular system. The system consisted of silicon tubing, a fluid reservoir, a peristaltic pump, and a following vessel to attenuate pump-induced pressure variability. The test fluid consisted of 1000\u2009ppm xanthan gum. Wall shear stress for 1000\u2009ppm Xanthan gum, a non-Newtonian power-law fluid [DiX AM-coated polypropylene sheets with applied oligonucleotides  was incubated with serum derived from fresh human blood for 15\u2009min, 24\u2009h, 48\u2009h, 72\u2009h, and 96 hours at 37\u00b0C. Serum was changed every 24 hours. Amount of oligonucleotide remaining on diX AM surface was determined.DiX AM coated with oligonucleotides . For confirmation of the results and better quantification, an ELISA system was developed, using an antifluorescein antibody  and an alkaline-phosphatase-conjugated secondary antibody . Incubation time was 2\u2009h at room temperature each. As substrate Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System for ELISA (Sigma) was used. 6 cells for 1\u20132.5\u2009h at 37\u00b0C, 5% CO2 in cell-corresponding or experiment-dependent medium or buffer on a rocking orbital shaker . Following medium exchange to remove nonadherent cells, Petri-dishes were inspected by phase-contrast microscopy . Cells in images with the same area were counted. An alamarBlue assay  was performed after 22\u2009h to determine the relative number of metabolically active adherent cells. 150\u2009\u03bcL alamarBlue reagent were added to the samples in 1.5\u2009mL medium and incubated for four hours at 37\u00b0C and 5% CO2. Two times 100\u2009\u03bcL of each sample were pipetted into a clear flat bottom microplate and measured at 530\u2009nm extinction and 600\u2009nm emission wavelength . Oligonucleotides were applied to marked areas or the whole surface of diX AM coated Petri-dishes or diX AM-coated 12-well plates. Petri-dishes were incubated with 0.05 \u2217 106 HUVECs at 37\u00b0C, 5% CO2 in medium containing FCS on a rocking orbital shaker . After one hour medium including non-adherent cells was removed. Adherent cells were fixed with 4% paraformaldehyde (Sigma) for 10\u2009min at room temperature. After a washing step with PBS (Lonza), PBS containing 2% FCS and 0.1% Triton (Sigma) was applied for 15\u2009min. Samples were incubated over night at 4\u00b0C with the primary antibodies , abcam, Cambridge, UK) in PBS containing 2% FCS and 0.1% Triton. After a washing step, the secondary antibody , abcam, Cambridge, UK) was applied for 1\u2009h at 37\u00b0C. Cells were stained with phalloidin  for 20\u2009min at room temperature following a further washing step with PBS. As a last staining step, cells were incubated with DAPI (Sigma) for 2\u2009min. Following staining of adherent cells, Petri-dishes were inspected by fluorescence microscopy at a magnification of 200x .Oligonucleotides were applied to marked areas of diX AM coated Petri-dishes. Oligonucleotide-coated and diX AM-coated Petri-dishes and tissue-culture Petri-dishes were incubated with 0.05 \u2217 104 HUVECs were seeded into each well. After 2.5\u2009h, incubation using continuous agitation  medium was exchanged and nonadherent cells removed. Cell viability was measured with the alamarBlue assay after 1, 2, 5, 8, 12, and 14 days.For viablity tests, standard ePTFE vascular prostheses either uncoated or coated with heparin  or ePTFE (FlowLine Bipore) coated with Parylene C, diX AM, and oligonucleotieds as described above were used. The three differently coated materials were cut into samples and glued with silicone paste into 48-well suspension culture plates. 3 \u2217 10\u03bcL supernatant was used for thrombin-antithrombin complex  and PMN-Elastase determination . 5.4\u2009mL blood from each group was added to 0.3\u2009mL of CTAD anticoagulant monovettes . Tubes were placed on ice for 15\u2009min. Following centrifugation at 2500\u2009g for 20\u2009min at 4\u00b0C 1.4\u2009mL, plasma from the middle fraction was aspirated and transferred to a new neutral monovette. After repeated centrifugation, 200\u2009\u03bcL plasma was used for determination of \u03b2-Thromboglobulin .12-well Polystyrene plates and ePTFE patches  were coated with diX AM and oligonucleotides as described above. ePTFE was cut into 12-well-sized samples and glued into 12-well suspension culture plates using silicone paste. Human blood samples were obtained from six healthy volunteers following giving informed consent . Whole blood from each volunteer was collected in heparin precoated monovettes . Two 12-wells in each group were incubated with 3\u2009mL donor blood each. After 1\u2009h incubation at 37\u00b0C with continuous shaking, the two blood samples of each group were pooled and further processed using the following protocols. 2.4\u2009mL blood from each group was added to an EDTA-monovette. Blood cell count, including platelet count, was performed using a Cobas Micros 60 S/N . 2.8\u2009mL blood from each group was collected in a Citrate-monovette. Monovettes were centrifuged at 2000\u2009g for 15\u2009min at room temperature. 600\u2009t-test was performed between each of two groups in case of a significant statistical difference existed. P values < 0.05 were considered statistically significant. All quantitative results were expressed as mean standard deviation. The differences among groups were analyzed with one-way ANOVA, and independent samples 2.Substrates were coated with parylene C as an intermediate layer and with diX AM as top coating. Coating thicknesses measured by ellipsometry on a simultaneously coated Si wafer were 550\u20132200\u2009nm for parylene C and 40\u2013120\u2009nm for diX AM, depending on charge deposition parameters. The elemental composition in percent of total number of atoms excluding hydrogen of the surface measured by XPS was as follows: N-6.2 at.% , C-90.7 at.%, Cl-1.9 at.%, and O-1.2 at.%. Chlorine is not contained in diX AM but makes up 14.2 at.% of Parylene C, which was the intermediate layer under diX AM. Thus, we presume the Cl signal measured by XPS to originate from Parylene C. As the diX AM coating was thicker than the XPS sampling depth of 5 to 10\u2009nm, we interpret the Cl signal as an indication that the diX AM coating was not completely closed. Concerning Oxygen content, amines are generally known to be prone to oxidation. St-Georges-Robillard  have recP < 0.001; To examine if changed surface wettability has an impact on cell adhesion, samples were analyzed by the captive bubble method. Samples coated by a single oligonucleotide sequence or by the oligonucleotide library had significantly lower contact angles resulting in increased hydrophilic surface characteristics compared to diX AM .To exclude degradation of the applied oligonucelotides by human blood serum, samples were incubated between 15\u2009min and 96\u2009h with human blood serum. The mean values of each time point differed by \u00b19.6% from the mean of the sample without serum incubation . No sign2 on each coating and cell type is shown. Cell number on oligonucleotide-coated surfaces was significantly higher than on uncoated diX AM or the HEPES control (P < 0.05). Thus, oligonucleotides enhanced the adhesion of all cell types compared to cell adhesion on diX AM or buffer-treated diX AM. Experiments (n = 9) containing controls on tissue culture (TC) surface were conducted with HUVECs. Cell adhesion on oligonucleotide-coated areas was significantly higher than on TC surfaces (P < 0.05), see Cell adhesion experiments were conducted with different cell types in FCS supplied medium . DiX AM samples where oligonucleotides with the same sequence, an oligonucleotide library, HEPES, or nothing of all was applied were used. Cells of a murine embryonic EPC cell line (eEPCs) adhered predominantly to areas coated with either the single oligonucleotide sequence or the oligonucleotide library . HUVECs 2+/Mg2+, PBS with Ca2+/Mg2+/Glucose or serum-free eEPC medium no significantly enhanced adhesion to oligonucleotide-coated areas could be detected .In a further experiment, it was investigated if the amount of oligonucleotide used for coating the diX AM surface plays a role in efficiency of cell adhesion. Different concentrations of the single oligonucleotide sequence and the oligonucleotide library were applied to a diX AM-coated 12-well plate. 5 \u00d7 10 visible . Also, tTo further characterize cell adhesion, samples were incubated with HUVECs for 1\u2009h and adherent cells were stained for Vinculin, CD49e  and F-Actin. Cells on oligonucleotide-coated surfaces showed a flat morphology and covered a bigger area than cells on diX AM control surfaces which showed a spherical morphology . Cell moViability of HUVECs over a period of 14 days was compared between two standard vascular prosthetic materials, ePTFE and ePTFE coated with heparin, and the newly developed coating, diX AM and oligonucleotides on ePTFE. Cell viability was significantly higher on oligonucleotide-coated ePTFE for all time points compared to ePTFE and ePTFE coated with heparin . 2.5\u2009h a\u03b2-thromboglobuline (\u03b2-TG) and PMN-elastase, respectively. \u03b2-TG is stored in the \u03b1-granules of platelets and released in large amounts after platelet activation. The release of polymorphonuclear- (PMN-)elastase takes place after activation of neutrophile granulocytes or collapse of these cells. It is broadly used to measure granulocyte activity during inflammatory response. Values for \u03b2-TG for oligonucleotide coated and uncoated dix AM surfaces did not differ significantly from negative control (P > 0.05). PMN-elastase was released to a higher extent by oligonucleotide-coated diX AM surfaces but there was no significant increase compared to the negative control (P > 0.05). Surface-dependent activation of the coagulation system was detected by measuring thrombin-antithrombin III (TAT). TAT is an inactive proteinase/inhibitor complex resulting from thrombin and its inhibitor antithrombin III. An increased TAT level is an indicator for thrombotic events. TAT level was not significantly increased for any of the samples (P > 0.05).Uncoated ePTFE, diX AM coated suspension cell culture plates and diX AM- and oligonucleotide-coated ePTFE samples were incubated with human blood for 1\u2009h at 37\u00b0C on an orbital shaker. As negative control, wells of a suspension cell culture plate were used. The experiment was repeated with six different blood donors. To quantify cell adhesion platelets, erythrocytes and leucocytes were counted in blood samples following incubation. ANOVA tests revealed no significant decrease in number and therefore no significant adhesion to any of the samples . SurfaceInterfaces between implant surfaces and recipient tissue or blood play a crucial role for long-term performance and patency of cardiovascular implants. This study analyzed a novel oligonucleotide-coated diX AM layer in terms of coating stability, EC and EPC adhesion and viability under continuous shear stress application and hemocompatibility. These criteria were chosen as representative key characteristics for cardiovascular implants. Application of the coating concept for other implants is also conceivable.in vivo endothelialization either by EC migration from adjacent native tissue or by EPC capture and adhesion from the blood stream. Stability of the oligonucleotides coated on the diX AM layer was excellent. The applied oligonucleotides withstand shear stress in the amount of shear stress occurring in blood vessels in vivo. Additionally, the oligonucleotides were not degraded by human blood serum during prolonged exposure of 96 hours. Maintained hemocompatibility was confirmed by testing for thrombocyte, granulocyte, and coagulation system activation.The oligonucleotide coating showed excellent adhesion properties for human EC and murine EPC under continuous shear stress application as well as good viability properties over a period of 14 days for HUVECs. Cell morphology of HUVECs after adhesion on oligonucleotide-coated surfaces was similar to cell morphology of cells adhered to tissue culture surfaces. This qualifies the material for A combination of two parylenes, with an intermediate layer of Parylene C and the outer layer of diX AM, an amino-modified parylene derivate, was chosen as a protection film between synthetic material and tissue or blood, as polymers of the parylene family are known to offer good elasticity, biostability and biocompatibility and can be coated easily to different substrate materials even of irregular shape. Parylene C coatings are currently used as protection films for biomedical devices, for example, metal stents, implantable electrode probes, and electronic circuitries , 26, 38.2 was applied to the samples, which exceeded the maximum shear stresses observed in blood vessels and heart valves [These oligonucleotides were robustly immobilized on the diX AM surface. Shear stress up to 9,5\u2009N/mt valves , 35, 40.t valves , 37. Shet valves . This waInterestingly, it did not matter whether a single sequence or an entire oligonucleotide library was used, both enhanced cell adhesion equally. The mechanism of enhanced attachment, therefore, is more likely to be dependent on the overall characteristics of DNA-oligonucleotides than on a specific sequence. As cells were only able to adhere, when they were added to the oligonucleotide-coated surface in FCS supplied medium or when the coated samples were preincubated in medium containing FCS, the mechanism of enhanced cell attachment maybe mediated through attachment-facilitating serum proteins deposited on the surface from FCS. It has been reported in the literature that deposition of serum proteins like fibronectin and vitronectin contained in serum-supplemented culture medium on cell culture material surfaces mediates initial cell attachment, spreading, and behavior , 42. No Viability of the adhered cells was measured over a time period of 14 days. As a high number of cells were used for seeding, samples were assumed to be confluent on day one. Viability on oligonucleotide-coated samples decreased to 43 \u00b1 5% on day 14. As the alamarBlue assays measure metabolic turnover, this might be due to less metabolic activity as further cell proliferation might not have been possible. Measured viability on ePTFE and ePTFE with heparin decreased more dramatically. In these cases, cell loss is probable. ePTFE coated with diX AM and oligonucleotides, therefore, showed considerably improved properties for cell growth. Additionally, the similar cell morphology of HUVECs adhered on oligonucleotide-coated surfaces like on tissue culture surfaces as shown by staining for Vinculin, CD49e, and F-Actin confirms the good properties for endothelial cell growth of oligonucleotide-coated surfaces.\u03b2-TG), granulocytes (PMN-elastase) or the coagulation system (TAT) by oligonucleotide-coated ePTFE. Values for all examined variables were in the same range as the values for clinically routinely used standard ePTFE material . Furthermore, the determined values were in a similar range as known values for materials with excellent hemocompatibility previously published: \u03b2-TG ranges around 500\u2009IU/mL for biopassive or active coated membrane oxygenators  [\u03bcg/L for coated membrane oxygenators [\u03bcg/L for star-PEG-coated surfaces [\u03b2-TG [\u03bcg/L for PMN-elastase [\u03bcg/L for TAT [\u03bcm2. To confirm the applicability of the presented DNA coating for in vivo endothelialization of cardiovascular implants, further experiments to clarify the adhesion properties of EC and EPC in human blood vessel environment conditions are required. As diX AM can be coated to substrates of various shapes and material, the coating process can be easily transferred to currently used vessel graft materials. In this work, we developed the successful coating of ePTFE with diX AM and oligonucleotides by surface activation using microwave plasma processing and subsequent chemical vapour deposition of diX AM and oligonucleotide adsorption.For blood contacting material, hemocompatibility is a further important point. Many surface modifications used for engineering of material interfaces for EC adhesion that bind EC also will bind platelets and initiate thrombosis. Here, we provide data, demonstrating that the adhesion of erythrocytes, thrombocytes, and leucocytes was low to the oligonucleotide-coated ePTFE. This is an indication that oligonucleotide-coated diX AM surfaces do not activate inflammatory or thrombogenic responses. The measured values confirmed this result by not showing any activation of thrombocytes (Germany) , glutaraGermany)  or star-Germany) . Values genators , 56. Datsurfaces  and nitisurfaces . Positives [\u03b2-TG \u201355, 450\u2013elastase , 56, and for TAT . Other s for TAT , 17, 18. for TAT , 16. Advin vivo endothelialization after implantation. Additionally, immobilization of oligonucleotides on other types of implants, for example, orthopedic tissues might enhance their incorporation into surrounding tissues.This study revealed DNA-oligonucleotides coated to diX AM surfaces as attractive coating materials for cardiovascular implants. They are shear resistant, nondegradable by human blood serum, offer excellent adhesion properties for EC, and are hemocompatible. Furthermore, they might support adhesion of circulating EPC or transanastomotic ingrowth of EC. This opens new opportunities for the manufacturing of \u201coff-the-shelf\u201d cardiovascular implants to achieve"}
+{"text": "Brassica napus seeds to investigate the mechanisms of aging initiation. B. napus seeds were exposed to artificially aging treatment (40\u00b0C and 90% relative humidity) and their physio-biochemical characteristics were analyzed. Although the treatment delayed germination, it did not increase the concentration of cellular reactive oxygen species (ROS). Comparative proteomic analysis was conducted among the control and treated seeds at different stages of germination. The proteins responded to the treatment were mainly involved in metabolism, protein modification and destination, stress response, development, and miscellaneous enzymes. Except for peroxiredoxin, no changes were observed in the accumulation of other antioxidant enzymes in the artificially aged seeds. Increased content of abscisic acid (ABA) was observed in the artificially treated seeds which might be involved in the inhibition of germination. Taken together, our results highlight the involvement of ABA in the initiation of seed aging in addition to the ROS which was previously reported to mediate the seed aging process.Plant seeds lose their viability when they are exposed to long term storage or controlled deterioration treatments, by a process known as seed aging. Based on previous studies, artificially aging treatments have been developed to accelerate the process of seed aging in order to understand its underlying mechanisms. In this study, we used  Successful germination of seeds is a prerequisite for plants to initiate their life cycle and to distribute their progeny, and is largely determined by the seed vigor   from Arabidopsis and lotus (Nelumbo nucifera) could enhance the seed vigor and longevity  plants were grown in green house under natural light condition in Wuhan, China. The non-dormant seeds were harvested in May of each year and used as experimental materials. Freshly harvested B. napus seeds were treated with high temperature and humidity according to Rajjou et al.  treatment and gibberellic acid (GA) recovery experiments, seeds were imbibed with 10The ion leakage was calculated as described previously  content was measured using a commercial kit  according to the manufacturer's protocol, which is based on the reaction between MDA and thiobarbituric acid to produce a red compound. In brief, 0.2 g of seeds were homogenized with 2 mL of ice-cold phosphate buffer and centrifuged at 1600 \u00d7 2O2 was carried out according to the method described before , and then centrifuged again at 6000 \u00d7 g for 15 min at room temperature. The absorbance of the supernatant was measured at 410 nm. The concentration of H2O2 was calculated based on the standard curve which was made with a series of H2O2 solutions with known concentration. All the measurements were conducted for three biological replicates.Measurement of H2H6] ABA, 50 ng/g) was added to each of the samples to serve as internal standards for the quantification. Then, the supernatants were sequentially passed through the tandem solid phase extraction (SPE) cartridges containing C18 adsorbent (50 mg) and SAX adsorbent (200 mg). Before SPE extraction, the tandem cartridges were pre-conditioned with 8 mL H2O, 8 mL methanol, and 8 mL modified Bieleski solvent. After sample loading, the C18 cartridge was removed and the SAX cartridge was rinsed with 2 mL methanol/H2O . After that, 3 mL acetonitrile (CAN) with 1% Hydrofluoric acid (FA) (v/v) was applied to elute the targeted ABA and the eluent was evaporated under mild nitrogen stream at 35\u00b0C followed by re-dissolving in 100 \u03bcL H2O. The resulting solution (100 \u03bcL) was then acidified with 10 \u03bcL FA, and extracted with ether (2 \u00d7 1 mL). The ether phase was combined, dried under nitrogen gas and reconstituted in 100 \u03bcL ACN. To the resulting solution, 10 \u03bcL triethylamine (TEA) (20 \u03bcmol/mL) and 10 \u03bcL 3- Bromoactonyltrimethylammonium bromide (BTA) (20 \u03bcmol/mL) were added. The reaction solution was vortexed for 30 min at 35\u00b0C and evaporated under nitrogen gas followed by re-dissolving in 200 \u03bcL H2O/ACN  for instrumental analysis. The calibration curve was constructed by comparing peak area ratio  to concentrations. The content of ABA was calculated according to the calibration curve. Three biological replicates were conducted.ABA concentration was measured using a derivatization approach coupled with nano-LC-ESI-Q-TOF-MS  as described previously -3-(4-nitrophenyl)-5--2H-tetrazolium (WST-1) with xanthine oxidase (XO) to generate O2\u2212 and formazan dye, which can be inhibited by SOD through catalyzing O2\u2212 into H2O2 and O2. SOD activity can be calculated by determining the absorbance of formazan dye at 450 nm.Total superoxide dismutase (SOD) activity was measured using a commercial WST-1 kit  following manufacturer's protocol. Briefly, seeds were powdered using liquid nitrogen and then homogenized in phosphate balanced solution buffer . The mixture was centrifuged at 12,000 \u00d7 2O2 was mixed with 5 \u03bcL of protein supernatant. The H2O2 was decomposed by CAT for 5 min, and the remaining H2O2 coupled with a substrate was treated with peroxidase (POD) to generate N-4-antipyryl-3-chloro-5-sulfonate-p-benzoquinonemonoimine. CAT activity was determined by calculating the decomposition rate of H2O2at 520 nm. Both enzymes were assayed for three replicates.Catalase (CAT) activity was assayed using commercial kit  as described previously  and vortexed for 20 min. Mixture was then centrifuged at 12000 \u00d7 g for 15 min at 4\u00b0C. After centrifugation, the supernatant phenol phase and intermediate denatured protein layer were collected. Phenol phase containing proteins was then mixed with 5 volumes of 0.1 M ammonium acetate in methanol and incubated at \u221220\u00b0C overnight. Pellet, obtained after centrifugation at 12000 \u00d7 g for 30 min, was washed 4 times with ice-cold acetone and vacuum dried. For two-dimensional electrophoresis (2-DE), the dried proteins pellets were dissolved in rehydration buffer containing 7 M urea, 2 M thiourea, 4% 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), 0.2% carrier ampholyte, and 65 mM DTT, and quantified according to Bradford's method  250 for 40 min, and then destained with 20% ethanol containing 10% acetic acid. The destained gels were scanned using Epson Perfection\u2122 V700 Photo scanner  at 800 dots per inch (dpi) resolution. The transparency mode was used to obtain a gray scale image. The images were digitized and analyzed with PDQuest\u2122 2-DE Analysis Software 8.0 . The relative volume of each spot, which is defined as is the ratio between the volume of the given spot and the total volume of all the spots displaying on the gel, was used to represent the corresponding protein abundance. To obtain reproducible result, three biological repeat experiments were conducted. All the 12 gels were digitized separately. Automatic matching between different gels was conducted with four spots as internal standards. After that, manual adjustment was carried out to avoid mismatching. Spots which were detected in all the three replicates were used for comparative analysis. The spots showing more than 2-fold changes in abundance were defined as differentially displayed protein spots.4HCO3 in 50% (v/v) ACN. After complete de-staining, excised spots were first dehydrated using 50 \u03bcL 100% ACN and then rehydrated with 10 pmol trypsin in 25 mM NH4HCO3 at 4\u00b0C for 1 h. Trypsin digestion was carried out at 37\u00b0C overnight. After digestion, the peptides were extracted according to the method described before  using MASCOT as a search engine  through BioTools (version 3.2) interface. The search parameters were set as follows: taxonomy, Viridiplantae; fixed modifications, carbamidomethylation; variable modification, methionine oxidation; MS tolerance, 50 ppm; MS/MS tolerance, 0.5 Da, peptide mass, monoisotopic. Only the significant hits (p < 0.01) with peptide scores >45 were accepted.Differentially displayed protein spots were excised from the gels and distained using 50 mM NHt-test when only two groups were compared, or with One-Way ANOVA followed by Tukey's multiple comparisons test for all other comparisons based on the three independent replicates.The statistical analyses were carried out using Student's B. napus gradually lose their viability during long term storage, which is defined as natural aging. To better understand the process of natural aging, germination percentages of 1 year aged seeds and freshly harvested seeds were calculated. Freshly harvested seeds started germination just after 6 h of imbibition and took less than 12 h for 50% percent of the seeds to germinate  are two important environmental factors that influence seed aging. High temperature and moisture accelerate the process of seed aging, leading to the rapid loss of seed vigor. The accelerated aging has been applied as an indicator of crop seed storability (Priestley, B. napus seeds, we did not detect any over accumulation of ROS (Figure The CDT resulted in the increased ion leakage Figure , which rS Figure , and anyS Figure . Since oS Figure , suggestS Figure . The CDT2O2 treatment preferentially regulates proteins majorly involved in redox homeostasis, metabolism, hormones related and RNA related, and it could invoke the changes of proteins with similar functions in different tissues (Wan and Liu, B. napus seeds is quite different with that indicating in case of pea and wheat seeds. Our results suggest that CDT treatment might initiate the aging of B. napus seeds through enhancing the biosynthesis of germination inhibitors and lately through the accumulation of ROS. Although there were 81 differentially accumulated proteins between CK and CDT seeds, only 49 spots were successfully identified. The identification rate is just about 60%, which might be explained by the un-sequenced genome of B. napus. Moreover, there seven spots were identified as two different proteins, which should result in difficulties to evaluate a real abundance of each protein within them. This is one of the limitations in 2-D gel based proteomic techniques. Future studies with gel-free systems might help to provide more information.Functional classification of the identified proteins showed that proteins related to protein modification and destination, development and cell structure were differentially modulated after CDT. However, previous studies on pea (Barba-Espin et al., B. napus seeds. Among the identified proteins, most of the proteins involved in metabolism and protein destination were decreased in the CDT seeds. These results indicate that the basic biological activities might be inhibited by the CDT treatment, which would result in the delayed germination. Interestingly, rubisco large subunit was increased in the CDT treated seed at 18 h after germination, which indicates the existence of feedback regulation. In contrast to the metabolism and protein destination related proteins, many cell structural proteins and miscellaneous enzymes, such as actin, mannose-binding lectin superfamily protein, glycosyltransferase, beta-glucosidase, were increased. All these proteins are related to the cell and cell wall structures. It might be interesting to know how these proteins affect seed aging.Since the CDT treatment delayed the germination not primarily because of accumulation of ROS, we speculated that there should be some other mechanisms which mediate the seed aging process in B. napus seeds showed that there was a sharp increase of ABA content in the CDT treated seeds (Figure B. napus seeds, is still elusive. Unfortunately, we did not detect any changes of the enzymes involved in the ABA biosynthesis and degradation. It is well known that ABA is synthesized in the seeds during desiccation which allow the survival of seeds in dry state (Tan et al., B. napus seeds during aging.Among all the internal factors, ABA has been reported to be one of the main factor that inhibits the seed germination (Gubler et al., s Figure . Althougs Figure . Furthers Figure . GerminaYX did the experiments; HD prepared the seeds and analyzed some of the data; YP designed the experiments and wrote 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."}
+{"text": "OsHSP18.2 transcript was found to markedly increase at the late maturation stage being highly abundant in dry seeds and sharply decreased after germination. Our biochemical study clearly demonstrated that OsHSP18.2 forms homooligomeric complex and is dodecameric in nature and functions as a molecular chaperone. OsHSP18.2 displayed chaperone activity as it was effective in preventing thermal inactivation of Citrate Synthase. Further, to analyze the function of this protein in seed physiology, seed specific Arabidopsis overexpression lines for OsHSP18.2 were generated. Our subsequent functional analysis clearly demonstrated that OsHSP18.2 has ability to improve seed vigor and longevity by reducing deleterious ROS accumulation in seeds. In addition, transformed Arabidopsis seeds also displayed better performance in germination and cotyledon emergence under adverse conditions. Collectively, our work demonstrates that OsHSP18.2 is an aging responsive protein which functions as a molecular chaperone and possibly protect and stabilize the cellular proteins from irreversible damage particularly during maturation drying, desiccation and aging in seeds by restricting ROS accumulation and thereby improves seed vigor, longevity and seedling establishment.Small heat shock proteins (sHSPs) are a diverse group of proteins and are highly abundant in plant species. Although majority of these sHSPs were shown to express specifically in seed, their potential function in seed physiology remains to be fully explored. Our proteomic analysis revealed that OsHSP18.2, a class II cytosolic HSP is an aging responsive protein as its abundance significantly increased after artificial aging in rice seeds.  Nelumbo nucifera was shown to increase seed germination vigor and seedling thermo tolerance in transgenic Arabidopsis  is a monocot model crop which feeds nearly half of the world population. Most of the cultivated rice varieties have a low or null dormancy and variable periods of storage life. Even under best storage conditions, rice seeds lose their viability and germination vigor  were imbibed overnight in dark and sown in glass bottles over moist cotton bed and a layer of germination paper. The bottles were then kept for normal growth in growth room 28 \u00b1 1\u00b0C and 65% humidity levels. For different stress treatments, 7 day-old seedlings were subjected to various stresses as described by Rice seeds (Arabidopsis seeds were imbibed for 1 h and then blot dried. The seeds were treated with a combination of high temperature (45\u00b0C) and high humidity (100% Relative humidity) for 6 days (for rice) and 4 days (for Arabidopsis) to induce artificial aging  as determined by Mascot were considered for further analysis.Artificially aged rice seeds (250 mg) were ground to fine powder and homogenized with 2 ml of extraction buffer . Homogenate was centrifuged at 15000 g for 15 min at 4\u00b0C and supernatant was collected as total soluble protein. Protein (500 \u03bcg) was precipitated with ice cold 100% acetone and pellets were solubilised in 250 \u03bcl of DeStreak Rehydration solution (GE) with 2% IPG buffer pH 3-10 NL. The protein sample was used to rehydrate IPG strips 13 cm, pH 3-10NL overnight. Isoelectric focussing (IEF) was performed with rehydrated strips on Ettan IPGphor 3 (GE) at 100V\u20132 h; 500V\u20131 h; 1000V\u20131 h; 8000V\u201330 min and finally at 8000V\u20133 h. After IEF, the strips were equilibrated first with 1% (w/v) DTT in equilibration buffer (10 ml of 50 mM Tris-Cl (pH 8.8), 2% SDS, 6 M urea and 30% glycerol) followed by 4% (w/v) iodoacetamide in equilibration buffer. The strips were then loaded on 12.5% polyacrylamide gels for SDS-PAGE. Gels were stained with PlusOne Silver Staining Kit (GE) and analyzed with Image Master Platinum seven software (GE). The protein spots were excised manually and in-gel digestion was performed using trypsin (Sigma). Peptide analysis was carried out on 4800 MALDI-TOF/TOF (Applied Biosystems/MDS SCIEX) and peptides were identified using Mascot search engine run on GPS explorer version 3.6 (Applied Biosystems) using the following parameters: 800\u20134000 m/z interval MS peak filtering, monoisotopic, MSDB version 20060831 , enzyme trypsin with maximum allowance of one missed cleavage, taxonomy Viridiplantae , \u00b1100 ppm peptide tolerance, \u00b10.3 Da fragment mass tolerance, with fixed modification as carbamidomethyl (C) and variable modification as oxidation (M). Protein samples with probability score above default threshold level  was obtained from Ensembl plant O. sativa indica group database available at http://ensembl.gramene.org and GenomeIndia\u2019s manually curated database of rice proteins respectively  following the manufacturer\u2019s protocol.CDS of OsHSP18.2 was subcloned into bacterial expression vector pET23b (Novagen) and transformed into Chaperone activity of OsHSP18.2 was assayed by the method of Citrate synthase activity was measured according to Seeds were stained with 1% solution of 2,3,5-triphenyltetrazolium chloride (Sigma) to differentiate between viable and non-viable seeds according to Seeds treated with or without CDT were stained with 3,3\u2032- DAB according to n = 50 seeds were used. Seeds were surface sterilized and plated on to either \u00bd MS or \u00bd MS supplemented with NaCl (150 mM), PEG (-0.4 MPa). For heat stress, sterilized seeds were incubated at 45\u00b0C for 1 h and then plated on to \u00bd MS. Seeds were considered germinated when radicle protruded beyond the testa and seedling establishment was considered positive with the emergence of green cotyledonary leaves . OsHSP18.2 is an intronless gene which encodes a protein of 166 aa with an isoelectric point of 5.61 and a predicted molecular mass of 18.2 kDa. Sequence analysis revealed a conserved 90 amino acid alpha crystalline domain, a characteristic feature of sHSP family  shows 59% sequence identity between them  as described by OsHSP18.2 transcript was found to be relatively low during initial stages of development, i.e., S1 till S4 stage (0\u201320 DAP) then strikingly increased in the late maturation stage S5 (21\u201329 DAP) and reached highest levels in dry mature seed . This data essentially revealed that OsHSP18.2 markedly increased at the later stages of seed development consistent with the time when seed actually acquires desiccation tolerance and longevity.To elucidate the function and mechanism of Figure 2C). Next, we investigated the correlation of transcript accumulation of OsHSP18.2 and aging in rice seeds. For this, rice seeds were subjected to CDT treatment for 6 days and transcript accumulation pattern was studied. Results clearly revealed a significant increase in transcript accumulation due to artificial aging even after 1 day of CDT which was further increased to more than 60-fold after 6 days of CDT . To examine the possible involvement of OsHSP18.2 in abiotic stress tolerance, transcript accumulation was checked in rice seedlings challenged with various stresses . As shown in Figure 2E, heat stress triggered maximum induction of OsHSP18.2 transcript as expected while other abiotic stresses had minor changes in transcript levels . This analysis clearly revealed that OsHSP18.2 is upregulated during seed maturation and upon aging and indicates its participation in maturation drying and seed longevity. Transcriptional induction of OsHSP18.2 in rice seedlings challenged with heat stress also indicates its participation in thermal stress tolerance. Investigation of the 5\u2032 upstream sequence of OsHSP18.2 revealed the presence of many interesting cis-acting regulatory elements which are possibly responsible for abiotic stress responsiveness and seed and embryo specific expression. A list of some of these cis-acting elements is shown in Table 1.Transcript accumulation was also studied during germination and data showed a sharp decline in transcript level as germination proceeds . Subsequently, 6X His tagged OsHSP18.2 protein was purified using nickel charged affinity chromatography. Size exclusion chromatography was used to check the oligomeric state of OsHSP18.2 and the purified protein was shown to elute as an oligomer of \u223c202 kDa thus having approximately 12 subunits which agrees to the dodecameric nature of wheat sHSP16.9 . The gel exclusion fractions were run on SDS-PAGE and revealed the presence of a single band in the 202 kDa peak fraction corresponding to the single subunit size of 18.2 kDa . In order to confirm this oligomeric association of OsHSP18.2, the purified protein was run on native PAGE  which revealed a band across 240 kDa marker band thus endorsing the gel filtration results.sHSPs are known to form multimers with varying number of subunits, most commonly between 2 and 48 subunits . In ordein vitro chaperone activity. Therefore, to assess the chaperone activity of OsHSP18.2, we used CS as a substrate for refolding experiments . Control without HSP and with lysozyme showed 20% remaining activity after just 20 min of heat stress which came down to almost zero after 60 min of heat stress whereas CS with OsHSP18.2 retained 30% activity even after 60 min of heat. After 60 min, all the combinations were shifted to 22\u00b0C, temperature permissive for refolding. Even at 22\u00b0C, control lacking HSP and lysozyme control did not display any regain in CS activity but CS with OsHSP18.2 showed a regain of 60% of native CS activity. This result clearly demonstrated that OsHSP18.2 functions as a molecular chaperone that facilitates protein folding and prevents thermal denaturation.Many small HSPs are known to have eriments . CS monoOsHSP18.2 was overexpressed in seeds in Arabidopsis thaliana using the napin promoter. Seed specific OsHSP18.2 transcript accumulation was examined through quantitative RT PCR and significant levels of transcripts were observed in seeds of transgenic lines. Based on this analysis, three independent homozygous T3 lines were selected and subsequently used to assess their germination vigor and longevity. To evaluate seed vigor and longevity, seeds were subjected to CDT and germination performance was analyzed. Under normal conditions, transformed and control seeds (empty vector transformed or WT) exhibited 100% germination , however, after CDT, control seeds showed <20% germination as opposed to OsHSP18.2 transformed seeds where remarkably >50% germination was observed in each line . In addition, TZ staining was carried out to examine the potential viability of these seeds  in contrast to control seeds which remain unstained or were stained pale red . Aging in seeds is also accompanied with ROS accumulation . These data clearly indicate that WT or empty vector transformed seeds subjected to controlled deterioration exhibited higher mortality, reduced germination and more H2O2 accumulation while seeds from transgenic lines overexpressing OsHSP18.2 exhibited less mortality, better germination and less H2O2, thus demonstrating the role of OsHSP18.2 toward maintaining seed viability and longevity during aging.To examine the functional implication of sHSP in seed vigor and longevity, se seeds . TZ precmulation . ROS advoduction . ResultsFigure 5). In normal conditions, OsHSP18.2 transformed and control seeds showed 100% germination. For heat stress, seeds were treated at 45\u00b0C for 1 h and then plated on \u00bd MS, results revealed that >90% of transgenic seeds could complete germination in all lines by the end of 5 days whereas only 70% seeds of WT and VC could germinate by this time . At 150 mM NaCl concentration, >90% seeds of transgenic lines could germinate compared to 80% of WT and VC . Dehydration stress was provided by -0.4 MPa PEG and as expected overexpression lines performed better than control lines . All seeds were monitored for 7 days and seedling establishment was checked which was taken as the emergence of green cotyledons over this period as indicated in previous study .Germination performance of these transgenic seeds under various stress situations were also evaluated, since seed vigor also implies the ability to complete germination under widely variable environmental conditions. Germination of WT, VC, and transgenic seeds was assessed under heat, dehydration, and salt stress and was considered complete when radicle emerged beyond testa. While moderate level of these stresses  did not much affect the germination (data not shown) but slightly elevated stresses revealed a significant difference in germination pattern among control and overexpressing seeds . In addition to this there are many abiotic stress and hormone responsive elements as well which include the heat stress response element HSE (GAANNTTCNNGAA) and well known dehydration responsive element CBFHV (RYCGAC), MYBCORE and MYCCONSENSUSAT thus explaining a higher expression of the transcript during heat and dehydration stress. Surprisingly, few low temperature responsive sequences were also present but our real time experiments revealed not much induction of OsHSP18.2 transcript during cold stress. DPBFCOREDCDC3 (ACACNNG) and DRE2COREZMRAB17 (ACCGAC) responsible for ABA inducible expression are also present. Expression data available on GENEVESTIGATOR also reveals slightly up regulation in response to ABA and Salicylic acid treatment . Studies on wheat sHSP16.9 also demonstrated a dodecameric structure of this heat shock protein although homologs from other species have been shown to have structures with subunits ranging between 2 and >48 subunits . This improved seed vigor and longevity correlates well with the reduced deleterious ROS accumulation in transformed seeds. Similar to our observation, heterologous expression of a sHSP from sacred lotus in Arabidopsis also lead to enhanced germination of transgenic seeds after accelerated aging treatment (CDT; OsHSP18.2 overexpressing lines. Collectively, our findings clearly establish that OsHSP18.2 is seed vigor and aging responsive protein which functions as a molecular chaperone in stabilizing the cellular proteins from irreversible damage and containing ROS accumulation during seed development particularly during seed maturation, drying and storage. Finally, our results also propose that this gene can be a good candidate to improve seed vigor and longevity in terms of better seed germination and seedling establishment in wide environmental stress conditions.Our subsequent functional studies essentially demonstrated that ent CDT; . In addient CDT; . Our resThe 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": "From this point of view, there are at least three sorts of seeing-as. First, in cases of ambiguity resolution (such as the duck-rabbit), the schematicity of the figure leaves us with a choice as to where in perceptual similarity space to place a mark (closer to the marks that represent rabbits or closer to the marks that represent ducks). Second, in cases where expertise affects perception , the accumulation of perceptual landmarks permits a more precise placement of a mark in perceptual similarity space. Third, extensive experience with an object  allows similarity to that object to serve as an acquired dimension in perceptual similarity space, which in turn affects the relative similarities of other objects.It is commonly supposed that perceptual representations in some way embed concepts and that this embedding accounts for the phenomenon of seeing-as. But there are good reasons, which will be reviewed here, to doubt that perceptions embed concepts. The alternative is to suppose that perceptions are  When one switches to seeing it as a rabbit, one\u2019s perception sheds the concept duck and takes on the concept rabbit.What happens in your mind when you see the duck-rabbit as a duck?beech leaf is built into one\u2019s very perception of the beech leaf.What happens when one acquires the ability to distinguish between various kinds of tree leaves just by looking at them? At first, one could not reliably distinguish between the leaves of a beech tree and the leaves of an elm tree, but then, later, one could. It is tempting to answer that one\u2019s perceptions come to be informed by concepts of the various kinds of leaves. It comes to pass that the concept aspect perception) may be taken to demonstrate the presence of concepts in perceptual representations. I will argue that there is a better way to conceive of the various phenomena of seeing-as. Perceptions are best conceived as marks in a perceptual similarity space that correspond to locations in an objective quality space. The relative locations in perceptual similarity space of the marks representing three objects can be taken as a measure of the perceived similarity of these three objects relative to one another. On this account, the various phenomena of seeing-as are distinct, but each brings to light an aspect of the recording of marks in perceptual similarity space. I will countenance three such phenomena, which I will call ambiguity resolution, heightened discrimination and dimension addition. But first, I will explain what I mean by concepts and then I will explain why I deny that concepts are built into perceptions.Thus, these different phenomena of seeing-as  words, \u201cEs gibt S\u00e4ugetiere, die Eier legen,\u201d he or she may thereby express the thought with the content that some mammals lay eggs. The communication is successful if the hearer, on the basis of understanding the words spoken, learns that the speaker thinks that some mammals lay eggs.The traditional conception of concepts is bound up with the idea that in speaking we express our thoughts. The process of communication supposedly begins when a speaker has a thought in mind that he or she aims to reveal to a hearer. The thought, together with a motive to express it, somehow issues in speech. The thought can be described has having a Concepts are the building blocks of the kinds of thoughts expressed in sentences, which stand to whole thoughts as words stand to whole sentences.In light of this theory of linguistic communication, concepts can be characterized as follows:some mammals lay eggs, then one of the concepts in that thought will be the concept mammal, which is expressed by the word \u201cS\u00e4ugetier\u201d.So if the thought expressed with the words, \u201cEs gibt S\u00e4ugetiere, die Eier legen\u201d is the thought that content of the thought expressed. According to the underlying theory of communication, speakers are able to succeed in communicating the contents of their thoughts to hearers, because the words they speak have a content too, which speaker and hearer both attach to the words spoken. So concepts in one sense are the components of shared content that correspond to individual words and phrases.  represent are properties that external objects really have. They are not properties of the perceptual or sensory experience itself. So on the low road the claim will be that a visual perception makes a claim to the effect that an object has a maximally specific objective color. But this is just not true. If you play around with a graphics program on your computer, you can create two brownish patches of color, one against a red background and one against a blue background, that appear to be exactly the same shade of brown, but which turn out to be very different colors when you take away the colored backgrounds (the one against the blue background being much greener than the one against the red background). Since the colors of the brown patches do not change when we place them against a uniform background, and they are clearly different colors when placed against a uniform background, they are different colors as well when placed against differently colored backgrounds, despite their looking the same. So when you are looking at the brown patches against the red and blue backgrounds, then since they look alike, although they are really different, your experience represents at most one of those patches as being the specific color it is. But since they look just alike, and the only difference in their presentation is the color of the background, there is no reason to say that the shade your perception represents is the one rather than the other.Nor is it plausible that in this case neither color is correctly represented, although in other cases maximally specific colors are correctly represented. We would say that only if we assumed that the background color misleads us. But there is no reason to pick out one background color  and say that only against that background do we perceive colors as they really are. So if we want to say in the case described that neither perception correctly represents the color of the surface perceived, then we should deny that perceptions ever correctly represent colors at all. Since there is no adequate account of what specific color we are representing on the assumption that our perception represents a completely specific shade, we should conclude that in fact we do not represent any maximally specific shade. It is just not true that the concepts that make up the conceptual content of our perceptions include maximally specific color concepts.scenario content, where a scenario content is roughly a way of filling space  is a relation that holds between elements of the scene . And we will have a function h such that, for each element e of the representation, h(e) is an element of the scene (such as a particular edge). If the mind literally contained maps, then \u03a0 could be a projection according to scale, but of course the mind does not literally contain maps, and so it cannot be that. We cannot demand that h be a homomorphism (with respect to \u03a0), because we have to allow that spatial configuration can be misrepresented. But we can suppose that h is approximately a homomorphism, and we can suppose that \u03a0 and h can be identified teleologically, in terms of the role they play in promoting the success of the species that these functions pertain to in promoting the reproductive fitness of that species.The representation of spatial configuration need not be thought of as conceptual. Rather, we can define the representation relation by means of a mapping of relations between representations and relations between things represented and a mapping between elements of the representation and elements of the scene represented. So we will have a relation, call it \u03a0, such that for each relation perceptual similarity space. My assumption is that perceptions can be modeled as marks in a perceptual similarity space. To explain what I mean, I need to start with the concept of an objective quality space. Objective quality space is a hyperspace with many, many dimensions that measure the location of an object along a variety of physical dimensions, namely, those that we are perceptually sensitive to. Distance in this similarity space (measured using the Pythagorean theorem) is inversely proportional to (perceptible) similarity. The dimensions of objective quality space will include not just the obvious ones, such as color , shape (its many dimensions), size, etc., but many others that less readily come to mind, such as jerkiness of motion. (The motion of a squirrel is jerkier than the motion of a cat.) There might be, for a given particular triangle, a dimension that measures degree of congruence to this particular triangle (and that dimension might be part of a subspace of dimensions that measure shape). Of course, many objects consist of a certain configuration of parts . As I said, we will need a separate account of the perceptual representation of the configurations of parts. But given that, we can suppose that spatial arrangements can be arrayed along the dimensions of a objective quality space in ways that represent the similarities and differences between the configurations. Things composed of comparable parts can be more or less similar to one another with respect to the arrangement of those parts.The other aspect of perceptual representation, which will be my focus here, can be modeled as a mark in a perceptual similarity space. Exposure to an object causes a mark and the mark is a measure of the location of the object along each of a number of dimensions of objective quality space. However, we can distinguish between the location that an object is actually at in objective quality space and a point in objective quality space that the mark, caused by exposure to the object, corresponds to. The perception \u2014 the mark \u2014 will be accurate only to the extent that the corresponding point in objective quality space is near to the location where the object that is the cause of the mark actually is.Perceptual representations can be modeled by means of a perceptual similarity space, which is a hyperspace consisting of dimensions that correspond to the dimensions of objective quality space. See Fig. . An indithe point in objective quality space at which the cause of the mark would have had to be located if the mark in perceptual similarity space had been recorded in the way that is biologically normal for the biological species to which the perceiver belongs. A further elaboration of this answer would take the form of an account of the biologically normal way of recording marks in perceptual similarity space, but I will not elaborate further here. . So my perception of it falls outside the region that is my concept bird. Then I learn that that animal is a bird . In that case, the region that is my concept bird has to expand to include the new perception of a bird. Since my concept bird is the region and the region has expanded, my concept bird itself changed. But on the contrary, when I learned that the new thing was a bird, what I learned is that the very same concept, viz., my concept bird, which I formerly did not apply to that thing, applies to that thing. So the concept was never the region in the first place.  Next, one could perform a multi-dimensional scaling to define a hyperspace in which x is closer to y than to z if and only if the judgment that x is more like y than like z is on the list. The last step would be to determine what the various dimensions of this space measure.In section There is no guarantee that the method will work. It could turn out that the various operationalizations do not yield consistent results. Or it could turn out that every time we add a new object to the sample and generate the relative similarity \u201cjudgments\u201d, the multi-dimensional scaling yields a new result . So my alternative conception of perceptual representation is nothing more than a hypothesis, which might turn out to be false, which is as it should be.In terms of the model of perceptions as marks in perceptual similarity space I can now describe three different phenomena that could be described as nonconceptual forms of seeing-as.Ambiguity resolution includes seeing Wittgenstein\u2019s duck-rabbit as a duck or as a rabbit. It includes as well the perceptual choices that other such ambiguous figures, such as the Necker cube, present us with. It includes also seeing animals in clouds. It does not, however, include what is better described as \u201cfilling in\u201d, such as seeing triangles in the Kanizsa triangles. That is probably better treated as a function of the perceptual representation of configuration. causes us to see it as a duck; rather, seeing it as a duck is nothing other than placing it closer to the marks representing ducks.My hypothesis regarding the duck-rabbit drawing is that, precisely because it is so schematic, we have a degree of choice as to where we place a mark representing it in perceptual similarity space. We can place it closer to the other marks representing ducks, in which case we see it as a duck, or we can place it closer to the other marks representing rabbits, in which case we see it as a rabbit. The claim is not that placing it closer to the other marks representing ducks duck or rabbit at all. We have to have some perceptual representations of ducks and some of rabbits, but we do not need to conceive of the things so represented as ducks or as rabbits.duck, then having placed our perception of the duck-rabbit closer to the perceptions of ducks, we can say, and can think, that\u2019s a duck! Moreover, the location of marks in perceptual similarity space may play a role in determining how concepts are applied.My claim is that we can exercise this choice without employing the concepts X or the concept Y. The objection could be either that without the concepts X and Y one could not exercise a choice, or that the very act of measuring the distance between the mark representing the drawing and the marks representing the X\u2019s and between the mark representing the drawing and the marks representing the Y\u2019s necessitates an exercise of the concepts X and Y. In either case, I would like to try to persuade the reader that that is not so, by asking the reader to engage in a little experiment.So the claim is that perceptual ambiguity resolution is not an application of concepts. Against this claim, someone might object that the similarity comparisons that I am invoking require the application of concepts. At this point I am taking for granted, for the reasons given in section duck or rabbit. Not only does one\u2019s perceptual representation not subsume them under any generic concept, but one does not immediately have any generic concept in mind at all. In particular, the reader does not have concepts of the maximally specific shapes of the objects depicted \u2014 because the drawings do not determine that much. Of course, the reader can describe these objects as I just did, for example, the one on the right as an object consisting of a tube coming out of a rectangular base. But, as we will see, that description does not provide a concept without which one could not represent the similarities between this object and others.Please examine the diagrams in Fig. If the reader thinks that he or she has a generic concept of the kind of thing the object is, then I would challenge him or her to say which of the objects in Fig. see as either one of them. See Fig. seen as either the box with a hole through it or as the tube sticking out of the base, despite the fact that the reader lacks generic concepts of the kinds of things that those objects belong to. The conclusion I draw is that the kind of seeing-as involved in the case of the duck-rabbit does not utilize concepts of genera.Notwithstanding the fact that the reader does not, as I will assume, have generic concepts of the kinds of things the objects in Fig. Another kind of seeing-as that might be explicated in terms of perceptual similarity spaces is what I call heightened discrimination. This is the effect on perception that comes with expertise. A person who does not know what a pine tree is and then learns to discriminate pine trees from deciduous trees might be thought to come to perceive pine trees differently than before.point in perceptual similarity space. But actually perception might often be something more like a fuzzily bounded region in perceptual similarity space. We may think of this region as an imprecise measurement of the qualities of the object of perception along the various dimensions.  and so on, serves to calibrate the scales for the dimensions that measure it. Thus, when a new tree leaf comes along, its location on each these dimensions can be more precisely pinpointed. The experience of taking in at a glance the tree leaf in all of its particulars and sharply distinguishing it from many others can be modeled as this capacity to pinpoint the location of the leaf in perceptual similarity space.Again, I am taking for granted at this point that perceptions do not have conceptual contents section . But as Jacksonpollockish and this concept comes to inform one\u2019s perceptions. But if one said this, one would be merely putting a label to an ability that one has no particular reason to describe as concept application. There was an evolution in Pollock\u2019s work, from the early landscapes to his famous drip paintings, but even within the drip paintings, which is the kind most frequently forged, one acquires a sense of a range of possibilities within the genus that the forgeries lie outside of. One\u2019s discriminatory ability might be grounded in a collection of paradigms, or one might form an impression of some central tendencies. The result might be described as a sense for the Jacksonpollockish, but nothing is added to the account by supposing that Jacksonpollockishness constitutes a kind representable in a concept.One might contest this ground for doubt by claiming that in learning to distinguish between Pollocks and forgeries one acquires the concept Perhaps there is a third kind of nonconceptual seeing-as, although I think ordinary experience does not reveal this as plainly as it reveals the first two kinds. Sometimes a particular object becomes very familiar to us. It might be the family dog. We come to recognize the dog in its many postures and moods. Or the object might be a favorite hand tool, such as a Swiss army knife with many blades and tools built into it. Or it might be a favorite sculpture by Jacques Lipschitz, a hard-to-grasp arrangement of shapes and surfaces. When this happens, we may be disposed to compare other objects we encounter to this familiar one. The familiar object might even become the origin, or zero, in a new dimension in perceptual similarity space. This dimension might be the product of collapsing several different dimensions into one: A dimension representing similarity to Fido sitting, a dimension representing similarity to Fido running, and so on, might be combined to produce a single dimension representing overall similarity to Fido.a, b and c, are all equally distant from one another, forming the vertices of an equilateral triangle. But if a new dimension is added, and a and b have the same value on this new dimension, but c has a different value, then a and b may qualify as overall more like one another than either is to c.When a new dimension is added to perceptual similarity space in this way, the relative similarity relations between objects is changed as well. In the old perceptual similarity space, it might be that three objects, For example, consider the four examples of Korean buncheong pottery in Fig. In short, if it is true that particular, familiar objects can introduce new dimensions into perceptual similarity space, so that the relative positions of our perceptions in perceptual similarity space can shift, then in this way too experience can alter the character of our perceptual experience.The phenomenon of seeing-as has been appealed to by those who want to claim that all visual perception involves the subsumption of objects under general concepts. The idea is that the classic cases of seeing-as, such as the duck-rabbit, in one way or another make the conceptual content of our perceptions obvious, either by demonstrating the possibility of a substitution of the determining concept or by demonstrating the possibility of a refinement in perception through the acquisition of refined concepts. The conclusion to be drawn is that all visual perceptual representations have conceptual content; but the cases of so-called seeing-as announce the presence of that conceptual content very loudly.Do my accounts of nonconceptual seeing-as likewise demonstrate that every act of visual perception is a case of seeing-as? On my account, the several forms of seeing-as are disparate; so they do not show that there is any one thing that happens in every act of visual perception. Visual perception on my account is not exclusively but always includes the placing of a mark in perceptual similarity space representing the location of an object, or arrangement of objects, in objective quality space. What I have called the phenomena of seeing-as are all different ways in which experience somehow affects the location of a mark in perceptual similarity space. In this light perhaps we should say that an infant\u2019s first visual perceptions are not cases of seeing-as; it has to make do with the innate structures of perceptual similarity space alone. But certainly by the time a normal human reaches several years of age, all visual perceptions representing particular objects and scenarios are shaped in this way by past experience. So we should say, yes, all of the visual perceptions of a normal adult human being are cases of seeing-as."}
+{"text": "Background. Changes in proprioception may contribute to postural instability in individuals with neurological disorders. Objectives. Evaluate proprioception in the lower limbs of patients with Parkinson's disease (PD) and the association between proprioception and cognitive ability, motor symptoms, postural instability, and disease severity. Methods. This is a cross-sectional, controlled study that evaluated proprioception in PD patients and healthy age- and sex-matched individuals. Kinetic postural proprioception of the knee was evaluated using an isokinetic dynamometer (Biodex\u00ae Multi-Joint System 4 Pro). Participants were evaluated using the Montreal Cognitive Assessment (MoCA), the Hoehn and Yahr rating scale and postural instability , and motor function (UPDRS-III) tests. Results. A total of 40 individuals were enrolled in the study: 20 PD patients and 20 healthy controls (CG). The PD patients had higher angular errors on the proprioceptive ratings than the CG participants (p = 0.002). Oscillations of the center of pressure (p = 0.002) were higher in individuals with PD than in the controls. Proprioceptive errors in the PD patients were associated with the presence of tremors as the dominant symptom and more impaired motor performance. Conclusion. These findings show that individuals with PD have proprioceptive deficits, which are related to decreased cognitive ability and impaired motor symptoms. Parkinson's disease (PD) is the second-most common neurodegenerative disease, and its motor characteristics include resting tremors, rigidity, bradykinesia, and postural instability .The mechanisms of postural instability are not well understood. It has been suggested that losses in proprioception may result from a lack of proper regulation of motor control and body reflexes . For exaFew studies have assessed proprioception in individuals with PD compared to healthy individuals \u201313. The The present study aimed to evaluate knee proprioception in individuals with PD and the association between proprioception and motor symptoms, cognitive status, postural instability, and disease severity.This controlled, cross-sectional study was approved by the Ethics Committee of the Universidade Federal de Ci\u00eancias da Sa\u00fade de Porto Alegre, number 988/12, and was performed according to the Code of Ethics of the World Medical Association (Declaration of Helsinki).This study consisted of individuals with Parkinson's disease group (PG) and a control group (CG) made up of healthy age- and sex-matched individuals.The number of participants needed for this study was determined using a calculation based on the results of the study by Duman et al. (2012) , which eAll participants who agreed to participate in the study signed an informed consent form and were scheduled for the procedure.The PG participants included individuals of both sexes with a clinical diagnosis of PD according to the London Brain Bank Criteria and were ranked between stages 1 and 3 according to the Hoehn and Yahr staging scale . For botIndividuals with a history of knee surgery or lower limb fractures in the past 6 months or amputation of any part of the leg, who presented with any restricting pain at the time of the assessment, who were not right-handed, or who had any other neurological disease or diabetes mellitus were excluded from both groups.PD patients were classified into the tremulous form or akinetic rigid type depending on the predominant symptoms, which were assessed using the UPDRS scale.All ratings were performed 1 hour after taking antiparkinsonian medication for patients in the ON phase.The participants in the PG were assessed through an interview and scales to assess cognitive ability  , motor fKinetic postural proprioception of knee flexion and knee extension were assessed using an isokinetic dynamometer (Biodex Multi-Joint System 4 Pro). The researcher who conducted the proprioceptive assessment of the participants was blinded to the other ratings, which were performed by a second evaluator.The CG participants responded to an anamnesis and MoCA scale , and theStatic balance was assessed by analyzing oscillations of the center of pressure (COP) using a baropodometer (FootWork\u00ae IST/AM3 Intermetique).This system is capable of statically and dynamically measuring plantar pressure. The baropodometer consists of a pressure platform that uses capacitive sensors with a thickness of 4\u2009mm, an active area of 490\u2009mm \u00d7 490\u2009mm, and a sampling rate of 40\u2009Hz. quasistatic posture on the baropodometer platform, and a constant gaze at a point on the wall located 3\u2009m in front of the person at eye level. Three measurements were performed with a 1\u2009min interval between each measurement, and an average COP value was calculated for the three assessments.The participants were instructed to remain immobile for 30 seconds using self-selected lateral spacing of the feet, aKinetic postural proprioception was evaluated with an isokinetic dynamometer according to the manufacturer's guidelines; the same method was used in both groups. Specifically, to test the position sense of the knee, the participants remained seated in a chair tilted to 70\u00b0 with their hips and knees flexed, and one leg was attached at the ankle using specific support equipment. For testing, the lower limb was stabilized, and the knee remained aligned with the mechanical axis of the dynamometer through a point marked on the lateral femoral condyle in the sagittal plane . A singlThe equipment was programmed for the proprioceptive assessment of the right and left knees with 45\u00b0 and 75\u00b0 of flexion as the testing parameters. The participants were blindfolded to prevent the visual system from influencing the test results. Initially, the lower limb was positioned at the reference angle (90\u00b0 for knee flexion). Subsequently, the target angle was reached with the help of a device at a speed of 10\u00b0/s, and the leg remained in this position for 15 seconds to allow the patient to memorize it.Once the patient memorized the position, the limb was passively returned to the reference angle and remained in this position for 10 seconds to rest. Then, the participant was asked to place the limb at the memorized target angle and press the stop button on the equipment to record the angle reached. The first round of this process was performed for the participants to become familiar with the test.After the training period, the participants performed three additional attempts, and we calculated the difference between the original target angle and the angle reached, the angular error, and the average proprioceptive error in both tests (45\u00b0 and 75\u00b0) for the three attempts for each lower limb and for bilateral proprioception.Only nonparametric tests were used for the data analysis, eliminating the requirement of a normal distribution. To compare the control and patient groups, we used the Wilcoxon test for paired samples. To compare the variables within the group of patients, we used the Mann\u2013Whitney and Kruskall-Wallis tests, for comparisons between two or more variables, respectively. The Spearman correlation was used to assess the relationship between two quantitative characteristics of the patients. The analyses were performed using SPSS software, version 18. The significance level was set at 5%.The sample consisted of 40 participants (20 PD patients and 20 age- and gender-matched controls). The sociodemographic characteristics of the sample are presented in p = 0.072). The mean duration of disease among the individuals with PD was 6.1 years .p = 0.002).With respect to the analysis of the center of pressure, oscillations of the COP were higher in patients with PD compared to controls (p = 0.002). PD patients had impaired proprioception performance compared with the control participants, which was observed by analyzing both the side less affected by the disease (p = 0.050) and the side more affected by PD (p = 0.004).PD patients had higher angular errors in the proprioceptive assessments than the participants in the control group (p = 0.040 for the right side and p = 0.006 for the left side).The significant differences between the PG and the CG were observed even when the side with the initial symptoms of PD was analyzed  that most interfered with proprioception, patients with tremor as the predominant symptom had larger angular proprioceptive errors (p = 0.017).Disease Staging by Hoehn and Yahr. We verified that there is a statistically significant correlation between proprioceptive deficits and the degree of staging of the more affected side  and in the bilateral analysis .Functional Motor Scale (UPDRS). Impaired proprioception was observed in both sides and was significantly correlated with the degree of functionality as assessed using the UPDRS .Other Variables. The duration of the disease, presence of dyskinesia, on/off phenomenon, oscillations in the COP, and pull test results did not influence the proprioceptive angular errors.In the present study, PD patients had higher angular errors in proprioception than healthy individuals. PD patients also presented with higher postural instability when evaluated using oscillations of the COP. PD patients showed greater impairment in reaching the target knee angle than did the controls. This deficit in reaching known targets has also been found in other studies \u20136, 8. ThAt the time of the proprioceptive assessment, all parkinsonian individuals were in the ON state of their dopaminergic medication. The influence of antiparkinsonian medication on proprioception is not well established. Some authors have suggested that antiparkinsonian medications may negatively affect the proprioceptive system in PD , 17, 18.We observed that participants who had tremor as the first noticeable symptom of PD had the largest proprioceptive errors. To date, no previous studies have linked the first motor symptom of PD with proprioceptive changes caused by the disease. Bradykinesia and postural instability are the most common motor symptoms associated with deficits in proprioception , 19, 20.One possibility for the absence of a correlation between the COP results and the pull test with angular errors of proprioception could be the compensation of the visual system in the postural stability tests. It is known that the visual system aids in minimizing proprioceptive deficits and postural reactions because vision compensates for instabilities, preventing falls. Therefore, the results of the postural instability test would likely have been different if the tests were conducted without the influence of vision, in which case there might have been a relationship between postural instability and the proprioceptive alteration of parkinsonian participants.Postural instability in PD is not fully understood; however, it is believed to be the result of a complex interaction between compensatory strategies and the impairment caused by the disease at different levels of the nervous system , 22. SevThe impaired motor ability of the participants in the PG  and more severe disease staging was significantly correlated with higher angular errors in the proprioceptive assessment. This relationship between impaired motor symptoms and higher deficits in proprioception was also observed in previous studies , 12, 20.Although the severity of the disease influenced the proprioceptive aspects in the sample, the time of diagnosis was not significantly associated with proprioception. This finding may be because sensory deficits increase with duration of disease, and the mean disease duration of the study participants was relatively low .From the findings of this study and based on previous studies, we observed that there was a proprioceptive deficit in PD and, therefore, in the joint position sense. However, the real origin of this alteration is still unknown because the function of the muscle spindles, particularly the receiver of the joint position sense, appears to be normal in PD. In addition, there is no evidence of the existence of an alteration in the ascending somatosensory pathways in PD.Some authors argue that proprioceptive deficits in PD may be associated with dysfunction in the sensory integration of cues used to guide movements within the basal nucleus (BN) because intact BN circuitry is essential for the perception of joint position and movement, and damage to this circuit can result in kinesthetic deficits. Therefore, the BN could be responsible for alterations in proprioception in individuals with PD. However, if the BN was responsible for the proprioceptive alterations in PD, studies that evaluate the effect of dopaminergic medications on proprioceptive deficits should find similar results for all patients, regardless of disease severity. Therefore, some authors , 12, 26 To corroborate this hypothesis, Jacobs and Horak  hypothesIn this study, we did not find any significant relationship between proprioceptive errors and disease duration, the presence of dyskinesia and motor fluctuations, oscillations of the COP, and the pull test. However, these results may have been significant in a larger sample.The present study showed that the participants with PD have higher angular proprioceptive errors, which supports the hypothesis that PD affects the proprioceptive system. There were also significant differences in fluctuations of the center of pressure between individuals with PD and healthy controls, indicating that individuals with PD tend to have greater postural instability than healthy people.The analysis of the factors that have a relationship with proprioception showed that tremors were the dominant symptom and that impaired motor ability significantly influenced the presence of proprioceptive alterations in the parkinsonian individuals.In this study, the postural instability in individuals with PD and the relationship between proprioceptive deficits and motor skills and the severity of PD highlight the importance of focusing on these aspects during the rehabilitation process. Based on our results, we suggest that physiotherapy for PD should include proprioceptive and balance exercises to improve motor skills and, in turn, improve the functionality and quality of life of individuals with PD."}
+{"text": "Preparation in the business of medicine is reported to be poor across a number of specialties. No data exist about such preparation in gynecologic oncology training programs. Our objectives were to evaluate current time dedicated to these initiatives, report recent graduate perceptions about personal preparedness, and assess areas where improvements in training can occur.Two separate surveys were created and distributed, one to 183 Society of Gynecologic Oncology candidate members and the other to 48 gynecologic oncology fellowship program directors. Candidate member surveys included questions about perceived preparedness for independent research, teaching, job-hunting, insurance, and billing. Program director surveys assessed current and desired time dedicated to the topics asked concurrently on the candidate survey. Statistical analysis was performed using Chi-squared (or Fisher\u2019s exact test if appropriate) and logistic regression.p\u00a0=\u00a00.01) and billing (p\u00a0<\u00a00.01). Compared to their current schedules, program directors desired more time to teach how to write an investigator initiated trial (p\u00a0=\u00a00.01). 94% of program directors reported having career goal discussions with their fellows, while only 72% of candidate members reported that this occurred (p\u00a0=\u00a00.05).Survey response rates of candidate members and program directors were 28% and 40%, respectively. Candidate members wanted increased training in all measures except retrospective protocol writing. Female candidates wanted more training on writing letters of intent (LOI)  contains supplementary material, which is available to authorized users. Gynecologic oncology training programs have evolved much over the last ten years, and the volume of information that fellows are required to master has significantly increased. As it is has been reported that matriculating fellows are often ill-equipped for the rigors of a gynecologic oncology fellowship , there aApproval was obtained from the University of Miami Institutional Review Board. Two separate de novo surveys were created  to the Accreditation Council for Graduate Medical Education (ACGME), program directors were identified by a query of both organizations. Associate program directors were not included. Length of the program was ascertained. The survey then inquired about current number of didactic hours dedicated to each of the following topics over the length of fellowship training: protocol writing, drafting a grant proposal/letter of intent, being an effective teacher, billing/coding, medical-legal concerns, financial planning, disability, and the Affordable Care Act. Program directors were then asked how many hours they would The surveys for both program directors and candidate members were released via email in January, 2017, with a link to the questionnaire. The survey link was active for six weeks, and three separate email invitations to participate were sent. A statement of implied informed consent was included in the survey instructions. Responses were collected anonymously and stored in a RedCap database.p-values <0.05 were considered statistically significant.Statistical analyses were completed using STATA IC . All data was used, even if the surveys were not completed in toto. Summary statistics were generated to describe the cohort. Chi-square testing (or Fisher\u2019s exact when appropriate) was used to analyze proportional associations between groups. Logistic regression was used to estimate associations between binary variables. All tests were two-sided, and Of the 183 candidate members contacted, fifty-one (28%) completed the survey. Demographic characteristics are shown in Table\u00a0Ninety-two percent of respondents felt comfortable writing a retrospective protocol and 74% felt comfortable drafting a letter of intent, but only 26% reported comfort writing a grant proposal. Even fewer (14%) felt comfortable writing an investigator initiated therapeutic trial. There were no differences in reported comfort levels by gender, age, time since fellowship, or length of fellowship.Figure\u00a0p\u00a0=\u00a00.04). Academic candidate members were also more likely to want additional training in drafting IIT, though this did not reach statistical significance . Age, length of fellowship, and time since completion of fellowship were not associated with reported interest in more education about any topic queried. Compared with males, a greater proportion of females wanted more teaching on billing and coding  and writing letters of intent . Males were less likely to want more training on disability insurance  and grant writing , though these did not reach statistical significance. Formal education in certain subject areas was negatively associated with a desire for further training on those subjects, including how to be an effective teacher (OR 0.11 [95% CI 0.03\u20130.45), p\u00a0=\u00a00.002), the Affordable Care Act , and disability insurance . Of those who did not receive any teaching on the Affordable Care Act, 89% reported wanting more training.Figure\u00a02\u00a0=\u00a06.11, p\u00a0=\u00a00.01).Of the 48 fellowship program directors contacted, 19 completed the survey for a response rate of 40%. Sixty-five percent of the respondents directed three-year programs, while 35% directed four-year programs. The current didactic time for each program dedicated to the non-clinical practice of medicine is reported in Table\u00a02\u00a0=\u00a03.89, p\u00a0=\u00a00.05) and that they received encouragement from their program directors to review employment agreements with a lawyer . Notably, those who were encouraged to review their contracts with a lawyer were more likely to actually do so compared with those who did not .Table\u00a0With increasing pressure on fellowship programs to train competent gynecologic oncologists in an era of rapidly advancing surgical and chemotherapeutic evolution, it is important to also recognize the concerns of trainees about adequate preparation for the practical aspects of being independent physicians. Our study shows that while recent fellowship graduates indicate a high level of comfort with some aspects of both the academic and business components of post-training employment, there remains a significant disparity in the perceived preparation for many of these important facets of practice.The findings reported here are not unique to gynecologic oncology. In fact, other surgical specialties have reported a recognition of the importance of training in the business of medicine and a lack of trainee knowledge in this arena. Fakhry et al.  publisheThere are limited data on the importance of education in the more traditionally academic components of employment, such as preparation for protocol development and teaching. A survey of maternal fetal medicine fellows demonstrated that the percentage of respondents who felt they had adequate training to apply for grants was 20% , which iThe amount of time dedicated to certain topics varies by program, and it is apparent that program directors have different opinions about the need for time spent to achieve competency. This is true of surgical training programs, as well, and is apparent in the diverse approach to education in the practice of non-clinical medicine. A number of strategies have been proposed to increase competency with contract negotiation, coding compliance, and financial planning. Jones, et al.  reportedThere are several weaknesses to this study. The survey that was released was developed de novo, and is not validated, so its reproducibility may be in question. The response rates of 28% and 40% for candidate members and fellowship directors, respectively, are low, but are in line with other studies requiring physician response to survey invitations. There is also a likely component of bias, as more than two-thirds of candidate respondents had less than three years of experience, and about 90% of had a university appointment or were in a community/academic hybrid practice. Relative overrepresentation of this population may skew perceptions on the need for additional academic-based professional training, but demonstrates that there are clearly concerns regarding tools for success as an academic gynecologic oncologist. Additionally, candidates were not queried about advanced training in research, business, or administration, which may have influenced their responses. Despite these limitations, this is the first study within the gynecologic oncology subspecialty to evaluate perceived preparation for measures outside of the usually studied surgical and medical oncology realm, and as such provides new insight into areas in which changes in didactic programs may be made. Larger investigations across multiple subspecialties, both on the residency and fellowship level, may assist in clarifying perceived additional educational need, with consideration being given to including such training as a core measure for trainee professionalism.At our institution, we have initiated a more comprehensive program of didactics based on the current findings, now including lectures from risk management, billing and coding, and the institutional review board. Our approach, however, may not be universally applicable to all gynecologic oncology programs. In fact, since preparation in the business and academic practice of medicine is not a component of any core competency for training, creativity in curricula development should be encouraged. It is clear, though, that new graduates from gynecologic oncology fellowships feel inadequately prepared for some of the responsibilities they will have post-training. It remains incumbent on those entrusted to educate them to continuously strive to improve their programs and maintain open communication with the fellows so that a truly collaborative teaching environment can be developed.Recent gynecologic oncology fellowship graduates want more training in the non-clinical, academic and business practice of medicine. Modifying curricula to more effectively prepare graduates for professional lives should be considered. Improved dialogue between trainer and trainee will be a crucial component of such a curricular evolution.Additional file 1:Candidate Member Survey. (DOCX 16\u00a0kb)Additional file 2:Fellowship Program Director Survey. (DOCX 14\u00a0kb)"}
+{"text": "Peptides were radio-iodinated and evaluated in vitro for binding specificity, cellular retention, and affinity. In vivo specificity for all heterodimers was studied in PC-3 (GRPR-positive) and LNCaP (PSMA-positive) xenografts. [125I]I-BO530 was evaluated in PC-3pip (GRPR/PSMA-positive) xenografts. Micro single-photon emission computed tomography/computed tomography (microSPECT/CT) scans were acquired. The heterodimers were radiolabeled with high radiochemical yields, bound specifically to both targets, and demonstrated high degree of activity retention in PC-3pip cells. Only [125I]I-BO530 demonstrated in vivo specificity to both targets. A biodistribution study of [125I]I-BO530 in PC-3pip xenografted mice showed high tumor activity uptake (30%\u201335%ID/g at 3 h post injection (pi)). Activity uptake in tumors was stable and exceeded all other organs 24 h pi. Activity uptake decreased only two-fold 72 h pi. The GRPR/PSMA-targeting heterodimer [125I]I-BO530 is a promising agent for theranostics application in prostate cancer.Gastrin-releasing peptide receptor (GRPR) and prostate-specific membrane antigen (PSMA) are overexpressed in most prostate cancers. GRPR expression is higher in early stages while PSMA expression increases with progression. The possibility of targeting both markers with a single theranostics radiotracer could improve patient management. Three GRPR/PSMA-targeting bispecific heterodimers , (CH Prostate cancer remains one of the most commonly diagnosed and deadliest cancers in men despite the major advancements made to improve the survival and management of patients . DespiteSeveral prostate cancer-associated molecular targets have been identified over the past few decades . Two of l-\u03b1-glutamyl-urea targeting PSMA have been studied in clinics +, 1483.746 [M + Na]+, 1499.720 [M + K]+.Peptide azide ng block . Yield: l-2-Nal-\u03b5-l-Lys-ureido-l-Glu (4) was synthesized as described previously \u2212: 706.309; observed: 706.308 [M \u2212 H]\u2212.Propynoyl-tranexamyl-eviously ,39, withl-2-Nal-\u03b5-l-Lys-ureido-l-Glu (5) was synthesized via SPPS using dec-9-ynoic acid as the N-terminal amino acid. Scale: 120 \u00b5mol. Formula: C43H59N5O10. Expected [M \u2212 H]\u2212: 804.4184; observed: 804.419 [M \u2212 H]\u2212.Similarly, dec-9-ynoyl-tranexamyl-l-2-Nal-\u03b5-l-Lys-ureido-l-Glu (6) was synthesized via SPPS using 4,7,-dioxa-dec-9-ynoic acid as the N-terminal linker. Scale: 113 \u00b5mol. Expected [M \u2212 H]\u2212: 808.3769; observed: 808.379 [M \u2212 H]\u2212.4,7,-dioxa-dec-9-ynoyl-tranexamyl-3  and alkyne 6  according to Cu(I)-catalyzed alkyne/azide cycloaddition +: 2271.14902; observed: 2271.148 [M + H]+, 2293.130 [M + Na]+, 2309.104 [M + K]+.Heterodimer BO530 was synthesized from azide addition ,41. A gl3  and alkyne 5 , in an isolated yield of 3.4 mg (1.5 \u00b5mol). Expected [M + H]+: 2267.19049; observed: 2267.190 [M + H]+, 2289.172 [M + Na]+, 2305.146 [M + K]+.Similarly, heterodimer BO535 was synthesized from azide 3  and alkyne 5 , in an isolated yield of 4.3 mg (2.0 \u00b5mol). Expected [M + H]+: 2169.08094, observed: 2169.080 [M + H]+, 2191.062 [M + Na]+, 2207.036 [M + K]+.Following a similar procedure, heterodimer BO536 was synthesized from azide v/v TFA) in water over 10 min. Purification was performed under similar conditions with a gradient from 30% to 50% acetonitrile over 20 min. The product fraction was collected and solvent removed using a Sep-Pak C8 Vac 1cc cartridge . The product was eluted in 90% v/v ethanol in water (120 \u00b5L). The heterodimers were radiolabeled with I-125 using Pierce Iodination Reagent (IodoGen) . LoBind Eppendorf tubes were coated with IodoGen (50 \u00b5g) as previously described . PBS , the mixture was centrifuged, and the supernatant was analyzed by instant thin layer chromatography (ITLC) eluted with 0.2 M citric acid.n-octanol were added in an Eppendorf tube containing radiolabeled peptide. The tube was vortexed for 2 min and centrifuged for 20 s. Fractions of each phase were collected and their activity was measured.The octanol\u2013water distribution coefficient was determined as described earlier . Briefly5 cells/well) were pre-treated with 200 nM of either NOTA-PEG4-RM26 (to block GRPR) or PSMA-617  (to block PSMA) for 10 min at room temperature. The radiolabeled heterodimer (1 nM) was added to all wells (including non-treated sets) and the plates were incubated for 1 h at 37 \u00b0C. After media aspiration, the wells were treated with trypsin\u2013EDTA . The detached cells were measured for the activity content.An in vitro binding specificity assay was performed using PC-3 (specificity to GRPR), LNCaP (specificity to PSMA) and PC-3pip (specificity to GRPR and PSMA) cells. Cells in six-well plates (5 \u00d7 1050) values were determined according to Varasteh et al. [111In]In-NOTA-PEG4-RM26, and for PSMA in concentration range of 0\u20131620 nM of the heterodimer using PC-3pip cells and 1 nM of [111In]In-PSMA-617 (GRPRs were blocked with 200 nM of unlabeled NOTA-PEG4-RM26). Cells were incubated at 4 \u00b0C for 5 h. After incubation, the cells were detached with trypsin\u2013EDTA and the activity in cell samples was measured. Data were plotted using nonlinear regression.Half maximal inhibitory concentration . PC-3, LNCaP, and PC-3pip tumor xenografts were established by implanting 5 \u00d7 104-RM26 or PSMA-617 was added to the radiolabeled heterodimer in order to block GRPR in PC-3 xenografted mice or PSMA in LNCaP xenografted mice, respectively. Animals were sacrificed by heart punctures exsanguination after pre-injection of Ketalar\u2013Rompun solution . The mice were dissected 1 h post-injection (pi) and the organs of interest were collected. The activity uptake in organs was measured.In vivo specificity for the radiolabeled heterodimers was tested on mice bearing PC-3 or LNCaP xenografts. The mice were injected with 40 pmol (30 KBq) of the radiolabeled heterodimer in 1% bovine serum albumin (BSA) in PBS. For the mice in the blocked group, 7.5 nmol of either unlabeled NOTA-PEG125I]I-BO530 were conducted on mice bearing PC-3pip tumor xenografts. The mice were injected with 40 pmol (30 KBq) of [125I]I-BO530 in 1% BSA/PBS. The radioactivity uptake in organs was studied 1, 3, 24, and 72 h pi as described above.Biodistribution studies for [125I]I-BO530 in 1% BSA/PBS. Whole body SPECT/CT scans were performed using nanoScan\u00ae SPECT\u2022CT  at 3, 24, and 72 h pi. CT scans were acquired at the following parameters: 50 keV, 670 \u03bcA, 480 projections, 5 min acquisition time; SPECT scans were carried out using I-125 energy window (25.56 keV\u201331.24 keV), 256\u2009\u00d7\u2009256 matrix, 1 h acquisition time. The CT raw data were reconstructed using Nucline 2.03 Software . SPECT raw data were reconstructed using Tera-Tomo\u2122 3D SPECT .Mice bearing PC-3pip xenografts were injected with 40 pmol (500 KBq) of [125I]I-BO530, [125I]I-BO535, and [125I]I-BO536, respectively.The heterodimers were successfully synthesized and radiolabeled with I-125 with radiochemical yields 69% \u00b1 1%, 73% \u00b1 4%, 73% \u00b1 3% for BO530 , BO535, The in vitro binding specificity test  demonstr50) were determined for the non-iodinated heterodimers by using either [111In]In-NOTA-PEG4-RM26 as the binding competitor to GRPR, or [111In]In-PSMA-617 as the binding competitor to PSMA. The values  showed constant release of cell-associated activity (14% \u00b1 2% at 24 h of incubation). On LNCaP cells (PSMA positive) cell-associated activity rapidly decreased during the first hour and remained stable between 1 and 24 h. Cell associated activity was 60% from initial at 24 h. Additionally, we applied a common assay allowing discrimination of membrane-bound and internalized activity for radio-iodinated heterodimers [The cellular retention of activity after interrupted incubation on PC-3pip cells A showed aP cells B. The cerodimers  and foun125I]I-BO530 showed significant difference in activity uptake by tumors and GRPR-expressing organs (pancreas and stomach) between the non-blocked and blocked groups. Heterodimers [125I]I-BO535 and [125I]I-BO536 failed to demonstrate significant difference in activity uptake in GRPR positive tumors between the blocked and non-blocked groups. However, activity uptake in GRPR expressing normal organs  was significantly lower in groups co-injected with non-labeled RM26 for both heterodimers. In mice bearing LNCaP xenografts (125I]I-BO530 and [125I]I-BO536 demonstrated significant difference in tumor activity uptake between the non-blocked and blocked groups while the significant difference in kidney activity uptake (PSMA expressing) was shown for all tested radiolabeled heterodimers. Activity uptake in lungs after injection of radio-iodinated heterodimers depended on the xenograft model, e.g., for [125I]I-BO530 activity uptake in PC-3 xenografted mice was significantly higher than uptake in LNCaP xenografted mice. Co-injection of non-labeled RM26 or PSMA-617 influenced activity uptake in a mixed pattern, while activity uptake of [125I]I-BO530 increased in both models, uptake of [125I]I-BO535 was blocked only by co-injection of PSMA-617, and was not changed for [125I]I-BO536.In vivo targeting specificity was tested for all the radiolabeled heterodimers in mice bearing either PC-3 (GRPR positive) or LNCaP (PSMA positive) xenografts. In vivo binding of radio-iodinated heterodimers to GRPR and PSMA was blocked by co-injection of non-labeled 7.5 nmol of either unlabeled RM26 or PSMA-617, respectively. It was found that in mice bearing PC-3 xenografts  [125I]I-nografts , only [1125I]I-BO530 over time  increased with time . Tumor-t125I]I-BO530 8-linker was the most lipophilic (BO535), as expected. Good radiochemical yields were achieved when the heterodimers were radiolabeled with I-125 using a mild oxidant (IodoGen). All the radiolabeled heterodimers bound specifically in vitro to both GRPR and PSMA on PC-3 (GRPR positive), LNCaP (PSMA positive), and PC-3pip (GRPR and PSMA positive) cells. All three heterodimers had higher affinity for GRPR than for PSMA. The IC50 values for the three heterodimers towards GRPR were in the low nanomolar range while the IC50 values for PSMA were around 100 nM; [125I]I-BO536 appeared to be the most potent of the three heterodimers with the best IC50 values for both GRPR and PSMA. The previous efforts done to target both GRPR and PSMA with a single tracer have used GRPR targeting motifs possessing agonistic functions. Owing to the internalization characteristics of GRPR agonists and triggered by the potential side effects and extensive downregulation of GRPR, we have incorporated antagonistic RM26 as the GRPR targeting motif in our heterodimers in order to maximize tumor uptake and slow down internalization via this receptor. The PSMA inhibitor PSMA-617 is known to possess favorable kinetics and was therefore used as the PSMA targeting motif in the heterodimers. The tyrosyl was incorporated as the binding site for radio-iodine. The synthesized variants of heterodimer differed in the linkers used to couple PSMA binding moiety and hence the lipophilicity. The less lipophilic variant as expected contained hydrophilic PEG linker (BO530), while the variant containing (CH125I]I-BO530 in PC-3 (GRPR positive) and LNCaP (PSMA positive) cells. The results showed that [125I]I-BO530 cleared more rapidly in PC-3 cells while it was highly retained by LNCaP cells over time. This indirectly indicated that cellular processing of the new heterodimers is mainly determined by interaction with PSMA. For comparison, GRPR agonists [18F]F-FB-[Lys3]BBN and [18F]F-FB-Aca-BBN(7\u201314) were rapidly internalized  followed by excretion of 75%\u201385% of internalized activity within 3 h [It has previously been demonstrated that radio-iodinated glu\u2013urea derivatives targeting PSMA could have long retention over time . The radthin 3 h .125I]I-BO530 demonstrated in vivo specificity to both targets. While [125I]I-BO536 had the best affinities for both targets, it did not demonstrate in vivo specificity for GRPR in tumors. Also [125I]I-BO535 failed to demonstrate in vivo specificity to both targeted xenografts despite its prominent affinity for GRPR and PSMA. However, their uptake in the pancreas and kidneys showed significant differences between the non-blocked and blocked groups. Partially this finding could be explained by high lipophilicity of [125I]I-BO535 that manifested in high activity uptake in blood and liver and low uptake in kidneys in comparison with the less lipophilic heterodimer. Off-target sequestering of radiotracer could have resulted in lower bioavailability and therefore lower uptake in tumors which additionally have worse penetration. The activity uptake in the GRPR tumors was significantly higher for [125I]I-BO530 (4.3% \u00b1 0.6%) than for [125I]I-BO535 (2.2% \u00b1 0.6%) and [125I]I-BO536 (3.0% \u00b1 0.3%) and was in agreement with data reported for heterodimers labeled with Ga-68 and Lu-177 reported previously [125I]I-BO530 (10% \u00b1 3%) and [125I]I-BO536 (11% \u00b1 3%) in LNCaP tumors was significantly higher than for [125I]I-BO535 (3% \u00b1 2%) and also in agreement with previously reported heterodimers labeled with radiometals. In vivo specificity of the three radiolabeled heterodimers was studied in mice bearing PC-3 or LNCaP xenografts. Only I-BO530 led to its selection for further evaluation in PC-3pip xenografts expressing both GRPR and PSMA. The biodistribution results were in good agreement with the in vitro observations. The biodistribution study showed a high tumor uptake at 1 h pi which continued to increase up to 3 h pi. Interestingly the tumor uptake remained stable up to 24 h pi. At 72 h pi, the tumor uptake decreased only 2-fold demonstrating promising indications for the potential use of [125I]I-BO530 for therapy. The initial blood uptake was high which reflected in a later increase in the tumor uptake indicating that the high blood uptake is not owed to radiocatabolites. The elevated kidney uptake is owed to the normal expression of PSMA in kidneys; however the renal uptake decreased more rapidly than the uptake in tumors. For comparison, ratio of activity uptake in kidneys and in tumors for [125I]I-BO530 was better than reported for 177Lu-PSMA-617 that nowadays is actively used for treatment of prostate cancer patients: initial uptake of 177Lu-PSMA-617 in kidneys was at least 10-fold higher than in tumors [The aforementioned results for [n tumors .125I]I-BO530 at 24 and 72 h pi were high with relation to the main sites of prostate cancer metastases. The mice were imaged at 3, 24, and 72 h pi and the images were in good agreement with the ex vivo findings providing a high contrast between the tumor and background 24 and 72 h pi.The tumor-to-organ ratios substantially increased with time, reaching satisfactory ratios for the majority of organs 24 h pi. At 72 h pi, the ratios further increased for most organs as the activity continued to clear from normal organs. It has been established that the most common site of prostate cancer metastases is the lymph nodes and bones, followed by liver, and the lungs . The tum125I]I-BO530 for theranostic applications in prostate cancer. The synthesized compound BO530 was radio-iodinated with high radiochemical yields and high radiochemical purities at high molar activities. The heterodimer showed in vivo targeting specificity to GRPR and PSMA and long activity retention in tumors while being cleared from normal organs providing images with high contrast. The findings demonstrate that while [125I]I-BO530 is a promising agent for the theranostics of prostate cancer, continued development towards improved radiotracers with reduced kidney uptake is necessary.In conclusion, in this study we reported the development of radio-iodinated GRPR/PSMA-targeting bispecific heterodimer ["}
+{"text": "Wistar rats were randomized into two groups: control (C) and obese (Ob). Group C was fed a standard diet, while group Ob was fed an unsaturated high-fat diet for 27 weeks. Adiposity, hormonal and biochemical parameters, and systolic blood pressure were observed. Concentration response curves were performed for leptin or acetylcholine in the presence or absence of Akt and NOS inhibitor. Our results showed that an unsaturated high-fat diet promoted a greater feed efficiency (FE), elevation of body weight and body fat (BF), and an adiposity index, characterizing a model of obesity. However, comorbidities frequently associated with experimental obesity were not visualized, such as glucose intolerance, dyslipidemia and hypertension. The evaluation of the endothelium-dependent relaxation with acetylcholine showed no differences between the C and Ob rats. After NOS inhibition, the response was completely abolished in the Ob group, but not in the C group. Furthermore, Akt inhibition completely blunted vascular relaxation in the C group, but not in the Ob group, which was more sensitive to leptin-induced vascular relaxation. L-NAME incubation abolished the relaxation in both groups at the same level. Although Akt inhibitor pre-incubation reduced the leptin response, group C was more sensitive to its effect. In conclusion, the high-unsaturated fat diet-induced obesity improved the vascular reactivity to leptin and does not generate endothelial dysfunction, possibly by the increase in the vascular sensitivity to leptin and increasing NO bioavailability. Moreover, our results suggest that the increase in NO production occurs through the increase in NOS activation by leptin and is partially mediated by the Akt pathway.Experimental studies show that the unsaturated high-fat diet-induced obesity promotes vascular alterations characterized by improving the endothelial L-arginine/Nitric Oxide (NO) pathway. Leptin seems to be involved in this process, promoting vasodilation via increasing NO bioavailability. The aim of this study was to test the hypothesis that unsaturated high-fat diet-induced obesity does not generate endothelial dysfunction via increasing the vascular leptin/Akt/eNOS signaling. Thirty-day-old male  Obesity, a disease characterized by excess body fat (BF), is recognized as a global epidemic since it affects virtually all age groups and social classes in both developed and developing countries . The mosEndothelial dysfunction associated with obesity has been considered an independent risk of developing cardiovascular diseases . For insThe mechanisms and factors involved in the vascular disease associated with obesity are uncertain, especially in the vascular endothelium. Several factors, such as insulin resistance and dyslipidemia, have been cited as possible factors that are responsible for cardiovascular dysfunction in models of obesity . FurtherLeptin is an adipocyte-derived hormone, which is elevated in obese humans and rodents . In addiOur laboratory has revealed that obese rats induced by an high-unsaturated fat diet (20% fat) show an increase of vascular L-arginine/NO pathway in order to protect the integrity of vascular function in obesity, possibly mediated by elevated levels of plasma leptin . Thus, tWistar rats (\u2248150 g) were obtained from the animal facility of the Federal University of Esp\u00edrito Santo. Rats were individually housed, at constant room temperature (24 \u00b1 2\u00b0C), humidity (55 \u00b1 5%) and light cycles (12-h light/dark), and had free access to water.Thirty-day-old male All experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the United States National Institutes of Health, and were approved by the Ethics Committee on Animal Use of the Federal University of Esp\u00edrito Santo (protocol 027/2014).n = 20), which were fed standard rat chow ; and obese , which were fed a high-unsaturated fat diet  for 27 weeks. The dietary ingredients used to prepare the high-fat diet were sodium chloride, casein, powdered milk, soybean protein concentrate, whole corn, cracker flour, dicalcium phosphate, calcium carbonate, additives emulsifier, antioxidants, and cheese flavoring, along with a vitamins and minerals mixture  and decapitated. After median thoracotomy, fat pads of adipose tissue were dissected and weighed. This method allowed the accurate and consistent assessment of the amount ofBF. The adiposity index, used to assess obesity, was calculated with the following formula: adiposity index [BF/final body wt] \u00d7 100. BF was measured from the sum of the individual fat pad weights as follows: BF = epididymal fat + retroperitoneal fat + visceral fat.After 27 weeks of the experimental protocol, a 95% confidence interval (CI) was built for the adiposity index from the Ob and C rats and was adopted as the separation point (SP) between the groups, as previously described . After tObesity developed by a high-fat diet may have changes in the cardiovascular, metabolic and hormonal profile, such as hypertension, glucose intolerance, systemic insulin resistance, dyslipidemia, hyperglycemia, hyperinsulinemia, and hyperleptinemia. Thus, these were evaluated in all groups.Systolic blood pressure was determined by using the non-invasive tail-cuff plethysmography method  in conscious rats. The rats were pre-warmed at 35 \u00b1 2\u00b0C for 5 min and placed in a cylindrical acrylic tube. At the proximal region of the animal\u2019s tail, a cuff was connected that was inflated to 200\u2013250 mmHg; signals were sent and recorded in the computer to obtain pressure data. At least three measurements were performed for each animal to obtain an average record.Following 27 weeks of treatment, all rats fasted for 6 h, and a blood sample from the tip of the tail of conscious rats was collected. The basal blood glucose level of each animal was immediately determined using a handheld glucometer . Subsequently, an injection of glucose solution (2 g/kg body weight) dissolved in water was administered intraperitoneally, and blood glucose levels were measured after 15, 30, 60, 90, and 120 min. Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was calculated according to the following formula: fasting glucose (mmol/l) \u00d7 fasting insulin (\u03bcU/ml)]/22.5.\u00ae, Belo Horizonte, Minas Gerais, Brazil) and analyzed by biochemical analyzer BS-200 . Serum insulin and leptin levels were measured using an enzyme-linked immunosorbent assay (ELISA) using specific kits . The reading was carried out using a microplate reader .For biochemical and hormonal analysis, animals fasted for 12\u201315 h, then were anesthetized with chloral hydrate  and sacrificed by decapitation. Blood was collected in Falcon tubes, centrifuged at 3,000 \u00d7 g for 15 min and stored at -80\u00b0C. Serum levels of glucose, triacylglycerol (TG), total cholesterol (T-Chol), protein and high-density lipoprotein (HDL) were determined using a specific kit . Cardiac remodeling at the macroscopic level, which identifies the presence or absence of cardiac hypertrophy, was determined by analyzing the following parameters: heart weight (HW), left and right ventricles (LV and RV) and atrium (AT) weights, HW, LV and RV/tibia length ratios.4 1.2, CaCl2 1.6, K2HPO4 1.2, NaHCO3 25 and glucose 11, and aerated with a carbogenic mixture containing 5% CO2 and 95% O2. The connective and adipose tissues were removed, and the artery was divided into sections of approximately 3.5 mm in length. Each vascular ring was mounted in an isolated organ bath system  containing 5 mL of Krebs-Henseleit solution heated to 37 \u00b1 0.5\u00b0C and was continuously gassed with the carbogenic mixture. Changes in vessel diameter were recorded by a force transducer connected to a data acquisition system . During the 45 min stabilization period, the aortic rings were subjected to a resting tension of approximately 1 g and adjusted when necessary. To test the functional integrity the rings were contracted with KCl 75 mM, and after washing and returning to the baseline values, the rings were contracted with 10-7 M phenylephrine and relaxed with 10-5 M acetylcholine to test the endothelial integrity.The animals were anesthetized with a mixture containing ketamine and xylazine  and sacrificed by decapitation. The chest was opened, and the descending thoracic aorta artery was removed and immersed in a modified Krebs-Henseleit solution composed of (in mM/L) NaCl 118, KCl 4.7, MgSO-10 to 10-4 M), and the data was shown as a percentage of contraction corrected by the maximal contraction induced by KCl. Prior to the concentration-response relaxation curves, all the rings were pre-constricted with 10-7 M of phenylephrine. The endothelium-dependent relaxation curves with acetylcholine (10-11 to 10-4 M) and leptin (10-14 to 10-6 M) were obtained. To analyze the role of Akt and NO in response to leptin and acetylcholine, aortic rings were exposed to the 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-Omethyl-3-O-octadecyl-sn-glycerocarbonate \u201cAkt inhibitor\u201d  and N\u03c9-Nitro-L-arginine methyl ester  30 min prior to the concentration-response curves. To evaluate the baseline NO production in the vascular constriction, the vessel rings of control and obese rats were exposed to L-NAME (100 \u03bcM) before the constrictor response curve to phenylephrine.The concentration-response constriction curve was obtained using increasing concentrations of phenylephrine , followed by Tukey post hoc test, was performed to evaluate the change in body weight. In addition, the concentration-response curves were analyzed using two-way ANOVA, followed by Fisher post hoc tests. The p-value was considered significant when <5%.Data on general characteristics, nutritional assessment and comorbidities were reported as mean \u00b1 standard error of the mean (SEM) and submitted to the Kolmogorov-Smirnov test to determine adherence to normality. Comparisons of groups were analyzed by Student\u2019s There were no significant differences in comorbidities associated with obesity, since the SBP, TG, Cholesterol, LDL, HDL, and protein were similar between the groups . The glup > 0.05). The concentration-response curve to phenylephrine showed a significant reduction in the constrictor response obtained from vessels of Ob animals compared to C  the high-fat diet led to the development of obesity; (ii) a reduction in the vasoconstrictor response to phenylephrine in obese animals; (iii) endothelial dysfunction was not observed in this model of obesity, since thoracic aorta from obese rats were still responsive to leptin effects; (iv) a significantly higher vasodilator response to leptin was observed in the obese group, which was abolished when exposed to NOS blockade in both groups; (v) there was a reduction in the vasodilator response to leptin in both groups, compared with the respective control, when the rings were exposed to Akt inhibitor.Experimental models that mimic the eating habits of the human population have been widely used to elucidate the mechanisms of obesity and metabolic disorders . In thisIn addition to absence of changes in the metabolic abnormalities, the model proposed did not lead to an elevation in blood pressure levels. This finding corroborates studies that have verified a significant reduction in SBP in obese animals . Some stThe results from the post-death morphological analysis revealed obesity-induced cardiac hypertrophy, evidenced by elevation of absolute LV weight, as well as LV/tibia length. These data corroborate several studies that indicate obesity induced by a high-fat diet for 15 and 30 weeks promotes cardiac hypertrophy; these authors suggest that cardiac remodeling occurs due to the long period of obesity , which iObesity constitutes a major cause of CVD, including hypertension, atherosclerosis, ischemic heart disease and heart failure. It is widely known that a high intake of saturated fat increases the risk of cardiovascular diseases, while diets enriched with unsaturated fatty acids seem to lower this risk . Thus, sAnother effect of obesity induced by a diet rich in unsaturated fat for 27 weeks was the significant reduction in the vascular adrenergic response in Ob animals. This result was in accordance with our previously published results , 2017. HOn the other hand, Wistar Kyoto rats. In addition, it was verified that insulin potentiates this response by increasing the Akt phosphorylation at the sites of Ser473 and Thr308  diet-induced obesity improved the vascular reactivity to leptin and does not generate endothelial dysfunction, possibly by the increase in the vascular sensitivity to leptin and increasing NO bioavailability. Moreover, our results suggest that the increase in NO production occurs through the increase in NOS activation by leptin and is partially mediated by the Akt pathway.AL-L, VR, and AL participated and conceived the research design. EC, VdS, JC, LD, MdC, AL-L, and AL performed the experiments and data analysis. VR, EC, MdC, HM, TBdN, AL-L, and AL interpreted and discussed the data. AL, VR, EC, MdC, and AL wrote the manuscript. HM, TBdN, AL-L, and AL refined the final draft and revised the manuscript. All authors read and approved the final version 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."}
+{"text": "The Scientific Teaching (ST) pedagogical framework encompasses many of the best practices recommended in the literature and highlighted in national reports. Understanding the growth and impact of ST requires instruments to accurately measure the extent to which practitioners implement ST in their courses. Researchers have typically relied on students, instructors, or observers to document course teaching practices, but it remains unclear whether and how these perspectives differ from each other. To address this issue, we modified our previously published instrument to generate the Measurement Instrument for Scientific Teaching-Observable (MISTO), which can be completed by students, instructors, and observers, and we investigated the degree of similarity between these three perspectives across 70 undergraduate science courses at seven different institutions in the USA.We found that the full MISTO and Active Learning subcategory scores showed the highest correlations among the three perspectives, but the degree of correlation between perspectives varied for the other subcategories. Match scores between students and instructors were significantly higher than observer matches for the full MISTO and for the Active Learning, Inclusivity, and Responsiveness subcategories.We find that the level and type of agreement between perspectives varies across MISTO subcategories and that this variation likely stems from intrinsic differences in the course access and scoring decisions of the three perspectives. Building on this data, we recommend MISTO users consider their research goals, available resources, and potential artifacts that may arise when deciding which perspective best fits their needs in measuring classroom teaching practices.The online version of this article (10.1186/s40594-018-0128-1) contains supplementary material, which is available to authorized users. Undergraduate science education is in the midst of broad-scale efforts to shift teaching and learning approaches from traditional lecture-style instruction to more active, evidence-based strategies that foster student success (American Association for the Advancement of Science (AAAS) In previous work, we developed a taxonomy of observable ST practices to identify and delineate the various pedagogical goals and instructional techniques of ST , which includes only the ST practices and frequencies that can be detected in video samples from 1\u00a0week of class sessions. We measured (1) to what degree student mean, instructor, and observer MISTO scores correlate with each other across subcategories, (2) how closely these three perspectives estimate the amount of class time devoted to active learning, and (3) how closely the three perspectives match on individual items and whether agreement varies across subcategories. Understanding the relationships between these perspectives will help researchers, instructors, and administrators better interpret course measurement data and identify the perspective that aligns most closely with their goals.We collected survey data and video recordings from 70 courses at seven U.S. institutions Table\u00a0. We firsTo produce a version of MIST amenable to class observation, we first identified and removed 12 items referring to practices that occur outside of class time, such as out-of-class homework  with no a priori assumption of which perspective would serve as the reference point. Thus, we derived \u201cmatch scores\u201d to estimate how closely aligned responses were between two perspectives. Match scores for an item were calculated using the following equation:1 and score2 represent the scores assigned by each perspective. For students, each item score was the mean student response for a course. Match scores were then averaged for the full MISTO and each subcategory for each course. Match scores occur on a scale of 0\u20131 with a higher match indicating closer agreement between perspectives.where scoreOnce the video scoring rubric was formalized, we developed a training procedure to achieve acceptable agreement between observers. Initially, two observers co-coded 1\u00a0week of videos from a \u201ctraining set\u201d of eight courses. These two observers monitored their agreement and discussed any disagreements to consensus. Two additional observers separately scored the eight courses from the training set, progressing from more guided scoring to more independent scoring across the eight courses. The observers monitored agreement with the consensus scores for these videos and discussed any disagreements to consensus.Following training, all observers were tested for acceptable agreement. One observer first coded 1\u00a0week of videos from five new courses. Next, each of the other three raters coded three of these courses, and all three of the observers achieved an average match score above 0.75, which we considered sufficient for independent scoring. Again, any disagreements were discussed to consensus.The videos from the remaining courses in this study were each scored by one of the four observers. After all initial course observations were completed, two of the observers co-coded a set of ten courses to check whether acceptable agreement had been maintained, achieving an average match score of 0.94 across the ten courses.g, were estimated using the cohen.d and hedges.correction functions in the effsize package , CU (project ID 15-0297), and all other participating institutions.r\u2009=\u20090.59\u20130.74, p\u2009<\u20090.001; Fig.\u00a0r\u2009>\u20090.7, p\u2009<\u20090.001), with the highest correlations occurring between students and instructors  to low (r\u2009<\u20090.3) correlations between perspectives, and these levels varied by pairings  between all perspectives . The instructor\u2013observer match was significantly higher than either student match for Reflection (p\u2009<\u20090.001), and no significant differences were observed between perspectives for the Experimental Design, Data Analysis, and Cognitive Skills subcategories.To better understand agreement between perspectives, we also used the \u201cmatch scores\u201d described in the methods to determine how closely the perspectives scored each item relative to its own scale. All three perspectives showed relatively high matches, with most pair-wise comparisons matching above 0.75. The relative match level varied between perspective pairs for several MISTO subcategories Fig.\u00a0. Studentp\u2009=\u20090.07\u20130.89). We also found no significant influence of class size or course level on agreement (p\u2009=\u20090.21\u20130.87).We also investigated the effect of several course and instructor characteristics on match scores  high correlation\u2013high match, (2) moderate correlation\u2013high match, and (3) low correlation\u2013low match.r\u2009=\u20090.91). Previous studies have raised potential concerns regarding the shortcomings and limitations of sampling teaching practices from each of the perspectives measured in this study  as well as the highest match scores for the full MISTO Figs.\u00a0 and 5. WOverall, our results highlight the nuanced nature of alignment between student, instructor, and observer responses for the full MISTO and subcategory scores. Given these results, we recommend that MISTO users consider their research goals and the resources available when deciding which perspective (s) to use as a measure of ST implementation.Students provide the benefit of larger sample sizes from which to elicit a measure of central tendency to estimate ST levels. Students represent a universally available resource for all courses, and collecting student responses can help mitigate potential conflicts with instructor motivations. For example, if an instructor wished to collect documentation of their teaching practices for promotion and tenure purposes, data from their students would likely be seen as more credible than self-reported data. As the ultimate target of educational programs, student perspectives also carry a certain primacy of importance. If students report a particular set of practices  at a lower frequency than instructors or observers, one would want to further investigate the reasons behind this phenomenon. On the negative side, student data tends to be noisy on the individual student level, and collecting data from students requires some additional coordination and potentially institutional review board (IRB) considerations.Instructors represent the most practical and accessible option for gathering data, especially under conditions of limited resources or when data collection spans multiple institutions. Since instructor surveys can be collected without any student involvement, the study coordination and IRB approval processes are substantially more streamlined. This may be a major motivation driving the use of instructor self-reports to measure teaching practices in many studies  will have greater potential for agreement between perspectives than those with more subjective and nuanced practices . We also note that practices associated with the Experimental Design, Data Analysis, and Reflection subcategories were implemented quite infrequently in our sample. These practices are likely to be important features of science curriculum, so their low levels of implementation warrant further research for the broader field.As transformation efforts in undergraduate science education continue, measurements of teaching practices are needed to gauge the status of the field, track how teaching changes over time, and determine the impact of specific strategies , CU (project ID 15-0297), and all other participating institutions.Additional file 1:MIST items removed from MISTO. This file lists all the MIST survey questions that were removed from MIST in creating MISTO, generally because the associated ST practices were not observable or were inconsistently observable in video recordings of classroom sessions. (DOCX 14 kb)Additional file 2:MISTO video scoring workbook. This file contains a multi-sheet Excel workbook where observers record teaching practices on up to three scoring sheets. Those records are then translated into observer MISTO survey responses and their corresponding scores for data analysis see Fig. . Additional file 4:Effects of course and instructor characteristics on match scores. Match pair indicates the perspectives being compared: IO is instructor\u2013observer, SO is student\u2013observer, and SI is student\u2013instructor. (DOCX 16 kb)Additional file 5:MISTO scoring template. After obtaining MISTO responses either through the online survey or the MISTO video scoring workbook, this Excel template can be used to calculate MISTO and MISTO subcategory scores for each perspective measured. Note: This template is designed for use with the MISTO question set (not the full MIST question set). (XLSX 2499 kb)Additional file 6:MISTO Qualtrics file. This qsf file contains the MISTO survey, which can be administered to students or instructors using the online Qualtrics platform. This version of the survey should only be used when asking students or instructors to reflect on a one week sample of class sessions or when comparing these perspectives to observers. For other purposes, we recommend using the original MIST survey qsf file, which is available in the supplement of the original publication (Durham et al."}
+{"text": "Unlike other members of the thermoTRPV channel family, TRPV3 sensitizes upon repeated stimulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show \u03b1-to-\u03c0-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker \u03c0-helix during ligand-dependent gating.Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. Mutations in  Transient receptor potential vanilloid channel 3 (TRPV3) responds to temperature and sensitizes upon repeated stimulation with either heat or agonists. Here authors present the cryo-EM structures of apo and sensitized human TRPV3 and describe the structural basis of sensitization. The vanilloid TRP (TRPV) subfamily consists of six members (TRPV1\u2212TRPV6), four of which (TRPV1\u2212TRPV4) possess an intrinsic capability to respond to temperature and are therefore referred to as thermosensitive TRPV (thermoTRPV) channels. In addition to heat, thermoTRPV channels are also modulated by synthetic and natural ligands as well as lipids8. Although the thermoTRPV channels share >50% homology, each channel possesses a distinct functional and pharmacological profile that enables integration of a multitude of different stimuli into a finely tuned response10.Transient receptor potential (TRP) channels are a superfamily of cation-selective ion channels that are involved in numerous physiological processes13. Notably, TRPV3 sensitization is inherent to the channel and independent of the modality of the stimulus. The channel undergoes hysteresis, which irreversibly lowers the energetic barrier for activation, resulting in a faster rate of activation and higher open probability13. TRPV3 is most abundantly expressed in epidermal and hair follicle keratinocytes where it plays a key role in the maintenance of normal skin physiology14. Activation of TRPV3 in keratinocytes has been shown to lead to release of proinflammatory messengers, and several \u201cgain-of-function\u201d mutations in trpv3 have been found to cause the human congenital skin disorder Olmsted syndrome, characterized by bilateral mutilating palmoplantar keratoderma, periorificial keratotic plaques, and hair loss with follicular papules22. Therefore, TRPV3 plays essential roles in epidermal proliferation, differentiation, survival, hair growth, and the development of itch sensation23. Several small-molecule inhibitors, such as FTP-THQ and GRC 15300, of TRPV3 have demonstrated analgesic properties in inflammatory and neuropathic pain models25.In contrast to other thermoTRP channels, TRPV3 sensitizes, rather than desensitizes, upon repeated stimulation with either heat or agonists30. However, as the structure of TRPV3 has yet to be determined, a large gap currently exists in our mechanistic understanding of thermoTRPV channel sensitization in activation and regulation. To fill this knowledge gap, we determined the cryo-EM structures of human TRPV3 in apo and sensitized states at ~3.4\u2009\u00c5 and ~3.2\u2009\u00c5 resolution, respectively. We additionally determined three conformationally distinct states of TRPV3 in the presence of the agonist 2-aminoethoxydiphenyl borate (2-APB), at resolutions ranging from ~3.5\u20134\u2009\u00c5, where we observe a departure from the canonical fourfold symmetry previously reported for most thermoTRPV structures. Together, these structures illustrate the importance of \u03c0-helical segments in the transmembrane domain of TRPV3 in dictating the conformational landscape during sensitization and gating.High-resolution structures of thermoTRPV channels captured in various states along the gating cycle have been critical to understanding the molecular underpinnings that govern both general and individual functional modalities of thermoTRPV channelsExtensive screening of various TRPV3 homologs and mutants identified human TRPV3 containing the point mutation T96A as optimal for structural studies (see Methods). Characterization of both T96A and wild-type channels confirmed that the T96A mutation did not significantly alter the ligand-dependent gating profile of the TRPV3 channel Fig.\u00a0. Whole-cT96A, hereafter simply referred to as TRPV3, in the closed apo state to ~3.4\u2009\u00c5 using cryo-electron microscopy (cryo-EM) (see Methods) , is a fourfold symmetric homotetramer. Each monomer contains an amino- (N-) terminal cytosolic ankyrin repeat domain (ARD) and a domain-swapped transmembrane domain (TMD) comprising six transmembrane helices (S1\u2212S6). The TMD and ARD are connected via the coupling domain (CD), which consists of the linker domain, pre-S1, and the carboxy- (C-) terminal domain (CTD). The TMD consists of a voltage sensor-like domain (VSLD) containing helices S1\u2013S4, and a pore domain comprising helices S5, S6 and the pore helix. The VSLD and the pore domain are connected via an S4\u2212S5 linker, which mediates the domain-swap configuration. In addition, the channel contains a TRP domain  Figs.\u00a0. The TRPain Fig.\u00a0. The stahelices S\u2212S6. The 27 (PDB 3J5P), TRPV228 (PDB 5AN8) and TRPV430 (PDB 6BBJ) channels reveals several notable features. First, the high quality of the TRPV3 EM density map has allowed us to build the most complete thermoTRPV CTD to date (residues 707\u2013754), a region that was not well resolved in the cryo-EM structures of TRPV1, TRPV2, or TRPV430. The most N-terminal region of the CTD, which extends from the TRP domain into the cytosol, forms a short \u03b1-helix similar to that observed in TRPV430, but in contrast to the loops observed in TRPV1 and TRPV229 . A single cryo-EM dataset yielded three distinct conformations, TRPV32-APB 1, TRPV32-APB 2, and TRPV32-APB 3  symmetry , with only the S4\u2212S5 linker deviating significantly. This suggests that the S4\u2212S5 linker is the origin of the rotation observed in subunit B of TRPV32-APB 2 , indicating that this region is the source of C2 symmetry in the TRPV32-APB 2 structure  of TRPV1 and TRPV228 analogous to glycine and proline hinges in K+ channels46. However, in contrast to the hinges in K+ channels which are engineered into the amino acid sequence, the \u03c0-helix hinge was predicted to form depending on the functional state of the channel28. While this manuscript was under review, structures of the mouse TRPV3 (mTRPV3) in apo (closed) and 2-APB bound (open) states were published47, allowing us to draw comparisons to our data. Most strikingly, the open mTRPV3 channel possesses a \u03c0-helix in the S6. We therefore reason that our putative sensitized state, which contains a \u03c0-helical segment in S6 and has a closed gate, illustrates the hallmarks of a sensitized channel that is closed but requires less energy for activation than the na\u00efve channel  which appears to be rotated with respect to the VSLD. Indeed, the position of the pore domain of the apo human TRPV3 at the extracellular side aligns better with the pore domain of the open mTRPV3 channel than its apo counterpart. This might be indicative of inter-species differences, suggesting that the pore domain of the human TRPV3 channel does not undergo independent movement during gating  of the C4-symmetric channel, which was performed to estimate possible global protein conformations of TRPV3, resulted in trajectories that recapitulated the experimentally observed adoption of the C2-symmetric states  insect cells (ATCC) identified the human TRPV3 as the most suitable candidate for structural studies. To further stabilize hTRPV3, we searched a random mutant library of mouse TRPV33 for clones that exhibited either a large 2-APB response or a high baseline in a Ca2+-dependent fluorescence assay. We aimed to find mutants with higher expression levels due to increased stability. Fifty clones were sequenced, and we introduced 29 point mutations into their corresponding sites in the hTRPV3  and broken by sonication (3\u2009\u00d7\u200930 pulses). 40\u2009mM dodecyl \u03b2-maltoside  and 4\u2009mM Cholesteryl Hemisuccinate Tris salt  were added to the lysate for extraction at 4\u2009\u00b0C for 1\u2009hour. Unsolubilized material was removed by centrifugation , and anti-FLAG resin was added to the supernatant for 1\u2009hour at 4\u2009\u00b0C. For preparation of the apo sample, the resin was loaded onto a Biorad column at 4\u2009\u00b0C and washed with ten column volumes buffer B ) and the protein eluted in buffer C , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1\u2032-rac-glycerol) (POPG), 10\u2009mM DTT, 10\u2009mg\u2009ml\u22121 FLAG peptide). For preparation of the sensitized TRPV3, the anti-FLAG resin was loaded onto a Biorad column and washed with five column volumes Buffer B , followed by five column volumes Buffer B supplemented with 1\u2009mM 2-APB. This was repeated ten times, before the protein was eluted in five column volumes buffer C . For preparation of the TRPV32-APB sample, the same procedure was followed except buffer C was supplemented with 1\u2009mM 2-APB. Following size exclusion chromatography, conducted at 4\u2009\u00b0C, the protein peaks were collected, mixed with Poly  substituted with 3-(Dimethylamino) Propylamine  (1:10 w/w ratio) and incubated overnight at 4\u2009\u00b0C with gentle agitation. Detergent was removed with Bio-Beads SM-2 (15\u2009mg\u2009ml\u22121) for 1\u2009hour at 4\u2009\u00b0C. The reconstituted protein was further purified on a Superose 6 column at 4\u2009\u00b0C in buffer D . In preparation of the TRPV32-APB sample, Buffer D was supplemented with 1\u2009mM 2-APB. Following size exclusion, the protein peak was collected and concentrated to 2\u22122.5\u2009mg\u2009ml\u22121.A full-length human TRPV3 construct, containing mutation T96A was cloned into a pFastBac vector in frame with a FLAG affinity tag, and baculovirus was produced according to the manufacturers\u2019 protocol . For protein expression, Sf9 insect cells (ATCC) were infected with baculovirus at a density of 1.3\u00d710\u22121) was dispensed on a freshly plasma cleaned (Gatan Solarus) UltrAuFoil R1.2/1.3 300-mesh grid (Electron Microscopy Services) and manually blotted50 using a custom-built manual plunger housed in a 4\u2009\u00b0C cold room (>85% relative humidity). Samples were blotted for 4\u20135\u2009s with Whatman No. 1 filter paper immediately before plunge-freezing in liquid ethane cooled by liquid nitrogen.Cryo-EM grid preparation was performed similarly for each TRPV3 specimen. Briefly, 3\u2009\u03bcl of purified TRPV3  were used for CTF estimation using CTFFIND455 (whole micrograph CTF estimation) to remove those micrographs with a confidence of fit below 90% were eliminated from further processing. Difference of Gaussian (DoG) picker56 was used to automatically pick particles from the first ~100 micrographs and then subjected to reference-free 2D classification using multivariant statistical analysis/multi-reference alignment (MSA/MRA)57 . The best 2D classes that represented orthogonal views of TRPV3 were then used for template-based particle picking using FindEM58. A final stack of 361,244 particle picks were then subjected to reference-free 2D classification using RELION59 version 1.4  with the best 215,346 particles were subsequently 3D auto-refined using a 20-\u00c5 low-pass-filtered TRPV2 structure (EMDB-6455) as the initial model. Refined particles were then re-centered and re-extracted binned 2\u2009\u00d7\u20092  and 3D auto-refined using a scaled version of the binned 8\u2009\u00d7\u20098 map as the initial model. Particle movement and dose-weighting were then performed using the \u201cparticle polishing\u201d feature as implemented within RELION. A smoothened plot of the per-frame B-factor and intercepts  were then used to calculate the frequency-dependent weighting. These \u201cshiny\u201d particles were then 3D auto-refined to yield a 3.95\u2009\u00c5 resolution reconstruction (C1 symmetry). This reconstruction exhibited C4 symmetry. Subsequent no-alignment 3D classification using a mask generated against the full molecule low-pass filtered to 10\u2009\u00c5 yielded a single good class that refined to 3.6\u2009\u00c5 resolution (C4 symmetry). During the preparation of this manuscript the frame-alignment software MotionCor260 was released so the 282,136 particles contributing to the 3.6\u2009\u00c5 resolution reconstruction were then re-centered and re-extracted from micrographs that had been aligned and dose-weighted using MotionCor2 without binning (0.655\u2009\u00c5 per pixel) with per-particle CTF values as estimated using Gctf61. These particles were subjected to 3D classification where the best resolved class was subsequently 3D auto-refined to 3.5\u2009\u00c5 resolution (C4 symmetry). These particles  were then subjected to a no-alignment 3D classification using a soft mask of the full molecule. The best resolving class  was then 3D auto-refined (C4 symmetry) to a final resolution of 3.4\u2009\u00c5, as estimated by gold-standard FSC 0.143 criterion63.Movies of apo TRPV3 were collected using a Thermo Fisher Titan Krios transmission electron microscope (TEM) operating at 300\u2009keV with a Gatan K2 Summit direct electron detector (DED) operating in super-resolution mode  at a nominal magnification of \u00d722,500. Movies were collected over a 12\u2009s exposure with an exposure rate of ~9e- pixel\u22121\u2009s\u22121, resulting in a total exposure of ~67 e-\u2009\u00c5\u22122 and a nominal defocus range from \u22120.8 to \u22121.4\u2009\u00b5m. MotionCor2 was used to perform motion correction and dose-weighting. Unweighted summed images were used for CTF determination using CTFFIND4. FindEM was used for template-based particle picking using templates previously generated of apo TRPV3, yielding a stack of 559,206 picks that were binned 4\u2009\u00d7\u20094  and subjected to reference-free 2D classification using RELION 2.164. A total of 339,693 particles corresponding to the 2D classes displaying the strongest secondary-structural elements were input to 3D auto-refinement in RELION without symmetry imposed. The apo TRPV3 volume was low-pass filtered to 30\u2009\u00c5 and used as an initial model. The refined coordinates were used for re-centering and re-extraction of 2\u2009\u00d7\u20092 binned particles  and subjected to 3D classification (k\u2009=\u20096) using a soft mask generated from the full molecule. A single class exhibiting C4 symmetry  was 3D auto-refined with a soft mask and C4 symmetry applied to yield a ~3.7\u2009\u00c5 reconstruction. These particles were re-centered and re-extracted unbinned (0.915\u2009\u00c5 per pixel) and auto-refined to obtain a final resolution of ~3.24\u2009\u00c5 (C4 symmetry).Movies of sensitized TRPV3 were collected on a Talos Arctica (Thermo Fisher) TEM operating at 200\u2009keV. Movies were collected using a K2 Summit DED in counting mode at a nominal magnification of \u00d745,000 corresponding to a physical pixel size of 0.915\u2009\u00c5 per pixel. A total of 2982 movies (47 frames/movie) were collected using a 12\u2009s exposure with an exposure rate of ~4.7e- pixel\u22121\u2009s\u22121, resulting in a total exposure of ~60e-\u2009\u00c5\u22122 and a nominal defocus range from \u22121.2 to \u22122\u2009\u00b5m. All preprocessing  was performed as described above. A stack of 897,643 picks  were subjected to reference-free 2D classification in RELION. A subset of 589,656 particles corresponding to the best 2D classes were subsequently 3D auto-refined with C4 symmetry imposed, using the apo TRPV3 volume low-pass filtered to 30\u2009\u00c5 as an initial model. The particles were then subjected to 3D classification (k\u2009=\u20096) without alignment, using a soft mask generated from the full molecule. A subset of 397,536 particles were selected and subsequently re-centered and re-extracted unbinned  and 3D auto-refined with C4 symmetry imposed to yield a ~3.6\u2009\u00c5 reconstruction. An additional round of 3D classification (k\u2009=\u20096) without alignment and without symmetry imposed revealed one state exhibiting C4 symmetry and two distinct states exhibiting C2 symmetry, which subsequently refined to ~3.5\u2009\u00c5 , ~4\u2009\u00c5 , and ~4\u2009\u00c5 , respectively. Local resolution estimates of the final reconstructions were calculated using BSOFT65.Movies of TRPV3 in the presence of 2-APB were collected on a Talos Arctica equipped with a K2 Summit DED operating in counting mode. Images were collected at a nominal magnification of \u00d736,000 corresponding to a physical pixel size of 1.15\u2009\u00c5 per pixel. A total of 1908 movies (60 frames per movie) were collected using a 15\u2009s exposure with an exposure rate of ~5.3e- pixel66, using the structure of TRPV2 (PDB 5AN8) as a template for the transmembrane domains and the structure of the human TRPV3 ankyrin repeat domains41 (PDB 4N5Q) as a template for the ARD. The structure was real-space refined in Coot, with ideal geometry constraints where appropriate. The model was iteratively refined using the phenix.real_space_refine command line67\u00a0by global minimization and rigid body, while maintaining high weight on ideal geometry and secondary structure restraints. Molprobity68 (http://molprobity.biochem.duke.edu/) was used to identify problematic regions, which were subsequently rebuilt in Coot. The refined apo TRPV3 model was used as a template for building the remaining four models. Radius along the permeation pathway was calculated using HOLE69. All analysis and structure illustrations were performed using Pymol  and UCSF Chimera70.The apo TRPV3 model was built in the cryo-EM electron density map in Coot71 as recommended by the developers.Normal mode analysis was performed on the apo TRPV3 structure using the backbone (C\u03b1) atoms using the program ProDy2. Cells between passage 10\u201330 grown in 40-mm wells were transiently transfected at ~50% confluency with plasmids encoding for either WT or T96A TRPV3 and green fluorescent protein using FuGene6 (Promega). Twenty-four hours post transfection, cells were reseeded onto 12-mm round glass coverslips (Fisher) and used 12\u201324\u2009hours after for electrophysiological measurements.HEK293T cells (62312975\u2014ATCC) were grown in Dulbecco's Modified Eagle Medium supplemented with 10%\u00a0fetal bovine serum (Gibco), 1% penicillin/streptomycin (Gibco) and were sustained at 37\u2009\u00b0C in 5% CO2, 10\u00a04-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5\u00a0ethylene glycol-bis(\u03b2-aminoethyl ether)-N,N,N\u2032,N\u2032-tetraacetic acid, and adjusted to pH 7.2 (CsOH). Glass coverslips with adherent transfected cells were placed into an open bath chamber  and an extracellular solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES at pH 7.4 (NaOH) with and without (wash) 30\u2009\u00b5M 2-APB (Sigma) (prepared daily from dimethyl sulfoxide (DMSO) stocks (1\u2009M) stored at \u221280\u2009\u00b0C; final DMSO 0.03%) was focally applied with a pressurized perfusion system .Voltage-clamp recording were performed at room temperature (22\u2009\u00b0C) in the whole-cell patch configuration with electrodes pulled from borosilated glass capillaries (Sutter Instruments) with a final resistance of 2.5\u20135 M\u2126. Electrodes were filled with an intracellular solution containing (in mM) 150 CsCl, 1 MgCl2-APB sensitization experiments were performed with a 30-s continuously repeating protocol  in which the cell was first perfused with extracellular wash for 1\u2009s, followed by a 15-s application of 2-APB extracellular solution, then preceded by 14\u2009s of wash solution. The repeating recording protocol was then stopped when the 2-APB-elicited responses no longer exhibited potentiation of the peak current amplitude. Current responses were low-pass filtered at 2\u2009kHz (Axopatch 200B), digitally sampled at 5\u201310\u2009kHz (Digidata 1440A), converted to digital files in Clampex10.7 (Molecular Devices) and stored on an external hard drive for offline analyses I) from each stimulation where normalized to the maximum current (Imax) amplitude, and the fractional current (I/Imax) of each stimulation was plotted by stimulation number for each individual recording13. The stimulation time course of sensitization was fitted with a standard two-state Boltzmann equation:S the stimulation number, S50 the stimulation number corresponding to I\u2009=\u2009Imax/2, and the slope factor k, a fitted constant expressed in stimulation number representing the rate of change of sensitization. The fitted parameter values were obtained by fitting individual I/Imax vs. stimulation plots from biologically independent experiments and individual values were used to calculate the mean S50 and k for WT and T96A. The extent of sensitization was characterized by the relative increase in current obtained during the first (I0) and maximum current (Imax) stimulation (Imax/I0) and calculated as the mean from each biologically independent experiment.Sensitization parameters were evaluated by the stimulation-dependent increase in the peak current amplitude measured at the end of each 2-APB exposure. Current amplitudes (Supplementary InformationPeer Review FileSource Data"}
+{"text": "Interestingly, in our cohort, nuclear and cytoplasmic THR\u03b21 appeared to be independent markers either for poor (p = 0.0004) or for good (p = 0.048) prognosis, respectively. Altogether, these data indicate that the subcellular expression of THR\u03b21 may play an important role in oncogenesis. Moreover, the expression of nuclear THR\u03b21 is a negative outcome marker, which may help to identify high-risk BC subgroups. The aim of this study was to investigate the expression of thyroid hormone receptor \u03b21 (THR\u03b21) by immunohistochemistry in breast cancer (BC) tissues and to correlate the results with clinico-biological parameters. In a well-characterized cohort of 274 primary BC patients, THR\u03b21 was widely expressed with a predominant nuclear location, although cytoplasmic staining was also frequently observed. Both nuclear and cytoplasmic THR\u03b21 were correlated with high-risk BC markers such as human epidermal growth factor receptor 2 (HER2), Ki67 , prominin-1 (CD133), and N-cadherin. Overall survival analysis demonstrated that cytoplasmic THR\u03b21 was correlated with favourable survival ( Breast cancer (BC), the most frequent cause of cancer death worldwide , is highLinks between BC and expression of other NR have already been outlined by our lab and others ,7,8,9,10A significant association between thyroid hormone (TH) signaling and BC has already been demonstrated ,18,19. HBC signaling and progression is also influenced by the complex interplay between NR, their transcriptional coactivators, and corepressors that also have prognostic significance . Among tWhile THR\u03b21 clearly appears to be a key player in BC carcinogenesis, the importance of its subcellular localization remained to be elucidated. Therefore, purpose of this study was to analyze the nuclear and cytoplasmic localization of THR\u03b21 in a well-defined cohort of 274 primary BC patients, and to correlate the results with clinicopathological parameters and clinical outcome.The total cohort consisted of 274 samples from 271 primary BC patients . Approvan = 263 tumors stained) is presented in Expression of THR\u03b21 was analyzed by immunohistochemistry (IHC), using immunoreactive scores (IRS) as described in Material and Methods. Distribution of staining intensities and percentages of stained cells are presented in p < 0.05), although both means were quite low . Nuclear THR\u03b21 staining was present in 60.5% of the tumors, and cytoplasmic THR\u03b21 in 43.3%. Interestingly, nuclear and cytoplasmic THR\u03b21 was significantly and positively correlated with each other (r = 0.440 p < 0.01 using Spearman\u2013Rho test).THR\u03b21 distribution was then analyzed both in nucleus and in cytoplasm, and total expression (sum of nuclear and cytoplasmic IRS) was calculated . NuclearDistribution of tumors with negative or positive nuclear, or cytoplasmic, THR\u03b21 staining was analyzed for all 263 tumors stained . It appeUsing pairwise analysis, we first analyzed the correlation of THR\u03b21 expression with expression of other NR and coregulators  with preWe previously investigated the expression of two NR transcriptional coregulators, namely RIP140 and LCoR, and demonstrated that their sub-cellular localization may define their association with BC aggressiveness and survival . RegardiCorrelations between THR\u03b21 expression and known clinicopathological characteristics, besides ER and PR, were also analyzed. CD133, a widely used marker for isolating cancer stem cells ,32, and In order to analyze the correlation between THR\u03b21 and patient outcome, we performed Kaplan\u2013Meier analyses . Insteadp = 0.038 and 0.015, respectively). Analyzing total THR\u03b21 expression of (sum of nuclear and cytoplasmic IRS), no correlation with RFS (As shown in with RFS C nor OS with RFS F was fouTo determine the specificity of this result (opposite correlations of nuclear and cytoplasmic THR\u03b21 expression with OS), we performed the identical analyses with another THR, namely THR\u03b12, in the same cohort . As shown in The aim of this study was to characterize THR\u03b21 expression in a wide range of primary BC tissues, taking into account its intracellular expression, and to correlate the results with clinicopathological parameters and patient outcome.Our study confirmed that THR\u03b21 is expressed with a predominantly nuclear location, as previously described for most THR isoforms. Nonetheless, our results also demonstrate cytoplasmic localization of THR\u03b21 in BC. THs are able to modulate gene expression by binding to THR\u03b1 either in the cytoplasm or in the nucleus of the cells . It is an = 796) [The second major observation provided by our study is that nuclear and cytoplasmic forms of THR\u03b21 may exhibit opposite roles in breast tumorigenesis. Indeed, considering the correlation with patient survival , cytoplan = 796)  focused n = 796) . In our p = 0.021), but not in ER-negative ones (p = 0.161) (p = 0.035), but not in non-luminal ones (p = 0.142)  . Consequ= 0.142) . Yet, whOur results also suggest that the differential impact on outcome depending on nuclear or cytoplasmic THR\u03b21 localization is not a common feature for all THRs. In the present study, we also analyzed THR\u03b12 expression. Previously, we had demonstrated that nuclear THR\u03b12 expression tends to be an independent and favorable prognostic marker for survival in a small cohort of 82 invasive BC cases . This waIn summary, the present study confirms the complexity of the links between subcellular localization of the THR\u03b21 protein and its association with patient outcome. To our knowledge, it is the only study supporting the fact that the nuclear form of THR\u03b21, probably acting as a classical ligand-dependent transcription factor, may have tumor-promoting effects in BC. Our results emphasize the importance of more precise investigations of the subcellular localization of THRs in order to define their impact as potential biomarkers in breast cancer.A total of 274 formalin-fixed paraffin-embedded primary BC tissues were collected from 271 patients  who underwent surgery between 2000 and 2002 at the Department of Obstetrics and Gynecology of the Ludwig-Maximilians-University Munich, Germany. All patient data and clinical information from the Munich Cancer Registry were fully anonymized and encoded for statistical analysis. Research was approved by the Ethical Committee of the Medical Faculty, Ludwig-Maximilian-University (LMU), Munich, Germany  and informed consent was obtained from all patients. Union for International Cancer Control (UICC) TNM classification was performed to evaluate the size and extent of the primary tumor (pT), lymph node involvement (pN), and distant metastasis (M). Tumor grade was determined by an experienced pathologist (Dr D. Mayr) of the LMU Department of Pathology, according to a modification of Elston and Ellis grading proposed by Bloom and Richardson . ER, PR,\u00ae-Benchmark Unit  as previously described [Expression of ER\u03b1 and PR was determined at diagnosis in all BC samples of this cohort at the LMU Department of Pathology, Germany. ER\u03b1 and PR expression were evaluated by IHC, as previously described ,30. Sampescribed . The Ki6escribed . We perfescribed , as wellescribed ,30, and escribed . For THRescribed ,39,43. AAfter incubation with a biotinylated secondary anti-rabbit or anti-mouse IgG antibody, and with the associated avidin\u2013biotin\u2013peroxidase complex , visualization was performed with substrate and chromogen 3, 3-diamino-benzidine . Negative and positive controls were used to assess the specificity of the immunoreactions. Negative controls (colored in blue) were performed in BC tissue by replacement of the primary antibodies by species-specific (rabbit/mouse) isotype control antibodies . Appropriate positive controls (placenta samples) were included in each experiment. Sections were counterstained with acidic hematoxylin, dehydrated, and immediately mounted with Eukitt  before manual analysis with a Diaplan light microscope  with 25\u00d7 magnification. Pictures were obtained with a digital Charged Coupled Device (CCD) camera system . All slides were analyzed by two or three independent examiners.Expression of THR\u03b21 and THR\u03b12 was assessed according to IRS, determined by evaluating the proportion of positive tumor cells, scored as 0 (no staining), 1 (\u226410% of stained cells), 2 (11%\u201350% of stained cells), 3 (51%\u201380% of stained cells), and 4 (\u226581% of stained cells); as well as their staining intensity, graded as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong) (IRS = percentage score \u00d7 intensity score), as presented in p values below 0.05 (*), 0.01 (**), or 0.001 (***) were considered statistically significant. Differences in Statistical analyses were performed using software package used for interactive, or batched, statistical analysis (SPSS) 24 . For all analyses, p values of Spearman\u2013Rho test presented), using pairwise analysis. Survival times were compared by Kaplan\u2013Meier graphics and differences in RFS and OS were tested for significance using the chi-square statistics of the log rank test. Data were assumed to be statistically significant in the case of p-value <0.05 or <0.01. Kaplan\u2013Meier curves and estimates were then provided for each group and each marker. The p value and the number of patients analyzed in each group are given for each chart.Correlation analyses presented in p values and hazard ratios were indicated, knowing that the hazard ratios of covariates are interpretable as multiplicative effects on the hazard, and holding the other covariates constant.The multivariable analysis for outcome (OS) presented in Although THR\u03b21 was predominantly expressed in tumor cell nuclei in our primary BC cohort, cytoplasmic expression was also detected; its correlation with patient survival was inverse to that of nuclear THR\u03b21. Our results demonstrate that THR\u03b21 may have different roles in tumorigenesis according to its subcellular localization. A major conclusion is also that THR, particularly nuclear THR\u03b21, can exhibit tumor-promoting activities in the mammary gland, as demonstrated by its independent prognostic value."}
+{"text": "Emergency departments need to continuously calculate quality indicators in order to perform structural improvements, improvements in the daily routine, and ad-hoc improvements in everyday life. However, many different actors across multiple disciplines collaborate to provide emergency care. Hence, patient-related data is stored in several information systems, which in turn makes the calculation of quality indicators more difficult. To address this issue, we aim to link and use routinely collected data of the different actors within the emergency care continuum. In order to assess the feasibility of linking and using routinely collected data for quality indicators and whether this approach adds value to the assessment of emergency care quality, we conducted a single case study in a German academic teaching hospital. We analyzed the available data of the existing information systems in the emergency continuum and linked and pre-processed the data. Based on this, we then calculated four quality indicators . Lessons learned from the calculation and results of the discussions with staff members that had multiple years of work experience in the emergency department provide a better understanding of the quality of the emergency department, the related challenges during the calculation, and the added value of linking routinely collected data. Emergency departments (EDs) have the liability to deliver high-quality emergency care. This requires continuously performing structural improvements, improvements in the daily routine, and ad-hoc improvements in everyday life.However, EDs are barely capable of fully implementing this into the daily business. One reason is that many different actors across multiple disciplines within the emergency care continuum (ECC) collaborate to provide emergency care . As a consequence, patient-related data is stored in several information systems (IS). Due to this distributed storing of data, many EDs struggle to establish quality indicators (QI) that holistically assess the quality of emergency care. Most of the current QIs focus either on particular aspects of the ED , have to be calculated manually, or require a collection of additional data . To addrOur approach relates to other German and international initiatives on using routinely collected data to increase emergency care quality. An example of a German initiative is the TRUST project, which provides trend and structural analyses of the rescue service in Bavaria using routinely collected data  . Another(RQ1) Is it feasible to use routinely collected data within the ECC to calculate QIs that identify improvements areas orad-hocimprovements for an ED and.(RQ2) do QIs based on routinely collected data add value to the assessment of emergency care quality?Overall, first attempts exist that link data within the ECC to improve emergency care. However, to the best of our knowledge, no research uses routinely collected data within the ECC to assess QIs of an ED in order to derive general improvement potentials  or ad-hoc improvements in everyday life. Hence, in this research, we conduct a case study in a German ED and aim to answer the following research questions:Our case under consideration is a German academic teaching hospital with 771 beds treating 41,726 inpatient and 60,862 outpatient contacts in 2016. The hospital\u2019s ED with two shock and 26 treatment rooms is open 24/7. Admissions are centrally organized to combine all patient inflows and enable a structured triage. Hence, the head of the ED has an overview of the bed occupancy and patient flow. The corresponding reports are manually conducted by station nurses. In order to support processes and manage information, the ED uses four IS listed in Table Our research process comprises three steps. First, we select QIs that will be calculated within this study. Therefore, we use QIs from a systematic literature review and additionally conduct group discussions with relevant stakeholders within the hospital. Second, to answer RQ1, we combine routinely collected data extracted from various sources, preprocess it, and calculate the selected QIs. Finally, we gather our lessons learned from step two and discuss the results of the calculation and the lessons learned of our research process with subject matter experts. Based on this, we examine the added value of QIs that are based on routinely collected data to answer RQ2.As we aim to assess whether routinely collected data within the ECC can be used to calculate QIs that identify improvement potentials for an ED, we classify QIs as relevant if they are well-known in literature or demanded by practitioners and fulfill the following criteria: They 1) solely require routinely collected data for the calculation, and 2) support to identify general improvement areas or ad-hoc improvements in everyday life.Following this, we rely on the systematic literature review of S\u00f8rup et al. from 2013 , 13 to iFor the identification of QIs demanded by practitioners, we performed three group discussions with subject matter experts. Each group discussion consists of physicians working in the ED, and experts specialized in the digitization of hospitals and eHealth as well as members of the hospital\u2019s IT department. Every participant had multiple years of work experience in her or his respective job. The group discussions were prepared in advance and followed a specific structure . Furthermore, each of the discussions had an individual focus . All results were documented, presented to the participants for review, and approved by the participants. Based on the results, two further QIs have been selected that target the assessment of the diagnosis process and the identification of situations when EDs need to close due to crowding situations , 16. BotFurthermore, they rely on data stored in IS that is either used by the ED, the hospital, or the treatment report. Finally, especially OC aims to support physicians in the daily decision-making process, whether the ED should be closed due to an overcrowded situation or not.Thus, in the course of the first research step, we have selected LWBS, UR, DE, and OC as QI to be calculated in this case study. The selection is based on our previously defined criteria and the discussions with the physicians working in the ED. Hence, we do not claim that these QI are the most important or even the only relevant QIs in other settings.After having identified our QIs  during our first research step, the following step focuses on the calculation of these and starts with the preparation of the necessary data.Before we calculate the QIs, we first analyze the IS used within the ECC of the hospital. Therefore, we examined the four available IS regarding their actors, processes, and information technology in detail.IVENA. This supra-regional web-based software shows the current care situation and availability of resources of individual hospitals.The primary task of emergency medical services in Germany is to avert life-threatening conditions and other severe harms to health. All emergency medical vehicles are tactically led by dispatch centers and send to the patient as required. After the prehospital stabilization, the patient is transported from the site of the accident to the hospital. In order to identify an appropriate hospital for further care provision, the dispatch center uses NIDA, which is an integrated digital documentation platform to communicate with the hospital before the arrival. Thereby the paramedic sends the patients\u2019 data to the ED, which leads to earlier coordination of all medical specialties.At the arrival, a handover takes place between paramedics and ED staff. To support the handover, most emergency medical vehicles are equipped with portable devices that use E.Care. If there is a necessity that the patient is admitted to the hospital for continuing treatment in specialist departments, the hospital\u2019s information system Agfa Orbis is used. The resulting process of data detection is summarized in Fig.\u00a0Having arrived at the ED, the patient goes through a triage process  and the first treatments are applied. To document the triage and treatment process and to manage the ED, the staff members use the emergency admission and workflow management software NIDA and E.Care already share relevant data from patient contacts. This allowed us to focus on three instead of four IS for further preparation. In the course of this, we extracted seven relations with a total of 385 data fields. We preprocessed the extracted data by removing all attributes, respectively instances  that do not add value in order to comply with common data economy guidelines. To comply with ethical standards and existing privacy regulations, we conducted several discussions within the research team and with the hospital\u2019s data protection officer considering potential ethical issues and especially focusing on privacy. All data handling was conducted by employees of the hospital (not the other authors) in compliance with all applicable legal boundaries and internal policies. Table Having identified all available IS, we extract as well as preprocess the data to prepare the calculation. Some data is stored redundantly across multiple IS, other data  is only available in one IS. Additionally, we observed that The extracted and preprocessed data covered a total of 156,581 patient contacts in the years 2014 to 2016, while one patient can have caused multiple patient contacts within this period. In general, the patients\u2019 demographics are comparable to other German hospitals . Table 3patientID is a unique identifier for every patient, dossierID is a unique identifier for every patient contact. By using dossierID, we were able to link all data relations from the hospital\u2019s IS with all data relations from the emergency department\u2019s IS. The second approach made use of the timestamps  by grouping these into a predefined time frame. We were able to link two relations from the hospital IS . Each relation from the emergency department IS with the relation of the treatment report IS by dividing every day into fixed time frames (10\u00a0min in our case) and mapping all events  that occurred within the corresponding time frame.In order to link the data of the different IS, we investigate the IS\u2019 metadata  and applAfter preparing and linking the data, we conducted the calculation of the four QIs. In the following, we provide a short description of the QI, discuss the calculation process, and outline the results.LWBS is characterized by the percentage of patients without initial care by a physician . LWBS aiUR represents the percentage of unscheduled patients returning to the ED with comparable symptoms within 72\u00a0h , 22. ThiDE focuses on the quality of the diagnosis process , 25 and OC aims to predict upcoming situations when EDs might need to close due to overload. In Germany, heads of EDs legally have to continuously evaluate whether the current number of patients waiting for treatment can be handled with the available resources in a reasonable time, providing a sufficient level of quality. A QI that indicates whether a potential overload situation comes up helps them to take appropriate measures at an early stage . First, we analyzed the available data within the ECC of the hospital in order to exploratively identify relevant attributes helping to estimate the workload within the ED. Based on intensive discussion with physicians, we selected the following attributes as potential signals for an overload situation: number of administered medications, number of performed tasks, waiting time, and triage level of the patient contact. We used different time frames to aggregate these criteria. Also, we applied conventional data analytics methods to classify whether the ED has stopped the admission of new patients, which is stored in the treatment report IS. In order to build a prediction model, three common types of classifiers were used: logistic regression, multilayer perceptron, and decision tree \u201329. We fAll four QIs enables the hospital to identify areas of improvement regarding the EDs structure or routines, and especially OC supports physicians in everyday life. For example, we run into the issue that we were not able to determine the exact value of LWBS but were able to identify a possible explanation for this and then take appropriate measures. A staff member of the ED reported that some medical specialists are used to enter patient information into the hospital IS and are not willing to document the full initial medical examination in the emergency department IS. This shows us that QIs that could generally be easily calculated cannot be calculated due to a lack of availability of the necessary data or due to insufficient data quality.In order to answer the question of whether QI based on routinely collected data add value, we will further present our lessons learned and extend this with the results of additional expert interviews.In general, we can divide our lessons learned into two different topics. Our first lesson learned topic is that the use of routinely collected data increases the efficiency and effectiveness of the calculation process. On the one hand, this means that the indicators can be calculated faster or with less effort, as no manual steps or additional data collections are necessary, and preprocessing steps can be automated. However, this is only possible if the quality of the routinely collected data is sufficient. On the other hand, it is possible to add further contextual information enabling a better understanding of the QIs and, thus, support the identification of improvement areas. For example, by calculating UR and using other additional contextual information , we gain insights into unplanned-reattendance-prone patient demographics. We can better understand their medical condition urgency, which allows decision-makers in ED to tailor targets to the specific context and supports the implementation of measures. However, the calculation of QIs based on routinely collected data can also entail risks and requires the results to be critically examined. Although DE enables a better understanding of the trade-off between time constraints and effective diagnosis, the QI is quite sensitive to data quality issues, due to the critical role of the length of stay in the formula. This could, in turn, leads to a wrong interpretation of the indicator and hence, the implementation of wrong measures. A possible solution in the case of DE could be to apply further data pre-processing steps . It should be noted that the classification as outlier heavily depends on the examined diagnosis and should, therefore, not be performed without the consideration of medical expertise. This underlines the need for a continued development and validation of this indicator.Our second lesson learned topic is that using routinely collected data also makes it possible to calculate new QI that supports physicians in their decision-making-process in everyday life. An example of this was the prediction of whether an ED should be opened or closed for new patients. Although it still requires experienced ED staff members to interpret the QI and make the decision, the medical quality officer is surprised about the accuracy of the developed prediction model. He argues that this QI enables them to timely take appropriate measures in order to prevent potential overload situations.In general, all experts noted that the calculation of QIs using data pools within the ECC offers excellent potential for quality improvement collaboratives for prompt and informed decision making. Furthermore, they outline that this may only be the first step and that the case study only touches the surface of possible opportunities to increase the quality of an ED using routinely collected data. Despite significant quality improvement efforts to accelerate the fostering of emergency care, there are still treatment delays and failures, which could be eliminated through gaining a comprehensive understanding of routinely collected data within the quality improvement collaborative.The results of this study should be interpreted with consideration of the following limitations. First, we faced some issues during the calculation of the QIs. We only calculated four QIs that were selected by our literature search and the experts of the hospital. Even though we aimed to have a mixture of various types of QI, there exist many further QIs in literature. Second, since we calculated our QI in one teaching hospital in Germany evaluated by experts of this hospital, the external validity of our results is rather low. Although the academic teaching hospital is comparable to other hospitals in Germany in terms of its study population, it is also quite innovative with an almost paperless emergency department.We calculated different QIs within a single case study in a German academic teaching hospital in order to answer our research questions. Finally, lessons learned from our research provide a better understanding of the quality of the ED, the related challenges during the calculation, and the added value of linking routinely collected data. Besides this, our research contributes by identifying different types of IS and data pools used in Germany within the ECC. Furthermore, to the best of our knowledge, it was the first time that DE was calculated outside of a clinical study. One reason why DE has so far only been calculated in clinical studies could be that the indicator is difficult to calculate in practice, as the necessary information is spread across several IS. A linking of data, as in our case study, will also allow further research on DE to be facilitated and, thus, help to mature and internationally validate the indicator.Our research shows the potential of QIs based on routinely collected data from multiple IS within the ECC and paves the way for improved quality in EDs. Hence, the consistent networking of actors within the ECC has the potential to develop new measuring instruments and to depict the reality of care in a more targeted manner in order to improve the quality of care ultimately."}
+{"text": "Culture media were conditioned with the GuttaFlow\u00ae bioseal and AH26\u00ae pellets. MDPC-23 odontoblast cell cultures were treated with conditioned medium and serial dilutions. To evaluate the metabolic activity and cellular viability, the MTT and SRB assays were performed. To determine the production of reactive oxygen species, the DHE and DCF-DA probes were used. Cell cycle and cell-death types were assessed by cytometry, and to evaluate the mineralization capacity, the Alizarin Red S coloration was used. Statistical analysis was performed using analysis of variance (ANOVA) when normality was found and Kruskal-Wallis on the opposite case. For the comparison with normality values, the Student t-test was used. Cells exposed to the GuttaFlow\u00ae bioseal conditioned medium maintained high metabolic activities, except at higher concentrations. Likewise, viability was maintained, but a significant decrease was observed after exposure to the highest concentration (p < 0.001), associated with cell death by late apoptosis and necrosis. When cell cultures were exposed to AH26\u00ae, metabolic activity was highly compromised, resulting in cell death. An imbalance in the production of peroxides and superoxide anion was observed. GuttaFlow\u00ae bioseal showed higher biocompatibility than AH26\u00ae.The sealers used for root canal treatment should be biocompatible for the peri-radicular tissues, to evaluate the cytotoxic effects of GuttaFlow Root canal treatment involves disinfection and conformation, followed by the tridimensional obturation and sealing the root canal system. This therapy aims to avoid infection and to promote the repair of the peri-radicular tissues . FillingRoot canal filling materials must have the capacity to form an airtight barrier with dentin and be biocompatible regarding peri-radicular tissues ,3. The uThe root canal sealer composition is an essential factor for its biocompatibility ,8. Also,The hydraulic calcium silicate cements present dimensional stability, adequate sealing capacity, and good radiopacity, bind chemically to the dental structure, and induce regenerative response . They pr\u00ae bioseal is a new formulation of a silicon-based sealer combined with calcium silicate particles. It contains calcium and silicate, which seem to stimulate tissue regeneration and healing potential properties , with annexin V (labeled with fluorescence isothiocyanate) and propidium iodide as recommended by the manufacturer of the kit (Immunostep ANXVFKIT Immunotech and KIT Immunotech). After vortex and incubation for 15 min, at room temperature, in the dark, cells were analyzed by flow cytometry in the FACSCalibur cytometer .After incubating the cell cultures for 24 h with the conditioned media, the cells were detached, centrifuged, and washed with PBS. Cells were labeled in binding buffer [consisting of 0.01 M Hepes , 0.14 mM NaCl  and 0.25 mM CaClAfter 24 h of incubation with the conditioned media, cells were detached, centrifuged (2500 rpm for 5 min), and washed with PBS. The pellet was fixed in a vortex with 70% ethanol and incubated for 30 min at 4 \u00b0C. The suspension was washed with PBS and labeled with propidium iodide for 15 min, at 37 \u00b0C, in the dark. Cells were analyzed in the FACS Calibur cytometer.2-DA) were used. The cells were detached, centrifuged, and incubated with the probes in PBS. DHE was used in a concentration of 5 \u00b5M for an incubation time of 15 min. DCF-HA probe was added to make up a concentration of 5 \u00b5M and incubated for 45 min at 37 \u00b0C. After the incubation time, the suspension was washed by centrifugation, suspended in PBS, and analyzed in the fluorimeter using the excitation and absorbance wavelengths of 530 nm and 645 nm for the DHE probe and 485 nm and 528 nm for the DCFH2-DA.The probes dihydroethidium (DHE) and 2\u2032,7\u2032-dichlorodihydrofluorescein diacetate  for 20 min at 37 \u00b0C . Washing\u00ae SPSS\u00ae software version 20. The Shapiro-Wilk test was used to evaluate the normal distribution of the quantitative variables, given the sample size. The results of the treated cell cultures were compared with the control cell cultures using the one-way analysis of variance (ANOVA) in cases where there was a normal distribution and homogeneity of variances. When the contrary was found, the Kruskal-Wallis test was performed. Multiple post-hoc comparisons were then performed between experimental groups using appropriate parametric or non-parametric tests, with significance adjustment according to the Bonferroni correction. Comparisons with the normalization value of 100% were performed using the Student\u2019s t-test for one sample. A significance value of 0.05 was considered for all comparisons.The results are presented as mean \u00b1 standard error of at least three experiments performed in duplicate. Statistical analysis was performed using IBM\u00ae bioseal shows a lower cytotoxic profile compared to the AH26\u00ae sealer. The AH26\u00ae leads to loss of cellular viability, associated with the activation of types of cell death, in particular, late apoptosis and necrosis. The cytotoxicity of these materials is dependent on the time of exposure and concentration of the biomaterial, whose cytotoxic effect may be related to the production of ROS.In conclusion, GuttaFlow"}
+{"text": "Globally, fertility-related issues affect around 15% of couples. In 20%\u201330% of cases men are solely responsible, and they contribute in around 50% of all cases. Hence, understanding of in vivo germ-cell specification and exploring different angles of fertility preservation and infertility intervention are considered hot topics nowadays, with special focus on the use of human pluripotent stem cells (hPSCs) as a source of in vitro germ-cell generation. However, the generation of male germ cells from hPSCs can currently be considered challenging, making a judgment on the real perspective of these innovative approaches difficult. Ever since the first spontaneous germ-cell differentiation studies, using human embryonic stem cells, various strategies, including specific co-cultures, gene over-expression, and addition of growth factors, have been applied for human germ-cell derivation. In line with the variety of differentiation methods, the outcomes have ranged from early and migratory primordial germ cells up to post-meiotic spermatids. This variety of culture approaches and cell lines makes comparisons between protocols difficult. Considering the diverse strategies and outcomes, we aim in this mini-review to summarize the literature regarding in vitro derivation of human male germ cells from hPSCs, while keeping a particular focus on the culture methods, growth factors, and cell lines used.  Since the first derivation of human embryonic stem cells (hESCs) in 1998  into a hThe latest generation of several disease-specific hiPSC lines, e.g., Klinefelter syndrome ,4,5, MowThe lack of treatment options for men suffering germ-cell loss-related infertility as well as the still inadequate level of knowledge of human embryonic germ-cell development, mainly limited as a result of inaccessibility of experimental material, results in a big interest in in vitro derivation of germ cells from hPSCs ,10,11. DAs extrapolated from murine studies, specification of primordial germ cells (PGCs) from the epiblast in response to bone morphogenetic protein (BMP) signalling from extraembryonic tissue is the first step in human germ-cell development .Found in the wall of the yolk sac at the end of the third week of gestation, PGCs then migrate through the allantois and the hindgut to the primitive gonads and reside there at 5\u20136 weeks of gestation. Studies in mice suggest that, during this developmental period, PGCs undergo epigenetic reprogramming including erasure of genomic imprints, DNA methylation and chromatin remodelling, and X-chromosome reactivation in female germ cells, followed later during embryonal development by global re-methylation (for review see ).Although progress has been made, especially during the last decade, the exact mechanism of PGC proliferation in humans is not yet known. Nevertheless, extrapolation from the results of in vivo mouse studies and in vitro differentiation studies has shown that BMP4, leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) play major roles and can support or inhibit in a species-specific manner important pathways ,19,20,21sex-determining region Y (SRY) and SRY-box (SOX) 9 genes and mediated by fibroblast growth factor 9 (FGF9) and the degradation of retinoic acid (RA) ) OR )) AND ((cell differentiation[MeSH Terms]) OR (differentia*[Title/Abstract] OR deriv*[Title/Abstract]))) AND (((pluripotent stem cells[MeSH Terms]) OR stem cells[MeSH Terms]) AND (induced pluripotent stem cells OR embryonic stem cells)). Inclusion criteria were the use of hPSCs and differentiation into the bi-potential germ cells state as well as male germ cells, while studies involving the use of animal-derived stem cells and differentiation into oocytes were excluded. Considering only Web of Science, 725 publications could be extracted using the above-mentioned search terms and 1597 publications could be extracted from PubMed. Manual selection after removing duplicates and articles not meeting the eligibility criteria yielded 47 relevant articles which were used to summarize the most relevant protocols for this review.SRY gene, than the actual germ-cell karyotype. However, humans as well as other mammals, undergoing gonadal sex reversal during early embryo development  suffer from infertility, as their germ cells, although proceeding in maturation to a certain point, are eliminated by check-point mechanisms [Starting with the first intended in vitro germ-cell differentiation approaches in 2004, both female as well as male PSC lines were used, irrespective of the intended sex differentiation A. Nevertchanisms . Althougchanisms ,36,37,38As differentiation results often vary between different hPSC lines, even with the same differentiation protocol, drawing clear conclusions when comparing the results and efficiency of different protocols is difficult. Although we observed that the hESC lines H1, H7, and H9 were the most frequently used hPSC lines in the original articles covered in this review, most protocols involve the use of in-house-generated hPSC lines, which hampers protocol comparisons. As hESC lines H1 and H9 make up the majority of stem cell line requests from the National Stem Cell Bank, inclusion of commonly available hPSC lines in differentiation protocols could allow better comparison and reproducibility of protocols. Nevertheless, as Merkle and colleagues have shown , 5% of 1Heterogeneity and comparability of differentiation results is not only due to the use of a great variety of hPSC lines, but also to different culture approaches.deleted in azoospermia-like (DAZL) genes, developmental pluripotency-associated protein 3 (DPPA3), DDX4, and synaptonemal complex protein (SYCP) 3, in parallel with other somatic lineages within the EBs 12. Since then, EB differentiation protocols, providing a three-dimensional (3D) culture condition, have been commonly applied, either on their own or in multi-step approaches in combination with two-dimensional (2D) culture conditions using defined matrices or feeder cells. Such multi-step approaches were first introduced in 2010 by Richards et al., where hESC-derived EBs cultured on porcine ovarian fibroblasts, among other features, commonly led to the differentiation of DDX4- and BOULE-expressing cells [Over the course of time, in vitro germ-cell differentiation approaches have developed from relatively simple one-step protocols towards more complex multi-step approaches B. Starting cells . Thereafng cells , gelatinng cells ,44,45, ang cells . As the ng cells  and feedng cells , led to ng cells ,42,49,50ng cells . Howeverng cells  and Sasang cells , who shong cells . After nng cells .Although 3D differentiation approaches seem to show better differentiation efficiencies, the manner of culture, might not be as important as the somatic environment in which the differentiation takes place. As PGCs become enclosed by Sertoli cells in the seminiferous tubules or migrate to the basement membrane, being in direct contact with extracellular matrix (ECM) proteins such as laminins and collagens, direct cell-to-cell or cell-to-matrix interactions might be needed for more efficient in vitro differentiation.Although the initial attempts at in vitro germ-cell differentiation were achieved by way of spontaneous differentiation, the variety of factors used to enhance human germ-cell differentiation from hPSCs has increased ever since . BMPs ardeleted in azoospermia (DAZ) [DAZL [BOULE [DDX4 [PR/SET domain 1 (PRDM1) [DPPA3 [nanos C2HC-type zinc finger 3 (NANOS3) [From 2008 on, bFGF started to be used as an additive in differentiation protocols ,62,63,65ia (DAZ) ,49, DAZLZ) [DAZL ,49,63,65L [BOULE ,49, DDX4LE [DDX4 , PR/SET ) [DPPA3 , and nanSince the first in vitro germ-cell differentiations starting in 2004, the manner of monitoring hPSC differentiation into germ cells has become more and more diverse . While iDespite the broad range of protein- and gene-expression analyses we observed that DDX4 (expressed in germ cells from 6 weeks post conception), POU5F1 (expressed by PSCs and PGCs as well as gonocytes), and SYCP3 (as meiotic marker) were the three most common protein markers used for the identification of germ-cell differentiation. Further, DAZL  , cKIT, PA gold standard methodology to assess the functionality of germ cells generated from hPSCs as established for animals, including changing DNA contents during meiosis , as well as the production of zygotes resulting in viable offspring using the in-vitro generated gametes , is not In order to develop and improve in vitro germ-cell differentiation protocols from hPSC towards post-meiotic germ cells, an adequate understanding of the in vivo germ-cell development is crucial. Although the detailed events leading to PGC specification and subsequent maturational steps upon migration into the gonads are still not fully understood in human, recent advances in RNA sequencing (RNA-seq), especially single cell RNA-seq, are gradually increasing the understanding of maturational events as well as the trajectory of specific germ-cell stages from pre-natal to adulthood.As such, Irie and colleagues were not only able to demonstrate the differentiation of hPSCs towards human primordial germ-cell-like cells (hPGCLCs) but were further able to show the importance of specifically SOX17 in human germ-cell specification by RNA-seq analysis of both, hPGCLCs and human first trimester male germ cells . Similardark and Apale ), and thereby highlight the challenges related to the differentiation of PSCs into functional male germ cells, present in the post-natal testis.Nevertheless, RNA-seq is mostly applied in the investigation and understanding of in vivo hPGC states and it has been able to demonstrate a variety of germ-cell stages throughout development in human foetal , neonataA further advantage of RNA-seq, in addition to a detailed gene expression profile of different germ-cell types, is investigations of the somatic environment needed to build up the gonadal structure, including the SSC niche as well as the vascular system, and interact with germ cells. Sequencing studies of human foetal  and postOverall, the recent discriminations of distinct germ-cell populations will potentially allow for improvements of the current differentiation protocols, but most importantly help to determine the similarity of in vitro differentiated germ cells to distinct in vivo germ-cell states.As male germ-cell differentiation and maturation in vivo is a process spanning many years, starting from epiblast cells specializing and migrating as bi-potential PGCs into the gonad, becoming enclosed in forming seminiferous tubules, migrating towards the basement membrane to become spermatogonial stem cells and starting spermatogenesis after the onset of puberty, in vitro differentiation towards fertile spermatids is one of the most challenging tasks existing in research. So far, robust and efficient differentiation approaches are limited, especially when aiming for more advanced stages of male germ-cell differentiation. Further, comparison of various protocols is not only difficult because of the variety of culture approaches but also because of the variety of cell lines used for differentiation, all possessing a specific profile from the start. Nevertheless, great progress has been made during the last decade in the differentiation of hPSCs towards bi-potential PGCs, whereas differentiation towards more advanced stages seems still challenging. Although expression of mature male-specific markers as well as the generation of haploid cells has been shown, morphological similarities to cells at more advanced stages of spermatogenesis, and functionality, are still missing so far.Future in vitro differentiation approaches employing standardized culture conditions on ECMs, e.g., laminins, will most likely have to include multi-step approaches simulating crucial phases of germ-cell development such as the specification of na\u00efve PSCs towards epiblast cells. Those strategies require robust protocols to derive na\u00efve hPSCs, and transform primed hPSC differentiation towards PGCs, stimulated by BMPs. In addition, effects related to the embedment of PGCs between Sertoli cells such as the suppression of RA during male germ-cell specification need to be addressed as well. A further area of study is the migration of SSCs to the basement membrane with close contact to ECM proteins influencing internal signalling cascades. Finally, the onset of puberty leads to hormonal stimulation of Leydig and Sertoli cells, releasing factors such as testosterone, as well as anti-M\u00fcllerian hormone and inhibin B, respectively, allowing differentiation into mature and functional spermatids.Although in vitro germ-cell differentiation from hPSCs is often mentioned as a potential future option for infertility treatment, one has to bear in mind that the current protocols do not allow sufficient differentiation of fully matured spermatids, and even if possible, testing the ultimate functionality by fertilizing oocytes and producing offspring will present us with many ethical conflicts, which need to be addressed very carefully.However, strategies aiming in the establishment of models to study the fundamental aspects of germ-cell specification can be considered as crucial steps to gain more knowledge on germ-cell development as well as differentiation. Those are needed to establish strategies for fertility preservation techniques employing hiPSCs from patients without their own germ cells."}
+{"text": "Training and mentorship in research skills are essential to developing a critical mass of researchers in low- and middle-income countries (LMICs). However, reporting on the details of such training programs, especially regarding the cost of the training, is limited.This paper describes a year-long operational research training and mentorship course in Rwanda, implemented between 2013 and 2017.We describe motivations for the design of the Intermediate Operational Research Training Course (IORT) across four iterations. We also report outputs, evaluate trainee experiences, and estimate training and mentorship costs.Of the 132 applicants to the course, 55 (41.7%) were selected, and 53 (96.4%) completed the training. The ratio of female-to-male trainees in the course increased from 1:8 in 2013 to 1:3 in 2017. Trainees developed and co-first-authored 28 research manuscripts, 96.4% (n = 27) of which are published in peer-reviewed journals. For the 15 trainees who completed the post-course evaluation, 93.3% and 86.7% reported improvement in their research and analytical skills, respectively. The median cost per trainee to complete the course was US$908  and per research project was US$2,708 . The median annual training delivery and mentorship cost was US$47,170  for a course with a Rwanda-based senior mentor, junior mentor, and training coordinator. The total essential cost for a year-long IORT course with 16 trainees co-leading eight research projects and mentored by two senior and four junior mentors was US$101,254 .We attribute the high course completion rates, publication rates, and skills acquisition to the learning-by-doing approach and intensive hands-on mentorship provided in the course. IORT was costly and funded through institutional resources and international partnerships. We encourage funders to prioritize comprehensive research capacity-building initiatives that provide intensive mentorship as these are likely to improve the pool of skilled researchers in LMICs. Training and mentorship in core biomedical research knowledge and skills are essential for building individual and institutional research capacity in low- and middle-income countries (LMICs) 23. Capac214679Within sub-Saharan Africa, research output and the reported progress in authorship dynamics reflect improvements in only a small subset of countries, masking the continental inequity in research capacity 11. To br1213Partners In Health Rwanda (PIH/Rwanda) is an international non-profit organization that has supported the RMoH in health care delivery since 2005. PIH/Rwanda began a comprehensive research capacity building initiative in rural Rwanda in 2012 . As partResearch training courses in LMICs vary in terms of their structure, duration, and the research competencies they cover . The avaIORT is a learning-by-doing course that uses a deliverable-driven model to teach research skills. Learning-by-doing is a hands-on training approach in which the learner interacts with their environment or project to enable learning . This apIORT consists of alternating in-person sessions and practicum sessions Figure . During 20Each course is 8\u201312 months long. We shifted the frequency and duration of in-person sessions from seven two-day sessions in the first two cohorts to three one-week sessions in the latter courses  and high review costs . These challenges have also been reported elsewhere in LMICs 26. ProjeOur mentorship process respected trainee leadership of the research project and allowed for growth of both trainees and mentors. With each milestone, the trainees led the drafting of the initial research protocol, data analysis, and manuscript writing. Trainees then received and addressed feedback from all mentors, beginning with junior mentors. We used an iterative process for review, within set timelines, until trainees and their mentors agreed that a drafted research document was ready for feedback from the authorship team. Because of the intensive contribution of mentors in the research process, all mentors formed part of the trainee\u2019s authorship team, enhancing both their research careers and leadership skills .During course application, trainees stated their research background and interests and provided a letter of support from their employer or supervisor. Because all trainees were full time health or academic professionals, we selected trainees jointly with their institutional leaders. We selected trainees based on three criteria. First, the senior course mentors ranked trainees\u2019 readiness to complete the IORT course based on previous research experiences and training. Second, the project mentors ranked trainees\u2019 existing or potential links to the program or research proposed. Third, the employer or work supervisor ranked applicants from their organization based on their organizational priorities for research training. The contribution of all stakeholders in the trainee selection enhanced stakeholder commitment to the course and secured institutional support for trainees to both attend the training and to lead research within their organizations post-training.Because of limited funding and mentorship resources, we paired trainees on each research project to train more individuals while maintaining a reasonable number of research projects. Pairing facilitated peer-to-peer learning and engendered research partnerships across institutions. However, training teams are best limited to two individuals per project to enable tangible leadership contributions to the research project. Our trainees published as joint first authors.The selection of research projects in the course shifted from trainees\u2019 self-identified topics in 2015 to trainees selecting topics from a list submitted by the project mentors in later years , agree (score = 3), disagree (score = 2) and strongly disagree (score = 1) to measure trainee reactions. To measure training pace in the mid-course survey, we applied the scale as too fast (score = 3), just right (score = 2), and too slow (score = 1). We calculated the median Likert score and frequency of respondents with each median score.We collected information on annual programmatic training and research project expenditures from research, finance, human resources, and procurement teams. We collected both cash and in-kind expenditures per course, as well as the corresponding number of training participants and research projects. We grouped expenditures into three categories: trainee costs, research project costs, and training delivery and mentorship costs Table . We convBetween 2013 and 2017, 132 candidates applied for the IORT courses, 55 41.7%) of whom were selected based on their research background, experience with chosen research project, and institutional training priorities   had previously participated in a research project, often as an academic thesis  and analytical  skills Table . Most trFor individual trainees to complete the course, we spent a median cost of $908  per trainee, excluding the cost of laptops which the trainees either owned or borrowed from PIH/Rwanda Table . The medThe bulk of total training costs fell under training delivery and mentorship  (Table The high course completion rates in the IORT is comparable to the course completion rates for the SORT IT courses (~89%) 31. We at17242021222324The progressively more gender equitable trend in the course reflects programmatic decisions to intentionally select more qualified women in the course. In addition, the proportion of female applicants increased from 4.2% (n = 1) in 2013 to 36.4% n = 16) in 2017. However, we still had more male than female trainees. This gender imbalance, also observed by other research capacity-building programs, may be attributed partly to time-demanding family obligations that typically fall on women in this context  in 2017.3435. Of 34163333Over time, our course increased both institutional and inter-professional partnerships, which may promote future research and build research capacity in Rwanda. In our experience, the diversity in trainees\u2019 professional and institutional backgrounds enhanced knowledge and skills sharing among trainees. It also advanced the formation of a national network of Rwandan researchers. Trainees such as university lecturers have a greater potential for mentoring new students in research skills through their academic positions. We postulate that an annual meeting of a national network of IORT alumni and trainees may sustain these collaborations. Such a meeting should target the growth of additional research leadership skills such as grant writing. We plan to explore this in the future.There is a growing call for systematic evaluation of the impact of courses like IORT 37. IndivThrough the surveys, trainees critically reflected on the course, highlighting the short-term impact of the IORT course in improving their critical thinking, writing, and data analysis skills . The ava2830Few research training programs report their costs. For those that do, the costs vary from US$500 to US$20,000 per research project depending on the competencies covered, length of training, and location of training participants 26. TheseTo fund this course, we leveraged institutional resources and external partnerships. Institutionally, we relied on existing resources of PIH/Rwanda, including training facilities and vehicles for field travels. PIH/Rwanda also funded three of the four courses through operational funds and innovation grants. PIH/Rwanda\u2019s partnership with Harvard Medical School\u2019s (HMS) Global Health Research Core provided in-kind course delivery and mentorship support. HMS funded the time for one senior course mentor for all four courses, another senior course mentor for two courses, and one training coordinator/junior mentor for three courses. We also received a grant through Harvard Global Health Initiative Burke Global Health Fellowship Grant for the 2016 course. Project mentors with their project-affiliated junior mentors also provided in-kind mentorship, estimated at 5% and 10% of their time, respectively. We did not estimate the cost for project mentors given the diversity of these mentors.Our reliance on in-kind support for course delivery and mentorship, a key element of the course, affects the sustainability of IORT, especially in a context of declining grant support for international training. This is critical for IORT approaches such as learning-by-doing and intensive hands-on mentorship that are both resource and labor intensive. For example, we fully sponsored trainees, including providing research tools such as analytical software to make the learning-by-doing approach feasible. Our senior mentors experienced a high mentorship workload, which makes senior mentors\u2019 role unsustainable without dedicated funding. We hope that the outcomes of this course encourage funders to invest more in mentorship-oriented capacity-building approaches. We also encourage the government of Rwanda to adopt IORT and dedicate funding for sustaining and scaling the program in Rwanda.Although a key output for the training is a peer-reviewed publication, publication fees were often prohibitive for the course, as has been observed by others . We prioWe only evaluated our course with the 2016 cohort due to challenges with prior planning and recognizing the need for a system of evaluation on a course-by-course basis. Equally, a system for follow-up with the trainees on research engagement post-course, as well as tracking the uptake of research project results on practice and policy, would further demonstrate the value of IORT. This should also measure research capacity outcomes such as changes in publication output post IORT, access to research grants, and growth of trainees and junior mentors to research roles.IORT aims to develop a pool of Rwandan researchers who become critical and effective health research leaders and collaborators in Rwanda. Over the four years of this course, we have trained 53 health professionals who developed 28 research manuscripts, most of which have been published in peer-reviewed journals. We implemented the course iteratively, with annual reflections on what worked best and adaptations to address new and ongoing challenges. We believe there is \u2018more than one way to slice the cake\u2019  when it"}
+{"text": "These materials are employed as a host for green and red phosphorescent OLEDs. The white device based on DBTPIN:Ir(MDQ) For an OLED with stable emission the current efficiency (CE) is proportional to external quantum efficiency (\u019eex): power efficiency (PE) is determined by CE and operating voltage (V) [PE\u2009=\u2009\u03c0 CE/V]4. Iridium and platinum based phosphorescent complexes and TADF  exhibit high \u019eex, however, suffered with short lifetime and roll-off efficiency and also the production cost of phosphorescent materials are unfavourable for practical applications5. Therefore, low driving voltage with high brightness become the major issue to achieve efficient OLEDs6. In OLEDs, balanced hole: electron recombination leads to formation of CT exciton (charge-transfer) which undergo decay directly or relaxes to LE , thus, utilization of both CT exciton and LE provides efficient EL (electroluminescence). From 4-(dicyanomethylene)-2-methyl-6-[4-(dimethylaminostyryl)-4H-pyran] with CT state maximum efficiency have been harvested9. Donor\u2013acceptor (D\u2013A) compounds with low % CT leads to RISC (reverse intersystem crossing) process which results high singlet utilisation efficiency(\u019es), however, colour-purity is still poor due to broadened PL (photoluminescence) and EL (electroluminescence) spectra13. D-A architecture with high % LE state leads to higher efficiency because of maximum orbital overlap whereas high % CT provides low efficiency due to partial hole and electron overlap. However, because of small energy splitting (\u0394ES-T\u2009\u2248\u20090) CT state undergo RISC process results in enhanced \u03b7s12. These issues are overcome, by employing D\u2013A configured emissive materials with HLCT emissive state: stabilised LE and CT states results in \u03b7PL(photoluminance efficiency) and high \u03b7s.. Thus, construction of D\u2013A emitters with HLCT emissive state is novel strategy to design efficient blue emitters.Development of blue emitter is crucial in organic light emitting devices (OLEDs) to reduce power consumption effectivelyvia twisted arrangement which reduce the conjugation. The twisted structure with high thermal properties promotes blue emission which need for non-doped blue OLEDs15. The development of efficient host having high ET (triplet-exited state energy) with good carrier transport properties is critical for efficient PhOLEDs16. The triplet energy of diphenylphenanthrimidazole based hosts is lower compared to individual imidazole molecule owing to enlarged \u03c0- conjugation. Therefore, tuning the molecular architecture to highly twisted molecular conformation may be an effective strategy to achieve hosts with high ET. In this communication, we report NDBNPIN and DBTPIN composed of phenanthrimidazole and phenyltriphenylamine with naphthyl and thienyl as spacer components used as blue emitters and host for green and red PHOLEDs.One strategy for constructing blue emitters is the integration of high-energy emissive moieties Tg)/Td (decomposition temperature) of 115\u00b0/440\u00b0 & 121\u00b0/501\u2009\u00b0C were determined for NDBNPIN and DBTPIN, respectively  indicates the high resistance of fused aromatic ring on thermolysis and high Td could enhance the device lifetime17. These materials has the ability to form an amorphous glass with high glass-transition temperature (Tg) of 115\u2009\u00b0C \u2013 NDBNPIN and 121\u2009\u00b0C - DBTPIN which is beneficial for the formation of stable, homogeneous and amorphous film upon thermal evaporation. Absence of endothermic peak during the measuring process reveal that no phase separation of host\u2013guest system occur when used as host material.The structure of the emissive materials NDBNPIN and DBTPIN are confirmed by CHN analysis, NMR and mass spectral studies. The glass transition temperature , however, some orbital delocalised on entire molecule (LE) of their excited states shows presence of CT and LE components. i.e., HLCT emissive state. The energy of S1 (1.0376\u2009eV - NDBNPIN and 1.7013\u2009eV - DBTPIN) and T3 states (1.0274\u2009eV - NDBNPIN and 1.6617 eV- DBTPIN) are almost same. A wider energy gap (Eg) (T3\u2009\u2212\u2009T2/T1) for NDBNPIN (0.42\u2009eV) and DBTPIN (0.57\u2009eV) is because of same acceptor (phenanthrimidazole) group and the energy gap of DBTPIN is larger when compared to NDBNPIN. In both NDBNPIN and DBTPIN, very small \u0394EST\u2009\u2248\u20090 facilitates RISC (T3\u2009\u2192\u2009S1) with hot exciton due to HLCT state. Thus, DBTPIN show high \u03b7PL and high \u03b7S compared with NDBNPIN. The \u03b7EQE  of device with DBTPIN is increased due to high % LE. The HOMO as well as LUMO of NDBNPIN and DBTPIN exhibit partial separation which enhanced hole- and electron-transportation (bipolar nature) with electron/hole transfer integrals, NDBNPIN (0.24/0.47\u2009eV) and DBTPIN (0.21/0.41\u2009eV) and minimised the \u0394EST  around 276 and 378\u2009nm is attributed to \u03c0\u2009\u2212\u2009\u03c0* and CT transitions, respectively and strong absorption is due to CT from triphenylamine (donor) to acceptor (naphthonitrilephenanthrimidazole)18. The suppressed \u03c0-\u03c0* stacking in film induced red-shifted \u03bbabs relative to solution19 and the larger red shift supports the CT in twisted DBTPIN or NDBNPIN. From the onset absorption in film, optical Eg (band gap) is calculated as NDBNPIN (2.98\u2009eV) and DBTPIN (2.90\u2009eV). The emitters NDBNPIN and DBTPIN show emission maxima at 440 and 428\u2009nm, respectively  relative to excited state dipole moment (\u03bce). By employing DFT and Lippert-Mataga plot  and DBTPIN (8.11/26.1 D). Solvents with f\u2009\u2265\u20090.2, CT state is stabilised22  whereas solvents with f\u2009\u2264\u20090.1 LE state is stabilised. Transformation in the slope observed between butyl ether (f\u2009=\u20090.10) and ethyl acetate (f\u2009=\u20090.20) reveal that the emitters show HLCT emissive state i.e., intercrossed excited state of LE and CT [ECT\u2009=\u2009ELE]  of NDBNPIN (83/80%) and DBTPIN (92/90%) is due to co-emission from LE and CT which is essential for efficient blue OLEDs and the enhanced quantum yield is due to decreased non-radiative (knr) transition23 (Table\u00a0f) for \u03bbabs/\u03bbemi of NDBNPIN (gas phase-372 (f-1.5283)/383 (f-1.7692); CHCl3-368 (f-1.7982)/412 (f-2.0462) and DBTPIN  show that oscillator strength of these compounds in CHCl3 is high relative to gaseous phase due to higher luminance of HLCT state in CHCl3  on TPA and PPI are separated results in CT transition from HOMO (donor) \u2192 LUMO (acceptor). At 90\u00b0, twisted conformation of NDBNPIN and DBTPIN is less stable because of higher energy NDBNPIN (\u22480.6\u2009eV) and DBTPIN (\u22480.04\u2009eV) than at \u224840\u00b0 (stable conformation)23 Table\u00a0. The oscCl3 Fig.\u00a0. To furtCl3 Fig.\u00a024\u201326. ThET) estimated as 2.62\u2009eV (DBTPIN) and 2.74\u2009eV (NDBNPIN) which are sufficient for exciting red as well as green phosphorescent emitters. Also small \u0394EST is sufficient for energy-transfer from host29 triplet to green and red emitters. The charge transportation of DBTPIN and NDBNPIN, CBP:Ir(ppy)3, DBTPIN:Ir(ppy)3 and NDBNPIN:Ir(ppy)3 was examined by single carrier device fabrication 3 3 devices due to direct carrier- injection into HOMO of Ir(ppy)3 followed by hopping transport via DBTPIN/NDBNPIN sites \u03b7s of DBTPIN and NDBNPIN is not due to TTA or TADF process31. The similar PL and EL emission of DBTPIN and NDBNPIN shows that both PL and EL stems from same source with similar radiative route. The DBTPIN device exhibit superior performance ) than NDBNPIN based device ). Further, doped devices [ITO/NPB (40\u2009nm)/TCTA(5\u2009nm)/CBP:DBTPIN or CBP:NDBNPIN (20\u2009nm)/TPBi (50\u2009nm)/LiF (1\u2009nm)/Al (100\u2009nm): CBP-LUMO -2.7\u2009eV: HOMO -6.1\u2009eV] were constructed to examine the efficiencies and CBP:DBTPIN show maximum efficiencies  and the experimental \u03b7EQE is of 6.50% and 4.8%, respectively. Experimental \u03b7EQE\u2009>\u2009Theoretical \u03b7EQE because larger triplet exciton are converted to singlet exciton in EL process36. The \u03b7r calculated for DBTPIN (31\u201338%) and NDBNPIN (25\u201332%) indicates \u03b3 \u02c2 100% because of poor unbalanced carrier transportation in the emissive layer. Enhanced \u03b7IQE 25.2% (NDBNPIN); 32.5% (DBTPIN) and maximum \u03b7s 31.6% (NDBNPIN) and 38.2% (DBTPIN) [\u03b7s\u2009=\u2009\u03b7out \u00d7 \u03b7PL \u00d7 \u03b7res \u00f7 \u03b7EL] is because of retained CT % due to CN group in D-\u03a0-A compounds. Maximum \u03b7s breaking 25% limit: 6.6% (NDBNPIN) and 13.2% (DBTPIN) of triplet exciton converted to singlet exciton by RISC and remaining follow non-radiative process leads to high efficiency blue OLEDs. The \u03b7c and \u03b7p of DBTPIN device  and NDBNPIN device  are larger relative to TPA-PA , TPA-NzP  and mTPA-PPI  devices. The quantum yield of DBTPIN (92%) and NDBNPIN (83%) is larger when compared with (i) Cz-BzP (69.7%) and TPA-BzP (49.2%) (ii) CBI (21%) and MCB (24%) and (iii) PPI-pCNCz (54%). Thickness of LBPPI influences \u03b7c . The \u03b7c harvested in the current study with 30\u2009nm DBTPIN (7.0\u2009cd/A) and 30\u2009nm NDBNPIN (5.8\u2009cd/A) is higher than reported \u03b7c : 5 wt % Ir(ppy)3/TPBi (50\u2009nm)/LiF (1\u2009nm)/Al (100\u2009nm)]: ITO/NPB (40\u2009nm)/TCTA (5\u2009nm)/DBTPIN (30\u2009nm): 8\u2009wt% Ir(MDQ)2(acac)/NDBNPIN (30\u2009nm): 8\u2009wt% Ir(MDQ)2(acac)/TPBi (50\u2009nm)/LiF (1\u2009nm)/Al (100\u2009nm), Ir(ppy)3-fac-tris(2-phenylpyridine) iridium(III) and Ir(MDQ)2(acac)-bis acetylacetonate iridium(III) are emissive layers for green and red devices, respectively]. The device performances are presented in Fig.\u00a03: part of the exciton transferred to Ir(ppy)3 triplet state and part of exciton transferred to ground state (So) to generate phosphorescence and fluorescence, respectively32. At 5% doping concentration all the exciton were transferred to Ir(ppy)3 to generate phosphorescent emission /TCTA 5\u2009nm)/DBTPIN (30\u2009nm): 5 wt % Ir(ppy)\u2009nm/DBTPI3 shows maximum efficiency: \u03b7c of 29.0\u2009cd\u2009A\u22121 and \u03b7p of 35.4\u2009lm\u2009W\u22121 at 2.6\u2009V; \u03b7ex of DBTPIN: Ir(ppy)3 and NDBNPIN:Ir(ppy)3 are of 23.0 and 20.1%, respectively. Red device with DBTPIN: Ir(MDQ)2(acac) exhibits excellent efficiencies  with CIE  on comparison with NDBNPIN based device  with CIE . The enhanced efficiencies reveal that DBTPIN and NDBNPIN are the best host-materials for green and red PHOLEDs. At 0.4% doping concentration both devices based on DBTPIN: Ir(MDQ)2(acac) [448\u2009nm (sh)&590\u2009nm] or NDBNPIN: Ir(MDQ)2(acac) [450\u2009nm (sh)&582\u2009nm] show two peaks leads to white emission  triplet state to excite phosphorescence and another part of the exciton transferred to ground state to produce fluorescence. Increasing the doping concentration, generated exciton were completely transferred to Ir(MDQ)2(acac) which results in phosphorescent emission. The white device based on DBTPIN:Ir(MDQ)2(acac) exhibit maximum \u03b7ex\u2009\u2212\u200924.8%; \u03b7c\u2009\u2212\u200957.1\u2009cd\u2009A\u22121; \u03b7p\u2009\u2212\u200964.8\u2009lm\u2009W\u22121 with CIE  than NDBNPIN: Ir(MDQ)2(acac) based device . At doping 0.4% concentration, blue emission of the EL spectrum of NDBNPIN: Ir(MDQ)2(acac) device was broader than DBTPIN:Ir(MDQ)2(acac). More blue fluorescence contribution in device NDBNPIN: Ir(MDQ)2(acac) than device DBTPIN: Ir(MDQ)2(acac) leads to lower efficiencies of NDBNPIN:Ir(MDQ)2(acac) than NDBNPIN: Ir(MDQ)2(acac). Outstanding efficiencies indicates, DBTPIN and NDBNPIN are potential host materials and transfer of exciton is shown in Fig.\u00a01) exhibits deep-blue emission whereas another part of singlet exciton transferred to singlet green/red phosphorescent emitters (GS1/RS1) and then delivered to triplet green and red phosphorescent emitters (GT1/RT1) by intersystem-crossing process and shows red and green phosphorescent emission. Furthermore, host triplet exciton (HT1) is transferred to GT1/RT1 to enhance the exciton utilization : 5\u2009wt% Ir(ppy)ET) estimated as 2.62\u2009eV (DBTPIN) and 2.74\u2009eV (NDBNPIN) are sufficient for the excitation of green and red phosphorescent dopants. Efficient green and red PhOLEDs with EQE of 23.0%/20.1% and 24.1%/21.3% have been harvested based on DBTPIN/NDBNPIN doped OLEDs,, respectively. White device with DBTPIN:Ir(MDQ)2(acac) (0.4%) exhibit maximum \u03b7ex\u2009\u2212\u200924.8%; \u03b7c\u2009\u2212\u200957.1 cdA\u22121; \u03b7p\u2009\u2212\u200964.8 lmW\u22121 with CIE  than NDBNPIN:Ir(MDQ)2(acac) (0.4%) based device . We have reported structure modification strategy to harvest efficient full-color OLEDs by employing phenanthroimidazoles in doped, non-doped devices and red, green and white PHOLEDs.We have reported two deep blue emitting materials DBTPIN and NDBNPIN with dual charge transport properties and exhibit high EQE of 6.5% and 4.8% with CIE . The triplet energies (3)4 (0.1\u2009mmol) and 2\u2009M K2CO3 (10\u2009mL) was refluxed in toluene (30\u2009mL) at 90\u2009\u00b0C for 32\u2009h (Scheme S1). The reaction mixture was treated with CH2Cl2 and dried, crude TPAB-Br was purified by column chromatography to afford white solid [CH2Cl2\u2013petroleum ether (60\u201390\u2009\u00b0C) (1:15)], yield: 84%. MS (EI): m/z 399.1, 401.3 (M+)37.About 1.42\u2009g of 1-bromo-4-iodobenzene (5\u2009mmol), 2.89\u2009g of (4-(diphenylamino) phenyl)boronic acid (10.0\u2009mmol), 50\u2009mg\u2009Pd(PPh3)4  and 2\u2009M K2CO3 (15\u2009mL) was refluxed in toluene (30\u2009mL) at 105\u2009\u00b0C for 3 days with N2 stream. The reaction mixture was treated with CH2Cl2 and dried (Scheme S1).Yield 86.1%. 1H NMR : \u03b4 6.45-6.68 , 7.02 , 7.25 , 7.41\u2009\u2212\u20097.68 , 7.72 , 9.12 , 9.80 ; 13C NMR : \u03b4 122.71, 123.68, 126.98, 128.32, 128.59, 129.74, 131.50, 132.86, 133.43, 135.47, 135.95, 136.89, 139.51, 141.12, 143.53, 191.52. MS (EI): m/z 475.23 (M+).The TPAB-Br , 4-formylnaphthalen-1-yl-1-boronic acid  and Pd(PPh3)4  and 2\u2009M K2CO3 (15\u2009mL) was refluxed in toluene (30\u2009mL) . The reaction mixture was treated with CH2Cl2 and dried.Yield 85%. 1H NMR : \u03b4 6.46-6.67 , 7.15 , 7.23 , 7.54\u2009\u2212\u20097.68 , 9.68 ; 13C NMR : \u03b4 122.68, 128.41, 129.54, 130.51, 132.74, 136.53, 138.72, 139.92, 141.09, 143.21, 148.32, 182.83. MS (EI): m/z 431.21 (M+).The TPAB-Br , 5-formylthiophen-2-yl-2-boronic acid , Pd(PPh2 atmosphere). The solution was extracted with dichloromethane and dried. Anal. Calcd. for C60H38N4: C, 88.43; H, 4.70; N, 6.87. Found: C, 88.36; H, 4.62; N, 6.75. 1H NMR : \u03b4 6.43-6.52 , 6.61 , 7.01 , 7.22 , 7.32 , 7.44 , 7.54-7.61 , 7.80\u20137.88 , 8.12\u20138.20  8.93  : \u03b4 109.88, 116.13, 122.12, 122.93, 123.11, 123.45, 123.97, 124.96, 126.13, 126.65, 127.53, 128.32, 128.47, 129.67, 130.15, 131.54, 133.21, 133.61, 133.89, 134.59, 135.47, 136.08, 139.99, 141.15, 149.51 , 9,10-phenanthernequinone (5\u2009mmol), 4-aminonaphthalene-1-carbonitrile (6\u2009mmol) and ammonium acetate (61\u2009mmol) was refluxed in ethanol  Fig.\u00a0; 13C NMR.51 Fig.\u00a0. MALDI-T 31 Fig.\u00a0.2 streame). The solution was extracted with dichloromethane and dried. Anal. Calcd. for C54H34N4S: C, 84.13; H, 4.45; N, 7.27. Found: C, 84.05; H, 4.37; N, 7.18. 1H NMR : \u03b4 6.46\u20136.52 , 7.0 , 7.24 , 7.43 , 7.54\u2009\u2212\u20097.56 , 7.80\u20137.88 , 8.12\u20138.20 , 8.96  : \u03b4 109.87, 116.15, 121.69, 122.45, 122.68, 123.35, 125.64, 126.79, 128.84, 129.75, 130.54, 131.58, 132.98, 134.10, 136.51, 138.32, 139.51, 141.06, 141.75, 143.54 , 9,10-phenanthernequinone (5\u2009mmol), 4-aminonaphthalene-1-carbonitrile (6\u2009mmol) and ammonium acetate (61\u2009mmol) was refluxed in ethanol  Fig.\u00a0; 13C NMR.54 Fig.\u00a0. MALDI-T 25 Fig.\u00a0.\u22125 mbar). Organic materials and metal electrodes are evaporated at a rate of 0.4\u2009\u00c5\u2009s\u22121 for 1\u20134\u2009\u00c5\u2009s\u22121, respectively. Thickness of each layer was measured with quartz crystal thickness monitor. The EL spectra/CIE coordinates were measured with USB-650-VIS-NIR spectrometer . Current density-voltage-luminance (J-V-L) were measured by computer-controlled source meter (Keithley 2450) equipped with light intensity meter LS-110 under ambient atmosphere. The EQEs were determined from luminance, current density and EL spectrum.ITO glass (resistance 20 \u03a9/sq) was cleaned with acetone, isopropanol and deionised water and dried (120\u2009\u00b0C) followed by UV-zone treatment (20\u2009min). Fabrication was made by deposition method in a vacuum (4 \u00d7 103 or NDBNPIN (30\u2009nm):5wt % Ir(ppy)3/HATCN (8\u2009nm)/LiF (1\u2009nm)/Al (100\u2009nm); (III) ITO/HATCN (8\u2009nm)/ CBP (30\u2009nm):5 wt % Ir(ppy)3 or CBP (30\u2009nm): 5 wt % Ir(MDQ)2 (acac)/HATCN (8\u2009nm)/LiF (1\u2009nm)/Al(100\u2009nm) and electron-only:(IV)ITO/TPBi (8\u2009nm)/DBTPIN or NDBNPIN (50\u2009nm)/TPBi (8\u2009nm)/LiF(1\u2009nm)/Al (100\u2009nm);(V) ITO/TPBi (8\u2009nm)/DBTPIN (30\u2009nm): 5 wt % Ir(ppy)3 or NDBNPIN (30\u2009nm):5 wt % Ir(ppy)3/TPBi (8\u2009nm)/LiF (1\u2009nm)/Al (100\u2009nm);(VI) ITO/TPBi (8\u2009nm)/CBP (30\u2009nm): 5 wt % Ir(ppy)3 or CBP (30\u2009nm): 5 wt % Ir(MDQ)2 (acac)/TPBi (8\u2009nm)/LiF (1\u2009nm)/Al (100\u2009nm) were made  ITO/HATCN (8\u2009nm)/DBTPIN or NDBNPIN (50\u2009nm)/HATCN (8\u2009nm)/LiF (1\u2009nm)/Al (100\u2009nm);(II) ITO/HATCN (8\u2009nm)/DBTPIN (30\u2009nm): 5 wt % Ir(ppy)ade Fig.\u00a0\u2013S338\u201367.1H and 13C NMR and mass spectra were recorded at 298\u2009K on Bruker 400\u2009MHz spectrometer and Agilent (LCMS VL SD), respectively. Absorption (solution and film) were recorded on Perkin-Elmer Lambda 35 and Lambda 35 spectrophotometer with integrated sphere (RSA-PE-20), respectively. PerkinElmer LS55 fluorescence spectrometer and fluorescence spectrometer Model-F7100 with integrating sphere was employed to analyse PL and absolute quantum yield, respectively. Thermogravimetric analysis (TGA) and differential scanning calorimetric (DSC) were recorded with PerkinElmer thermal analysis system and NETZSCH-DSC-204, respectively . Lifetime was estimated with time correlated single-photon counting (TCSPC) method on Horiba Fluorocube-01-NL lifetime system. Cyclic voltammetry was performed with potentiostate CHI 630\u2009A electrochemical analyzer. The HOMO [EHOMO\u2009=\u2009\u2212(Eox\u2009+\u20094.8\u2009eV)] energies and LUMO [ELUMO\u2009=\u2009(Ered\u2009\u2212\u20094.8\u2009eV)] energies were calculated using oxidation and reduction potentials, respectively.68 and multifunctional wavefunction analyzer (Multiwfn)69 (Figs.\u00a0The ground and excited state analysis were studied by using Gaussian 09 program69 Figs.\u00a0\u2013S14.Supplementary information."}
+{"text": "Prostate cancer (PCa) is the most common cancer in men and the fifth leading cause of cancer death worldwide. Unfortunately, castration-resistant prostate cancer (CRPCa) is incurable with surgical treat and prone to drug resistance. Therefore, it is of great importance to find a new target for treatment. LSD1 is up-regulated in PCa and related with prognosis. The high-expression LSD1 has been shown to be a potential target for treatment and is widely studied for its demethylase-activity. However, its demethylation-independent function remains to be elusive in PCa. Recent study shows that LSD1 can destabilize cancer suppressor protein FBXW7 without demethylation-function. Hence, we hope to investigate the impact of non-canonical function of LSD1 on PCa cell survival. We over-expressed FBXW7 gene through plasmid vector in LNCaP and PC3 cell lines and the result shows that up-regulated FBXW7 can suppress the viability of PC cell through suppressing oncoproteins, such as c-MYC, NOTCH-1. After FBXW7 function experiment on PC cell, we knock-down LSD1 gene in the same kinds of cell lines. In western blot assay, we detected that down-regulation of LSD1 will cause the increasing of FBXW7 protein level and decreasing of its targeting oncoproteins. And mRNA level of FBXW7 did not change significantly after LSD1 knock-down, which means LSD1 may destabilize FBXW7 by protein-protein interactions. Moreover, exogenous wild type LSD1 and catalytically deficient mutant K661A both can abrogate previous effect of LSD1 knock-down. Consequently, LSD1 may promote PC cell survival by destabilizing FBXW7 without its demethylase-activity. Next, we compared two kinds inhibitors, and found that SP-2509 (Allosteric inhibitor) treatment suppress the cancer cell survival by blocking the LSD1\u2013FBXW7 interaction, which is an effect that GSK-2879552  cannot achieve. This work revealed a pivotal function of LSD1 in PCa, and indicated a new direction of LSD1 inhibitor research for PCa treatment. As of 2018, global cancer statistics show that prostate cancer has become the second most common cancer in men and the fifth leading cause of cancer death .LSD1 is overexpressed in many human cancers and is associated with poor prognosis . IncreasProstate cancer is also a cancer that highly expresses LSD1. However, the interaction between LSD1 and FBXW7 has not been reported in prostate cancer. Our study investigated the expression of LSD1 and FBXW7 in PCa specimens, and correlated their expression levels with clinicopathological data in the patient cohort. At the same time, the LSD1 inhibition was used to observe the changes in FBXW7 protein levels. Our research has shown that LSD1 destabilizes FBXW7 in a way that does not depend on demethylase, thereby promoting the survival of CRPC cells.Fifty two pairs of prostate cancer tissues and adjacent normal tissues were obtained from patients undergoing urological surgery in Wuhan University People\u2019s Hospital from 2015 to 2018. All research protocols were approved by the Ethics Committee of Renmin Hospital of Wuhan University. Sign informed consent before surgery and collect relevant clinical data of patients. All tissue samples were immediately frozen in liquid nitrogen and then stored at \u221280\u00b0C until Immunohistochemical analysis.(IHC) using specific antibodies for LSD1 , FBXW7 . Serial sections (thickness 5 \u00b5m) were cut from the tissue blocks, deparaffinized in xylene, and hydrated in a graded series of alcohol. Staining was then performed using the DAB chromogenic agent . Negative control experiments were routinely performed.The slides were scored independently by two experienced pathologists who were unaware of the origin of the slides. The semiquantitative scoring system suggested by Remmele and Stegner consideri2. Cells were treated with 1 \u03bcM GSK-2879552  or SP-2509  for 72\u00a0h.BPH-1, LNCaP, and PC3 cells were purchased from the ATCC . Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum  at 37\u00b0C with 5% CO5 cells/ml cells were transferred to cell plates, which were transfected by rLV-FBXW7 on the basis of MOI=20 the next day. Two days after transfection, lentivirus-infected cells were selected using complete medium  containing 10\u03bcg/ml of puromycin.Plasmid was extracted using a Small Plasmid Extraction Kit  after vaccination and amplification of single colonies and bacterial fluid was sequencing verified. 293T cells were co-transfected with pLVX-FBXW7-ZsGreen-Puro (recombinant plasmid) to obtain high titer lentiviral (rLV-FBXW7) containing target gene using a Lentiviral packaging kit . When cells reached 80\u201390% confluence, a total of 5\u00d710siRNA was constructed and obtained from Wuhan Viraltherapy Technologies Co. Ltd. siRNA (100nM) was transfected into cells using Lipofectamine RNA iMAX (Invitrogen) for at least 48h; cells were then harvested for RNA and protein preparation.3 cells/well in 96-well plates. After overnight culture, the cells were transfected with plasmid vector or vehicle to overexpress FBXW7. After this process, cells were incubated with CCK-8 reagent. Then, cell viability was evaluated by absorbance measurements using a microplate reader  at 450 nm.Cell viability was assessed using a CCK-8 assay  according to the manufacturer\u2019s instructions. Briefly, cells were seeded into 96-well plates at a density of 5 \u00d7 10The protein expression levels of LSD1, FBXW7, c-MYC, NOTCH-1, and GADPH were examined by Western blotting. Briefly, proteins were separated on 8% SDS-PAGE gels (50 \u03bcg/lane) and then transferred to nitrocellulose membrane . The membranes were blocked with 5% non-fat milk in TBST buffer  for 2\u00a0h and incubated with primary antibodies overnight at 4\u00b0C. Primary antibodies used here were polyclonal rabbit antibodies against LSD1 , FBXW7 , c-MYC , NOTCH-1 , and GADPH . After extensive washing with TBST buffer, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies . The proteins were detected using an enhanced chemiluminescence system  and captured on light-sensitive X-ray film . Optical densities were detected using ImageJ software.\u2212\u0394\u0394Ct method. All the primers are shown in Total RNA was isolated from cancer cell lines using TRIzol reagent . The purity of RNA was examined by spectrophotometry and the first strand cDNA was synthesized using reverse transcription Reagents  or the TaqManH MicroRNA Reverse Transcription Kit  following the manufacturer\u2019s instructions. QRT-PCR was performed using SYBRH Select Master Mix for CFX (Invitrogen) and using the CFX Connet TM real-time PCR system . All results were normalized to the expression of GAPDH or snRNA U6. The quantitative analysis was calculated by using 2All data are expressed as the mean \u00b1 SEM. Statistical analyses involved one-way ANOVA and Tukey\u2019s multiple comparisons tests. P<0.05 was considered to indicate a statistically significant difference.In previous reports, LSD1 is highly expressed in various type of prostate cancer , 11 whilPrevious study revealed that the expression of FBXW7 protein in prostate cancer was markedly reduced  comparedvs. SiFBXW7+SiLSD1). Results suggested that viability partly recovered after combination with FBXW7 knockdown . The result showed that cell viabilities were significantly reduced in LNCaP and PC3 cells . To furtTo further investigate the underlying mechanism that LSD1 regulate the expression of FBXW7, we explored the interaction between above two. Previously, LSD1 was deemed as a hypothetical AR cofactor, which can regulate target proteins mRNA level through histone demethylation . TherefoIn the past, it was believed that LSD1 played a role in prostate cancer by regulating the transcriptional activity of AR, because LSD1 can remove the methylation of the transcriptional repressive marker H3K9me1/2 \u201320. Howein vitro and in vivo assays are needed to confirm this research.In conclusion, we demonstrate a new role of LSD1 in prostate cancer in our study. First, we determined that LSD1 up-regulation is ubiquitous in lethal CRPC samples and FBXW7 level is down-regulated. Moreover, LSD1 inhibition resulted in an increase of FBXW7 protein levels, while FBXW7 mRNA levels did not increase. We demonstrate that LSD1 can promote the survival of prostate cancer cells by down-regulating FBXW7 protein levels. More importantly, the demethylase activity inhibitor GSK-2879552 cannot eliminate this effect between LSD1 and FBXW7, while the allosteric inhibitor SP-2509 is effective. These results indicate that demethylation function of LSD1 may be dispensable in CRPCa. However, the mechanism that SP-2509 reverse the effect of LSD1-FBXW7 interaction was not fully elucidated. It may be ascribed to the reduction of LSD1 or the alteration of LSD1 conformation due to the binding of inhibitor. These results showed that in the development of LSD1 targeted drugs, we should further look for inhibitors of demethylase-independent functions. However, these hypotheses were limited, as they were mainly based on a retrospective study. Better designed prospective studies and further experiments LSD1 is abnormally expressed in a variety of tumors and is often associated with poor prognosis, it is often considered as a potential anti-cancer treatment target. Accordingly, a line of LSD1 inhibitors have been in clinical studies, such as ORY-1001, RG6016, INCB059872, and so on. And most of these inhibitors are based on blocking its demethylase activity. However, recent studies showed that LSD1 is also involved in a series of protein-protein interactions that are independent of its demethylation function . The funThe 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 Renmin hospital of Wuhan University Ethics Committee. The patients/participants provided their written informed consent to participate in this study.X-kQ designed the study and experimental studies. YD edited and prepared the manuscript. X-hL was the guarantor of integrity of the entire study. LW performed the statistical analysis. All authors contributed to the article and approved the submitted version.This paper is funded by the National Natural Science Foundation of China (No. 81972408 and No. 82000639), the frontier project of Wuhan Applied Foundation (No. 2018060401011321), and Innovation Project of Medical School of Wuhan University (TFZZ2018017).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": "A significant telomere shortening parallels this finding in the post-COVID-19 cohort compared with COVID-19-free subjects (p < 0.0001). Additionally, ACE2 expression was decreased in post-COVID-19 patients, compared with the COVID-19-free population, while DPP-4 did not change. In light of these observations, we hypothesize that some epigenetic alterations are associated with the post-COVID-19 condition, particularly in younger patients (< 60 years).The SARS-CoV-2 infection determines the COVID-19 syndrome characterized, in the worst cases, by severe respiratory distress, pulmonary and cardiac fibrosis, inflammatory cytokine release, and immunosuppression. This condition has led to the death of about 2.15% of the total infected world population so far. Among survivors, the presence of the so-called persistent post-COVID-19 syndrome (PPCS) is a common finding. In COVID-19 survivors, PPCS presents one or more symptoms: fatigue, dyspnea, memory loss, sleep disorders, and difficulty concentrating. In this study, a cohort of 117 COVID-19 survivors (post-COVID-19) and 144 non-infected volunteers (COVID-19-free) was analyzed using pyrosequencing of defined CpG islands previously identified as suitable for biological age determination. The results show a consistent biological age increase in the post-COVID-19 population, determining a DeltaAge acceleration of 10.45 \u00b1 7.29 years  compared with 3.68 \u00b1 8.17 years for the COVID-19-free population ( SARS-CoV-2-infected people who develop adult respiratory distress syndrome (ARDS) often accumulate an excessive extracellular matrix deposition, causing pulmonary, cardiac, and nervous fibrosis that worsens organ function . Other cUpon SARS-CoV-2 infection, the angiotensin-converting enzyme 2 (ACE2) expression level in the vascular system tends to decrease . This enDipeptidyl-peptidase IV (DPP-4) is the receptor of MERS coronavirus (MERS-CoV) and has been reported, in some cases, to function as a coreceptor of SARS-CoV-2 . DPP-4 eIn humans, telomere shortening is associated in vivo with the aging process and, in vitro, with cellular replicative senescence . TelomerIn recent years, several studies aimed to identify biological or molecular markers of aging that correlate with chronological age and could therefore be helpful to estimate biological vs. chronological age . Some ofA positive DeltaAge is considered an acceleration of the biological blood clock, while a negative DeltaAge indicates a younger blood bioage than the chronological one. This parameter has proven helpful in evaluating the risk of the onset of cardiovascular and neurodegenerative diseases, cancer, and the occurrence of death by all-causes .Helicobacter pylori [In infectious diseases, the application of these methods is still limited. However, a DeltaAge acceleration has been observed in people infected by human immunodeficiency virus (HIV), cytomegalovirus, or bacteria such as r pylori . In postr pylori ,34. HIV r pylori . Similarr pylori . The lonThe present study investigates whether, in COVID-19 survivors, there is a DNAmAge alteration and a DeltaAge acceleration, which, in association with other molecular parameters such as the telomere length and ACE2 expression in peripheral blood, might typify a set of biomarkers valuable in other and future studies exploring the risk of PPCS-associated pathophysiological manifestations.p-value < 0.0001). No difference was appreciable in the controls  independent of an accelerated DeltaAge, suggesting that these two parameters might be regulated independently. Again, no significant differences emerged between females and males.TL shortening has been reported as a risk factor for developing more severe COVID-19 syndrome . We invep-value < 0.0001). The expression level of DPP-4 was unchanged  and DPP-4 (MERS-CoV receptor). The results are shown in nchanged B.p < 0.01). No differences were observed in the distribution of DPP-4 by DeltaAge . As shown in Telomere length is a marker of aging: progressive telomere shortening is a well-characterized phenomenon observed in older adults and attributed to the so-called telomere attrition. This condition is worsened by the absence of telomerase activity which is physiologically silenced in the early post-natal stage and throughout adulthood . An acceACE2 is a crucial component of the SARS-CoV-2 infection process. SARS-CoV-2 uses the ACE2 receptor to invade human alveolar epithelial cells and other cells, including cardiac fibroblasts . In infeThe two groups considered in this study were not significantly different in terms of age, sex, and known clinical conditions before SARS-CoV-2 infection, except for a relatively higher incidence of BMI > 30 (15.3% vs. 9%) in the post-COVID-19 population compared with controls, as well as a record of more frequent lung diseases , a heterogeneous group that included healthy, cardiovascular disease-affected, and obstructive sleep apnea-affected patients, and the post-COVID-19 group, which included all of the previous types of patients who had also been infected with SARS-CoV-2 (n = 117). The clinical features of both populations are summarized in The samples were classified into two groups: COVID-19-free (Blood samples collected in EDTA (200 \u03bcL) were used to perform the extraction using the QIAmp DNA Blood Mini Kit  associated with the automated system QIACube , according to the manufacturer\u2019s instructions. Subsequently, 2 \u03bcL of DNA was quantified with QIAxpert .One microgram of DNA was converted using the EpiTect Fast DNA Bisulfite Conversion Kit  associated with the RotorGene 2plex HRM Platform  and the QIACube automated system, following the manufacturer\u2019s instructions. Subsequently, 2 \u03bcL of converted DNA was quantified with QIAxpert.PCR reaction mixes were performed using the PyroMark PCR Kit , following the manufacturer\u2019s instructions. The sequences of primer used are available in the The amplicons were sequenced in order to check the level of methylation in each CpG site. PyroMark Q24 Advanced Reagents  were loaded in the PyroMark Q24 Cartridge , following the manufacturer\u2019s instructions, and 5 \u03bcL of PCR product was added to the reaction mix containing: Pyromark Binding Buffer (supplied in PyroMark Q24 Advanced Reagents kit), Streptavidin Sepharose High Performance , and DNase/RNase-free distilled water. Samples were shaken at room temperature for 15 min at 1400 rpm. Subsequently, the samples underwent the PyroMark Q24 Vacuum Station  procedure, in which the target sequences were purified and put into an annealing buffer containing the sequencing primer (0.375 \u03bcM). The sequences of oligos are available in the Bekaert\u2019s algorithm was applied to estimate the biological age of the population  as reporThe length of chromosome ends was quantified using a PCR Real-Time of Absolute Human Telomere Length Quantification qPCR Assay Kit , following the manufacturer\u2019s instructions.The total RNA was isolated from whole blood using a QIAmp RNA Blood Mini Kit  and an automatized extractor QIACube, according to the manufacturer\u2019s instructions. The RNA was quantified with QIAxpert.An Omniscript RT Kit  was used to convert total RNA into cDNA according to the manufacturer\u2019s instructions.Initial denaturation: 95 \u00b0C, 5 min;Denaturation: 95 \u00b0C, 15 s;Annealing: 60 \u00b0C, 30 s;Elongation: 72 \u00b0C, 30 s;Final elongation: 72 \u00b0C, 1 min.Real-time qPCR was performed on the RotorGene 2plex HRM Platform using RT2 SYBR Green ROX FAST Mastermix . The sequences of primers are available in the Denaturation, annealing, and elongation were repeated 45 times.p-values were calculated using two-sided T-tests.All data were analyzed with GraphPad Prism 8.4.3 and This study has many significant limitations, including the limited number of subjects investigated and the low number of CpGs considered. Although we used a valid forensic method to establish the biological age in the examined groups ,31, adopNevertheless, it was shown here that individuals belonging to a group of COVID-19 survivors exhibited a significant acceleration of their biological age, occurring mainly in the younger individuals. This information was correlated with TL shortening and the expression of ACE2 mRNA. It is too early to extrapolate whether relevant clinical indications may arise from this and other studies assessing the role of epigenetic changes in the COVID-19 syndrome ,47. Howe"}
+{"text": "The overall survival rate was 97.5% in the no transfusion group, compared to 95.9% in the 1\u20132 units group (HR 1.50 (1.22\u20131.83), p < 0.001), 92.0% in the 3\u20135 units group (HR 2.43 (1.87\u20133.15), p < 0.001), and 67.5% in the 6 or more units group (HR 6.81 (5.03\u20139.22), p < 0.001). Conclusions: Peri-transplant RBC transfusion was independently associated with the increased risk of renal allograft failure and death in KT patients.Background: Patients undergoing kidney transplantation (KT) often receive red blood cell (RBC) transfusion during admission for KT which may increase the risk of allosensitization. The association between peri-transplant RBC transfusion and graft survival was evaluated using a nationwide cohort. Methods: This retrospective study analyzed 13,871 patients who underwent KT in Korea between 2007 and 2015. The outcomes were graft failure rate and overall patient survival depending on the amount of RBC transfusion. Results: The overall graft failure rate was 15.5%. Compared to the graft failure rate of 13.5% in the no transfusion group, the graft failure rate was 15.4% in the 1\u20132 units group (sHR 1.06 (95% CI 0.97\u20131.17),  Kidney transplantation (KT) is the treatment of choice for end-stage renal disease (ESRD) with clear advantages over renal replacement therapy in survival and quality of life . Graft sBlood transfusion prior to KT was common practice  until thRecent data show that 52\u201364% of patients undergoing KT still receive blood transfusion ,11. HoweThis study was a retrospective cohort study and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Institutional Review Board of Seoul National University Hospital (1708-061-877). The clinical and research activities reported are consistent with the Principles of the Declaration of Istanbul as outlined in the \u2018Declaration of Istanbul on Organ Trafficking and Transplant Tourism\u2019.The NHIS database contains all claims data for more than 97% of the population who are covered under the National Healthcare Insurance (NHI) program and the Medical Aid program in Korea. The NHIS database is provided to researchers after de-identification for research purposes and generation of real-world evidence.Adult patients (age \u2265 19) who received KT between 2007 and 2015 were identified from the NHIS database by searching the NHI procedure code for KT during hospitalization. Patients with simultaneous NHI procedure codes for other solid organ transplantations such as pancreas, liver, or heart transplantation during the same admission for KT were excluded. After identification of adult KT recipients, underlying comorbidities including hypertension, diabetes mellitus, coronary artery disease, and cardiovascular disease were extracted from the database using ICD-10  codes. To adjust for the severity of illness, the Elixhauser Comorbidity Index, derived from 30 disease entities using ICD-10 codes and shown to correlate with hospital mortality, was used as a covariate.The two outcomes of interests were allograft failure (not including death with function) and all-cause mortality. Allograft failure was detected by NHI procedure codes for dialysis or retransplantation. Long-term mortality was detected when healthcare coverage by the NHI was terminated based on automatically reported death certificates to the NHI. In-hospital mortality, intensive care unit (ICU) length of stay, and hospital length of stay were also extracted. To analyze immunologic risk factors, the following data were extracted from the database: presence of donor specific antibody , regimens of induction treatment, delayed graft function , and acute allograft rejection (defined by NHI procedure code for anti-rejection therapy). The data on perioperative bleeding complications were obtained from the database using the NHI procedure codes for angiographic embolization and bleeding control operation.Patient characteristics were compared according to the number of transfused packed RBC units using the chi-square test or Fisher\u2019s exact test.A competing-risk analysis model (Fine and Gray model) was used considering death with functioning graft as a competing event to investigate the association between transfused RBC units and graft failure with adjustment of multiple covariates. The competing-risk analysis results were presented as a subdistribution hazard ratio (sHR) with a 95% confidence interval (CI). To analyze the association between transfused RBC units and mortality, a Cox proportional hazards model was used and the results were presented as a hazard ratio (HR) with a 95% CI.p-values were less than 0.05.The Kaplan\u2013Meier survival curve after KT depends on the number of transfused RBC units and performed the log-rank test to compare the survival curve. All analyses were performed using SAS 9.4 . Results were considered statistically significant when two-sided A total of 13,781 KTs were performed from 2007 to 2016 in Korea. Patient and center characteristics are presented in The overall graft failure rates at 1, 3, 5, and 7 years after KT were 7.0% , 14.1% (1150/8163), 18.3% (904/4942), and 21.3% (507/2381), respectively. There was a positive correlation between the number of RBC transfusions and graft failure rates at various time points after KT . p < 0.001).The competing-risk analysis for graft failure treating death with functioning graft as a competing event showed that the risk of graft failure was significantly higher in patients who received larger amounts RBC transfusion. Patients who received 6 or more units ) and patients who received 3\u20135 units ) showed higher risk of graft failure compared to the patients who did not receive transfusion. The risk of graft failure after KT in patients who received 1\u20132 units was comparable to patients who did not receive transfusion ) . Deceasep < 0.001, In the Cox proportional hazard model for all-cause mortality, adjusted HRs were higher in patients who received 6 or more units of RBC compared to patients who received 5 units or less . DeceaseOur results suggest that 3 or more units of RBC transfusion during the perioperative period for KT may be associated with an increased risk of graft failure after KT. The association between perioperative RBC transfusion and renal allograft survival in a nationwide large cohort may suggest the importance of adequate management for anemia in ESRD patients waiting for KT. As the incidence and prevalence of chronic kidney disease (CKD) grows, the number of patients on the waiting list for KT is also increasing. Death-censored kidney allograft survival has increased steadily over the past decade in both adult and pediatric patients . The SciMany factors affect the long-term outcome of KT, which is often defined as patient death or renal dysfunction leading to graft loss requiring dialysis . Donor aThe NHI program is the universal healthcare coverage system in Korea. The NHIS provides healthcare insurance to more than 97% of the population in Korea through the NHI program and is the single payer of the NHI program. The remaining 3% of the population with the lowest income are supported by the Medical Aid program . The broStudies prior to the 1980s suggested that pre-transplant RBC transfusion may improve allograft survival ,18. BaseThe impact of transfusion in KT has not been studied extensively and the results of the few studies that evaluated the impact of transfusion on renal allograft outcomes were inconsistent. Transfusion prior to KT was recently reported to be associated with the development of anti-HLA antibodies in patients with no previous organ transplantation or pregnancy . In contAlthough, our study focused on the effect of perioperative RBC transfusion on the renal outcome, transfusion after discharge from the admission for KT may have affected allograft outcomes. However, previous studies have shown that most blood transfusion (70 to 90%) occurred during the perioperative hospital admission period ,11. In bOur data suggests that perioperative transfusion may have negative effects on long-term graft survival after kidney transplantation. The Renal Association Clinical Practice Guidelines suggest that hemoglobin levels should be maintained in the range of 10\u201312 g/dL in CKD patients with ESA therapy . MaintaiPatients undergoing deceased donor KT recipients showed a higher transfusion rate compared to living donor KT recipients (63% vs. 50%), which is consistent with the findings of previous studies . Given tThis study has several limitations that requires consideration. First, due to the nature of administrative data, the NHIS database lacks several clinically relevant variables including laboratory data and detailed clinical information. Although data regarding perioperative bleeding complications were added to compensate for the lack of hemoglobin levels, comparing patients with similar hemoglobin levels may have provided a clearer picture. However, we believe that the clinical implications of our study are still considerable as specific clinically relevant outcomes were analyzed, adjusting for major comorbidities and immunologic factors. Second, data regarding the types of transfused RBCs such as leukocyte depleted or radiated RBCs were not available in our analyses. Although leukocyte depleted RBCs are commonly used to prevent alloimmune sensitization, HLA class I molecules are expressed constitutively on erythrocytes even though their expression levels are low. The content of HLA molecules within leukocyte depleted RBCs is sufficient to induce HLA class I sensitization. Leukodepletion of blood products is not sufficient to prevent the risk of allosensitization ,29. ThirDespite these limitations, our study provides some important clinical implications. Our study is the first large scale study to investigate the relationship between perioperative RBC transfusion and graft survival after KT. In addition, selection bias was minimized by using the NHIS database, which includes nearly all KT operations performed in the recent 10 years in Korea. In conclusion, KT patients who received more RBC transfusion over 2 units during the perioperative period showed lower graft survival compared to those who received no transfusion or less than 3 units."}
+{"text": "In post disastrous situations, coordinated and integrated interventions aimed at relief and rehabilitation not only help facilitate reaching out to the affected communities in a timely fashion but also pave the way to channel scarce and valued resources towards end users in an efficient and effective manner. This article attempts to trace the origins and gradual development of \u2018inter-agency collaboration\u2019 and the implications thereof for disaster management strategies in Pakistan through an analysis of relief and rehabilitation interventions undertaken by the Government of Pakistan in collaboration with local and international Non-governmental Organisations (NGOs) and relief agencies in the ex post of the 2005 earthquake. Data for this study were collected through structured and semi-structured interviews from government officials, representatives of NGOs and relief agencies and ordinary women and men in the earthquake stricken localities of Balakot and Mansehra districts of Pakistan. On the heels of the 2005 earthquake, both local NGOs and faith-based organisations in concert with international NGOs and relief agencies from around the world rushed to assist Pakistan in it\u2019s rescue and relief operations at a time when the country was faced with the twin dilemma of both the non-existence of peculiar institutional arrangements for disaster management and a lack of the necessary technical and financial resources. The aftermath of the 2005 earthquake offered opportunity to the Government of Pakistan and the NGOs and relief agencies alike to transform their individual interventions into a robust and organised \u2018inter-agency collaboration\u2019, which was later on realised in the form of establishment of a national disaster management organisation called the \u2018Earthquake Reconstruction and Rehabilitation Authority (ERRA)\u2019. The establishment of ERRA not only paved the way for avoiding duplication and wastage of resources but also ensued in reaching out to the affected communities in a timely fashion. The Pakistani case offers implications in terms of highlighting the salience of establishing \u2018inter-agency collaboration\u2019 in other settings. The early 21st century has witnessed a multitude of natural calamities ranging from massive earthquakes and cyclones through to disastrous tsunamis. The human and physical costs emanating from such disasters have been colossal  and international non-governmental organisations (INGOs) during 2005 earthquake response and recovery phases. The article specifically emphasises probing the effectiveness of inter-agency collaboration in disastrous situations such as the 2005 earthquake. The analysis in this article demonstrates how inter-agency collaboration ensued in radically overhauling existing institutional arrangements associated with disaster management in Pakistan and facilitated the collaborators to effectively cope with ravages of the October 2005 notorious calamity.Over the last two decades or so, the scale and scope of natural and man-made disasters have dramatically increased. The ineffectiveness of traditional disaster management approaches has given way to the emergence of decentralised emergency management systems. This shift is being prompted by the exigency to collaborate during various stages of a disastrous situation  and Emergency Service-Rescue 1122 was a pilot project initiated in 2004 in Lahore, the capital city of Punjab province and later on extended to other districts. The model is also replicated by other provinces in anticipation to cope with emergency situations  7.6  earthquake struck the capital, northern Pakistan and the Kashmir region. The epicentre of earthquake was located 100 km north-northeast of Islamabad. This devastating earthquake was followed by 1000 aftershocks of (Mw) of 5.0\u20136.0 in India-Pakistan Kashmir region. The Pakistan-administered Kashmir known as Azad Jammu and Kashmir (AJ&K) and the eastern districts of Khyber Pakhtunkhwa (KP) province were amongst the most severely affected regions , five respondents from Islamic Relief and eight officials were interviewed from ERRA. All interviews were conducted in person and each interview took one and a half hour; a prior permission was taken from respondents for recording and documentation. More than two rounds of semi-structured interviews were conducted with five respondents from ERAA, IRC and United Nations Office for the Coordination of Humanitarian Affairs (UNOCHA), two rounds of semi-structured interviews were conducted with three respondents from ERRA, Islamic Relief and World Vision whilst the rest of semi-structured interviews were conducted in one round. An interview guide was used when conducting semi-structured interviews. The open question of the interview guide helped to keep interviews within the scope of the study by directing the respondents towards talking about the inter-agency collaboration. The interview allowed the respondents to speak freely within the specific themes whilst explaining the inter-agency collaboration. During the data collection, the interviewer asked probing questions to clarify ideas and explore more in-depth information. The important aspects covered in interviews included, for instance, (1) initial condition (2) process dimension (3) structure and governance (4) contingencies and constraints and (5) outcomes and accountability. Secondary data were retrieved from published reports, government briefings and feedback from UN and donor agencies. Before the earthquake in 2005, FFC, Pakistan Agriculture Research Council (PARC) and many international organisations such as ActionAid, Catholic Relief Service, Concern Worldwide, Oxfam, United Nations Food and Agriculture Organization (UNFAO), United Nations World Food Program (UNWFP), World Health Organization (WHO), United Nations Children\u2019s Fund (UNICEF), United Nations Development Fund (UNDP) and Department for International Development (DFID) were engaged in the planning, implementation and oversight of programmes and projects for disaster risk mitigation NDMA .All the interviews were transcribed, and the data were analysed in specified themes under the research model. This method helped and contributed towards the understanding of establishing inter-agency collaboration in post-earthquake 2005 disaster management in affected areas of Pakistan.The 2005 earthquake stands out as one of the worst disasters Pakistan has ever witnessed. A large number of international relief agencies swiftly responded to grapple with the ravages of this catastrophic event ERRA . Given tIn Bryson et al.  model, tA]t that morning when this catastrophic event (earthquake) unfolded, everyone was taken aback. On the heels of the earthquake, provision of all sorts of public services and goods halted altogether.\u2019 \u2018[One of the representatives from INGOs in Balakot (the epicentre of the 2005 Earthquake) spoke about the initial conditions and highlighted that situation has worsened in the area. Just after the disaster the information received by local sources reported on large-scale human losses and damages to valuable assets and infrastructure. Respondents in the study area revealed that both the federal and provincial governments lacked the demonstrable capacity and vigour to properly respond to emergency situation, notwithstanding government officials, at large, were determined to identify and resolve the issues that ensued from the chaotic situation. The initial response of the government entailed the deployment of two Army Divisions for undertaking the rescue and relief operations in the disaster-stricken areas. This initial response pursued by government also brought into spotlight the incapacity and inefficiency of the relevant government departments. For instance, in Mansehra district alone, the relevant government departments grappled to respond to the situation.The 2005 earthquake had received considerable attention and coverage from both national media and international news agencies around the world, which brought into spotlight both the government\u2019s institutional incapacity and inefficiency and the need for undertaking measures aimed at establishing inter-agency collaboration:\u2018In compulsion the government had initiated the inter-agency collaboration for initial response \u2026 because, government alone could have barely responded to the situation. Furthermore, people in the earth stricken areas were also looking forward to seek assistance from relief agencies.\u2019 Discursively, a variety of factors account for engendering collaboration between government and aid providing actors such as coping with rescue and rehabilitation issues in the ex post of the disaster, efficient and effective exploitation of available resources, avoiding duplication and overlapping of activities and taking advantage of the prowess and expertise of international aid and relief agencies. Majority of the respondents, 18 of them, highlighted that major international organisations arrived with its full capacity but to step in on foreign land was a major challenge in offering relief assistance. For instance, the IRC, World Vision and Islamic Relief had both experienced and relief specialists who could facilitate people in affected areas. An interview conducted with the IRC representative in health project indicated that the IRC had organisational capacity, skilled manpower and good resources to respond to disastrous situation of 2005 earthquake. In Mansehra, the field coordinator of World Vision recalled that the organisation was capable of establishing liaison amongst key stakeholders and bringing them on one platform. Non-profit organisations were closely affiliated with other central actors such as, UN agencies, large faith-based entities, universities and public sector organisations towards collective involvement in relief efforts.Two respondents from ERRA described that although establishment of inter-agency collaboration was not free from challenges but the need for collaboration with capable players particularly with INGOs was felt within the government and it was realised that without involving these actors, government on its own could not reach to affected areas with full capacity. A dire need was felt between government and INGOs to establish regional level offices for coordination of the initial relief and rehabilitation interventions. In this way, the initial processes provided foundation for the formation of collaboration and core groups based on their experiences.At the outset, international organisations such as the IRC, Islamic Relief, World Vision, etc. carried out their rescue and relief activities independently at large and demonstrated relatively more vigour and robustness in serving the affected communities upon receiving the No Objection Certificate (NOC) from the Government of Pakistan. The rescue and relief teams  arrived at Balakot city just after disaster delivered the essential life support goods and services to the earthquake survivors. Moreover, relief operations such as setting up of field hospitals, rescue operations, temporary shelters and distribution of food items were made possible because of relaxed government policy. Majority of organisations had formal agreements Memorandum of Understanding (MoUs) with the government of Pakistan. Therefore, the IRC, World Vision and Islamic Relief continued to extend relief efforts in Abbottabad, Mansehra and Batagram in Hazara Division. The IRC team leader recalled:\u2018Operational manager called me after four days of earthquake and urged that collaborative working group will be needed to respond to this disastrous event.\u2019 Bryson et al.  discusseComponent of trustworthiness and legitimacy is the critical part of any collaboration. In the case of 2005 earthquake, the international organisations and IRC had working relationships with government in many development projects in different parts of the country. Therefore, these organisations had a good social image and track record, which put the government at ease to start collaborative initiative with partnering organisations. Managing conflict is a critical determinant of any inter-agency collaboration efforts, especially when majority of the partnering organisations belonged from diverse backgrounds. A total of 19 respondents maintained that all the collaborating partners have common agenda: protecting the earthquake affected communities with respect and dignity, minimising their suffering and bringing them back to normal life. The IRC representative revealed that in newly established collaborations, conflict among organizations usually arises due to the discrepancies associated with the organizations\u2019 commitment, mission, interests, values and resources. However, in inter-agency collaboration, managing conflicts across organisations starts from the fact that when each partnering organisation recognises the other organisation\u2019s commitment and values. This is particularly true in the case of post-earthquake 2005 collaborative initiatives, as many of private, public and non-profit organisations were securing their interests and mission but with passage of time these organisations realise that public needs are above their organisational interests and mission. Since ERRA had played a dominant role in the collaboration process, ERRA in tandem with its collaborative partners, thus, paid special attention to re-visiting the organizational commitment and values that were generally considered as antithetical to the common agenda.ex ante planning (if any existed) failed to respond to the situation. Consequently, the proposition for establishing inter-agency collaboration was welcomed particularly by INGOs:Bryson et al.  argued t\u2018Without inter-agency collaboration responding to earthquake could be a miracle. In such a turbulent situation resources were mobilized in hurly-burly way. Because, either unnecessary supplies were received, or some got more while others were waited even for life saving drugs and food for survival.\u2019 (World Vision representative)Majority of the respondents revealed that the process of inter-agency collaboration is usually affected by political constrains and administrative problems. Although reaching to a formal agreement for establishing inter-agency collaboration mostly takes longer time but in 2005 earthquake government showed openness to a greater extent and high level of cooperation. One of the key aspects of the inter-agency collaboration was the establishment of knowledge-based collaboration amongst actors. Government initiative provides foundation for the vast documentation of information on risk assessment, lesson learned from various sectors by different stakeholders and contingency planning for disaster management. Respondents from ERRA and many others from INGOs thought that 2005 earthquake was one of the first natural disasters of that scale, it opened a new possibility for establishing a state level resource centre for disaster management in the country.The structure and governance component of the model referred to the informal and formal membership, structural configuration and governance structure. Nineteen of the respondents highlighted that in establishing collaborative efforts, governance and structure have substantial importance. Earthquake Rehabilitation and Reconstruction Authority was the main officiating body responsible for all decisions including usages of funds and coordination and monitoring of resources in post-earthquake scenario. The Earthquake Rehabilitation and Reconstruction Authority had devised a mechanism which pave the way for devolving maximum powers to the Provincial Earthquake Rehabilitation and Reconstruction Agency (PERRA), the State Earthquake Rehabilitation and Reconstruction Agency (SERRA) and District Reconstruction Units (DRUs). This forum acted as a secretariat for provincial and state steering committees. The chief headed each committee, a secretary with representation from line departments and planning wing of ERRA. The committee had the mandate to approve the work plans and authority to accept the projects of up to 250 million rupees. Earthquake Rehabilitation and Reconstruction Authority was also responsible for ensuring required coordination and facilitations for other key actors including INGOs. The cluster forum paved the way for effective collaborative mechanism. The UN system particularly the UNOCHA was in close collaboration with government for integrated emergency response. As the UNOCHA aimed at strengthening collaborative partnerships, predictability and accountability of humanitarian assistance and defining the roles and responsibilities of humanitarian organisations, therefore, a platform was provided to international organisations for information sharing, mobilisation of resources, which minimise the overlapping and duplication of resources. UNOCHA has online centralised system for information sharing; all the implementing partners were responsible for uploading their intervention details on system. This system provided a multi-level information sharing platform to all relief assistance providing actors:\u2018At the outset, despite the availability of ample resources, an organized and integrated relief assistance mechanism was yet to be established. This could cause overlapping and duplication of interventions activities undertaken by different agencies. Given the dire need to make all relief assistance efforts integrated and relatively more organized a cluster system was unveiled which served as a platform for all the implementing partners to share as well as to get access to the necessary information concerning relief assistance in the earthquake stricken areas.\u2019 (IRC Representative)Majority of the respondents from INGOs noticed that in the case of INGOs much of the governance mechanism relies on each organisation\u2019s commitment and mandate in their respective areas. Moreover, the INGOs have the responsibility to regularly publicise their intervention details on website for instance, type of relief assistance provided, catchment area, number of beneficiaries covered and services in progress. However, some of the respondents from INGOs highlighted that majority of the information shared on websites on UNOCHA or organisation\u2019s own website is no longer available either online or in physical form. The online information sharing system was an innovative method for monitoring and evaluation (M&E) and provided valid data to other partner organisations. Whilst the system paved the way for making relief information transparent and widespread amongst all contributing organisations, still it was characterised by major flaws. For instance, gap in information shared by implementing partners and ground work, follow up of interventions and lack of trust amongst partner organisations.All the respondents note that every organisation has its own governance mechanism to operate its activities. In the case of 2005 earthquake, ERRA played an instrumental role in providing guidelines for defining the roles and responsibilities of aid providing actors. For instance, organisational values and commitments are valued by collaborating partners but ERRA guidelines were monitored and partnering organisations paid attention to these guidelines whilst operating in any disaster hit area. Majority of the respondents highlighted that in the inter-agency collaboration power was concentrated at the top, that is, ERRA and responsibilities were shared across collaborating partners. Each partner is anticipated to own the collaborative effort initiated by the government and implement its approved project within the time and share the progress of the operation on regular basis.Bryson et al.  model suAccording to INGOs representatives, the IRC, World Vision and Islamic Relief all were running their projects from international donations received for earthquake. These organisations were the custodian of donations and public charity received on the basis of humanitarian assistance. Power resides with top management to decide a project according to the need identified. Each partner is anticipated to take ownership of their projects and regularly reported their progress. Whilst ERRA, as the main collaborative body, provided facilitation to the partnering organisations, other organisations with abundant funding resources dominated the situation. During emergency response and recovery phase, publicity of donor organisations was mandatory for implementing partners. In both relief assistance and reconstruction projects the acknowledgement of funding agency was displayed on monument plaque.Outcome and accountability is the last category of the model. Outcome is discussed in three categories: public value; first, second and third order effects, whilst accountability pertains to input, process or outcomes. Bryson et al.  stated tAll the respondents explained that the disastrous event of 2005 not only attracted public, private and non-profit organisations at regional and global levels but also received wide coverage from national and international media. Therefore, a huge influx of bilateral and multilateral donors and dozens of non-governmental and private organisations were arrived. This creates an opportunity for establishment of new partnerships, extended the collaborative efforts outside the formal boundaries, learning from experiences and designing strategy for effective inter-agency collaborative efforts. The collaborative efforts initiated in post disaster scenario provided mechanism for channelising and mainstreaming of relief resources such as, charitable donations was discernible immediately after earthquake as first-order effects. Majority of relief providing agencies including INGOs covered most of the deprived and ignored segment of affected community.Our interviewees also explained that collaborative experiences had positive effects both at organisational and personal level. In essence, partner organizations steered such collaboration opportunities towards fostering capacity building such as improving communication skills and human resource development. The local staff found the inter-agency collaboration very productive and revealed that they had the opportunity to learn beyond their expectations from such collaboration. An ERRA official mentioned in an interview that such exposure was never been experienced in particularly how to expand the inter-agency partnerships in the time of extreme events when each organisation had come with its full capacity. The local organisations such as, community-based organisations (CBOs) also took advantages from this inter-agency collaboration. For instance, INGOs involved these organisations in implementation process, arranged workshops and trainings for their capacity building, which increase their capability to run small-scale projects. Consequently, big non-profit organisations created social capital for themselves. This form of new partnership emerged as second-order effects.Fourteen of the respondents explained that the system of accountability is prerequisite for successful collaboration. However, in time of disaster, the new partnership often creates coordination problems and the aspect of accountability may be challenging for partnering agencies. These problems range from competition and tension amongst collaborative partners. In the case of 2005 earthquake, initially, the challenge for the inter-agency collaboration lies in overall management of collaborating partners during rescue and relief phases. For example, our interviewees explained that instead of focusing on relief and rescue operations most of the organisations were interested to show-off their relief work and gain recognition for their efforts.Nine respondents three from ERRA and six from INGOs explained that novelty of inter-agency collaboration created few problems, which overall disturbed the accountability of collaborative efforts. Initially, because of lack of capacity and efficiency at government level, resources were not properly streamlined. Therefore, achieving accountability in relief work was a challenge for FRC. After the establishment of ERRA the principle of accountability was ensured in three ways. Firstly, policy regarding the provision of relief and reconstruction services were formulated and implemented. Secondly, relief resources were channelised and a selection criterion was established for funding applications. Thirdly, indicators based monitoring system and tracking of information through multilevel platform for the projects were employed. For instance, ERRA with the assistance of Department for International Development (DFID) had developed the M&E system for unified data system and for effective tracking of fund and its usages. The INGOs particularly, the World Vision, IRC and Islamic Relief had the capacity to implement and monitor their project on its own. Our interviewees explained that each organisation has its own M&E mechanism that ensures accountability of interventions undertaken. Moreover, the cluster system further strengthens the process by integrated approach and generated unified and valid data system that is to some extent ensure the accountability aspect of INGOs. Such complex mechanism of (M&E) and development of unified data system ensure the third-order effects of inter-agency collaboration.The current research is based on Bryson et al.  ICM by eThe current research endeavour indicated that regardless of government swift response and openness in initial stages administrative and operational issues were emerged from disaster response management. The major issues highlighted in interviews were the streamlining of resources and establishment of collaborative mechanism with relief providing actors. As the political leadership and civil administration were not prepared for such disastrous event therefore the coordination mechanism confronted with major administrative and operational problems. Although, respondents have mentioned the government willingness and involvement remained marvellous at all levels in establishing inter-agency collaboration, but it is interesting to note that UN system and INGOs played substantial role in terms of providing technical and financial support to the government. Therefore, ERRA had clear guidelines for setting up goals and mandates for the inter-agency collaboration.A lot of resources were invested in structuring effective inter-agency collaboration. Therefore, the cluster approach did increase the collaboration amongst relief providing agencies and limiting duplication of resources and played vital role as a facilitator within and amongst government and relief providing actors. Although field level coordination is a crucial part of collaboration efforts such as, efficient use of resources by expanding the service delivery to a greater number of affected communities. Therefore, adaptation of referral mechanism was amongst the driving forces which fierce the coordination process for instance, the IRC established traumatised centres and provided psycho-social support to number of patients referred by different organisations. It is also interesting to note that the novelty of cluster system confronted some major issue such as, less ownership of the coordination process at government level, lack of involvement of donors\u2019 agencies, unclear guidelines and under representation of INGOs/NGOs. The study also indicated that other deriving forces also hamper the coordination process such as, competition over funding between actors dwindle the coordination process as most of actors wanting to work in the area and show-off their work.Pakistan\u2019s disaster response strategy was mainly based on reactive approach. The West Pakistan Calamity Act (1958) was a legal remedy whilst ERC (1971) was institutional support at federal level dealt with disasters. Other network of emergency response institutions with overlapping roles and responsibilities were also being operational in the country. This lack of institutional capacity and efficiency exhibited the need for appropriate policy and institutional arrangements for disaster management at federal and provincial levels. Therefore, National Disaster Management Ordinance (2006) was the first initiative for integrated disaster response mechanism which further paved the way for the establishment of NDMA, Provincial Disaster Management Authority (PDMA) and Federally Administered Tribal Areas Disaster Management Authority (FDMA) in the country. The 2005 earthquake, exposed the existing institutional arrangements and deficiencies at all levels of government to deal with mega disaster. The post-earthquake 2005 inter-agency collaboration played instrumental role in mainstreaming the disaster management in development planning in the country.The findings of the study demonstrate that inter-agency collaboration serves as an integral component of disaster management and may turn out to be a magic bullet particularly in situations when institutional capacities in host countries are tenuous. On the heels of any disastrous situation, timely provision of services always poses challenges not only to the government at all levels but also to the aid relief providing agencies as well. An effective way to cope with such challenges is to seek for collaboration between different levels of government and the national and international relief agencies. Inter-agency collaboration in disastrous situations requires participation and ownership from all actors. However, governments shall preferably play a leading role and take all the requisite steps in order to institutionalise such initiatives, which may help facilitate the responding agencies both in the short- and long-term disastrous situations. Although such inter-agency collaboration may be a platform for achieving a set of common goals, sharing of information, pooling of various resources and the lessons learnt but it could also serve as a very useful tool for spelling out both the governments\u2019 and other collaborating actors\u2019 roles and responsibilities.The inter-agency coordination was established in a turbulent situation resulting from 2005 earthquake, the urgency and attention for the relief work was intensified whilst challenges remained and even increased with the passage of time. Therefore, inter-agency collaboration such as introduction of cluster system was an appropriate approach for creating connection within and amongst assistance providing actors. On initial stages some administrative and operational issues emerged from the inter-agency collaborative initiative as no framework existed in the country. Arguably, since its establishment, ERRA was the main pillar of inter-agency collaboration and was on the forefront of relief and recovery operations and it continues to play a vital role in the reconstruction and rehabilitation processes. The INGOs contributions in earthquake were paramount from rescue to reconstruction phases. As disaster management is a complex process that requires preparedness and capability at all levels, the 2005 earthquake thus not only exposed the ineffectiveness of different levels of government to deal with mega disasters but it also opened up windows of opportunities for institutionalisation of disaster risk management (DRM) and disaster risk reduction (DRR) strategies in the country. The post-earthquake 2005 inter-agency collaboration transformed the disaster management system in Pakistan in mainstreaming the disaster management in development planning.The findings also shed light on the relevance of Bryson et al.  framewor"}
+{"text": "Chytridiomycota and Blastocladiomycota fungi, and two groups of fungal allies: Oomycota and Microsporidia. The Oomycota (water moulds) are considered as a model biological control agent of mosquito larvae. Due to their shared ecological and morphological similarities, they had long been considered a part of the fungal kingdom; however, phylogenetic studies have since placed this group within the Straminipila. The Microsporidia are parasites of economically-important insects, including grasshoppers, lady beetles, bumblebees, colorado potato beetles and honeybees. They have been found to display some fungal characteristics, and phylogenetic studies suggest that they are related to fungi, either as a basal branch or sister group. The Blastocladiomycota and Chytridiomycota, named the lower fungi, historically were described together; however, molecular phylogenetic and ultrastructural research has classified them in their own phylum. They are considered parasites of ants, and of the larval stages of black flies, mosquitoes and scale insects.The relationship between entomopathogenic fungi and their insect hosts is a classic example of the co-evolutionary arms race between pathogen and target host. The present review describes the entomopathogenic potential of  The interaction between host and parasite is characterised on the one hand by the parasites developing more effective strategies of host exploitation, and on the other, by the hosts mounting increasingly robust defences though Red Queen dynamics or coevolutionary arms races. Furthermore, depending on gene flow and differences in selection pressure between sites, both host and parasite may demonstrate local adaptation to their counterpart or develop more general resistance or offensive traits , belonging to the Straminipila.Historically, the Oomycota are a genetically and morphologically-diverse clade that can form hyphae or exist as simple holocarpic thalli; the phylum contains at least 1500 species of fungus in 100 genera , Leptolegnia , Pythium , Crypticola (C. clavulifera and C. entomophaga), Couchia , and Aphanomyces (A. laevis)  but develops well at 25\u00b0C and below ; these in turn multiply by binary fission or multiple fission, depending on the species, to form multinucleate plasmodial forms. Finally, these forms undergo sporogony, with the meronts developing into sporonts, which produce two or more sporoblasts; these in turn undergo metamorphosis into spores.The life-cycle of microsporidia is characterized by three phases: an infective or environmental phase (spores), a merogony (proliferative) phase and a sporogonic or spore-forming phase  and microsporidiosis in aquaculture ; Nosema bombycis has also been found to infect the domesticated silkworm Bombyx mori. In addition, some microsporidia act as opportunistic pathogens of humans, especially in patients with AIDS , is commonly used as a biological control agent for grasshoppers in the commercial products Nolo Bait and Semaspore  transmission; these affect only one generation of insects and are not usually host or tissue specific  and vitellogenin (Vg), and upregulates Jun-related antigen Jra and pi3k  gene was found to be upregulated in Nosema-tolerant honeybees  Rozella spp., the earliest diverging lineage in kingdom Fungi; (2) Olpidium brassicae, the only species classified in Zygomycota;, and (3) the core chytrid clade, encompassing the remaining orders and families and most flagellated fungi, including those of Chytridiales, Spizellomycetales p.p., Monoblepharidales and Neocallimastigales . That species is an obligate endoparasite of various ant hosts with a global or wide-ranging specific distribution  and beetles (Coleoptera), where they are mainly found in the lumen  and two mosquito generations. Infection is also known to cause significant epizootics which can persist in larval populations for several years, resulting in mortality rates greater than 50%, and often higher than 90% (Scholte et al. The species of Myiophagus were initially considered to belong to Chytridiomycota (Blackwell and Powell Blastocladiomycota (Humber Myiophagus requires free water for dissemination (Cole The fungi placed in a Humber . Speciesa Humber , scale ia Humber  resultina Humber . As Myioion Cole , infectiion Cole .Biological control is an effective and environmentally-acceptable alternative to chemical insect control methods. Entomopathogenic fungi and their metabolites are believed to represent potential alternatives to chemical pesticides. When used as biocontrol agents, entomopathogens might offer several advantages over conventional insecticides, such as high efficiency and selectivity, safety for beneficial organisms, reduction of residues in the environment, and increased biodiversity in human-managed ecosystems. However, relatively little is known of their effectiveness in field applications and the potential side effects of their use. It is also important to emphasise that due to the global distribution and variety of insects, many as yet unknown entomopathogenic fungi may exist; as such, there is a clear need for further comprehensive studies of the biology and ecology of entomopathogenic organisms and of the mechanisms underlying their action on insect hosts."}
+{"text": "The primary objective of this symposium is to describe our integrated, interprofessional behavioral health training program in substance use and opioid use disorders (SUD/OUD) across the adult lifespan (19 to 80) within our clinical psychology graduate program in the Deep South. Due to the COVID-19 pandemic, our assessment, treatment, and prevention delivery has occurred via telehealth. The first paper describes our Clinical Training Model in two federally qualified health centers  and one residential drug and alcohol rehabilitation program. Graduate and undergraduate students provide prevention, assessment, and treatment with an emphasis on 1) mindfulness-based relapse prevention, 2) literacy-adapted treatment for chronic pain, and 3) trauma and recovery. The second paper describes the participant population. Specifically, participants (N = 105) receiving prevention, assessment and treatment services report high levels of substance and opioid use and are underserved, impoverished, and have low levels of education and health/mental health literacy. The third paper explores the relation of age, adverse childhood experiences, and PTSD symptoms within the context of substantial or severe SUD/OUD. The final paper describes issues surrounding telehealth delivery in the rural south with underserved populations. The discussant, an expert in integrated, interprofessional telehealth delivery across the adult lifespan, will provide insight on program sustainability and dissemination. Given the pronounced need for SUD/OUD treatment in underserved populations with attention to the intersection of age and urban/rural residence, this project is poised to make a substantive impact across the adult lifespan."}
+{"text": "None of the currently used diagnostic tools are efficient enough in diagnosing Mycobacterium tuberculosis (M.tb) infection in children. The study was aimed to identify cytokine biosignatures characterizing active and latent tuberculosis (TB) in children. Using a multiplex bead-based technology, we analyzed the levels of 53 Th17-related cytokines and inflammatory mediators in sera from 216 BCG-vaccinated children diagnosed with active TB (TB) or latent TB (LTBI) as well as uninfected controls (HC). Children with active TB, compared to HC children, showed reduced serum levels of IL-17A, MMP-2, OPN, PTX-3, and markedly elevated concentrations of APRIL/TNFSF13. IL-21, sCD40L, MMP-2, and IL-8 were significantly differentially expressed in the comparisons between groups: (1) HC versus TB and LTBI (jointly), and (2) TB versus LTBI. The panel consisting of APRIL/TNFSF13, sCD30/TNFRSF8, IFN-\u03b12, IFN-\u03b3, IL-2, sIL-6R\u03b1, IL-8, IL-11, IL-29/IFN-\u03bb1, LIGHT/TNFSF14, MMP-1, MMP-2, MMP-3, osteocalcin, osteopontin, TSLP, and TWEAK/TNFSF12 possessed a discriminatory potential for the differentiation between TB and LTBI children. Serum-based host biosignatures carry the potential to aid the diagnosis of childhood M.tb infections. The proposed panels of markers allow distinguishing not only children infected with M.tb from uninfected individuals but also children with active TB from those with latent TB. Mycobacterium tuberculosis (M.tb) remains a global health emergency with 10 million new cases and 1.5 million deaths annually. As estimated, almost 30% of the world population is latently infected with M.tb, among whom in approximately 5\u201310% active TB disease develops. Children belong to the group of individuals highly susceptible to TB. Epidemiological data show that the risk of developing active TB in children ranges from 20% to 40% and is the highest among children below 5 years of age.Tuberculosis (TB) caused by M.tb exposure , and a Bio-Plex Human Inflammation Panel 1 assay , purchased from Bio-Rad . In brief, 50 \u00b5L of mixed beads was added to prewet wells and washed twice. After the addition of 50 \u00b5L of a standard, in-house control or a sample, the plate was incubated for one hour. After the subsequent washing, 25 \u00b5L of a detection antibody mixture was added to each well and the plate was incubated for 30 min and then washed. In the next step, 50 \u00b5L of streptavidin-PE was added, and after 10 min incubation and washing of the plate, the beads were finally re-suspended in 125 \u00b5L assay buffer. Assay readings were made using a Bio-Plex MAGPIXTM Multiplex Reader (Bio-Rad) with the Bio-Plex manager 5.0 software (Bio-Rad version 5.0). Standard samples were analyzed using a Five-Parameter Logistic (5PL) regression curve fitting (Bio-plex software). For each cytokine the standard curve ran from 3.2 to 10,000 pg/mL.http://www.graphpad.com accessed on 15 November 2020) and R Version 3.6.0 . Non-parametric tests were used to compare protein levels between the diagnostic groups: Mann\u2013Whitney U test for two-group comparisons and Kruskal\u2013Wallis tests for multiple groups. Categorical variables were compared using two-tailed chi-square tests. For basic differential expression, the limma package was used. The protein expression levels were quantile normalized prior to testing. In linear models, we considered the expression level to be the dependent variable and the group assignment to be the independent variable. We did not control for additional covariates in order to avoid overfitting. To increase the power of the study, we used orthogonal contrasts between the three groups with: the first contrast measuring the difference between HC and (jointly) TB and LTBI, whereas the second contrasts measuring the difference between TB and LTBI. The ROC analysis was performed with the aid of package pROC in R, with the 3-class AUC estimated via the function \u201cmulticlass.roc\u201d. The most informative sets of features (protein expression profiles) were detected in a logistic model of penalized regression (elastic-net) by means of 5-fold cross-validation. We fitted regression models separately for Th17-related cytokines and inflammatory mediators due to limited sample size. We consider only binomial  models as the sample size did not guarantee robust results. Hence, we considered three different (in theory independent) models: HC vs TB, HC vs LTBI and LTBI vs TB. For the TST, only one model for Th17-related cytokines and one for inflammatory mediators was fitted with the same methods as above used for identification of informative features. The elastic-net models were fitted with the aid of \u2018glmnet\u2019 package in R. The violin plots were made with the ggplot2 package in R.Statistical analysis was performed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA;"}
+{"text": "Extracellular vesicles (EV) are vesicular vesicles with phospholipid bilayer, which are present in biological fluids and extracellular microenvironment. Extracellular vesicles serve as pivotal mediators in intercellular communication by delivering lipids, proteins, and RNAs to the recipient cells. Different from extracellular vesicles derived from biofluids and that originate from cell culture, the tissue derived extracellular vesicles (Ti-EVs) send us more enriched and accurate information of tissue microenvironment. Notably, tissue derived extracellular vesicles directly participate in the crosstalk between numerous cell types within microenvironment. Current research mainly focused on the extracellular vesicles present in biological fluids and cell culture supernatant, yet the studies on tissue derived extracellular vesicles are increasing due to the tissue derived extracellular vesicles are promising agents to reflect the occurrence and development of human diseases more accurately. In this review, we aimed to clarify the characteristics of tissue derived extracellular vesicles, specify the isolation methods and the roles of tissue derived extracellular vesicles in various diseases, including tumors. Moreover, we summarized the advances and challenges of tissue derived extracellular vesicles research. Extracellular vesicles (EVs) are nano lipid bilayer-delimited particles released by a variety of cells . MicroveBefore being released, EVs can package multiple lipids, nucleic acids and proteins. Owing to the stable lipid membrane of EVs, these cargoes are prevented from degradation . When EVEVs widely exist in almost all types of body fluids, including blood, urine, cerebrospinal fluid (CSF), saliva and tears. Thus they are appropriate biomarkers for the early diagnosis and prognosis in clinical application due to these body fluids are relatively easily to collect . As repoex vivo tumors  diseases related to brain function degeneration, including Alzheimer\u2019s Disease (AD), Parkinson\u2019s Disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Huntington\u2019s Disease . AlzheimIn acute myocardial infarction (AMI), the reperfusion can induce irreversible myocardial injury, which is also known as myocardial ischemia-reperfusion (IR) injury . To datevia delivering the cardioprotective miRNAs into the heart. This result showed the BAT-sEVs are critical mediator in the interaction between BAT and cardiomyocyte (Adipose tissue (AT) has been regarded as an endocrine organ, which can secrete adipokines, such as proinflammatory cytokines, anti-inflammatory cytokines and metabolism regulatory cytokines . Adiposeomyocyte . Besidesomyocyte .via EVs, thus accelerating atherogenesis  and endothelial cells within the plaque are able to release EVs into the extracellular space. This finding provides a novel aspect of intercellular communication in plaque environment . In anotogenesis . Further of VSMC .In the inner ear, cochlea is a vital organ that responsible for the auditory signal transduction. Cochlea develops during the period of embryonic day 9 to postnatal day 21 . Both thCompared with EVs isolated from cell culture supernatant and body fluids, Ti-EVs may play a more crucial role in different sorts of diseases. Due to Ti-EVs are located in the tissue microenvironment, they have abundant cell crosstalk information and can authentically reflect the intercommunication between cells within tissue microenvironment. Moreover, the contents of Ti-EVs are relative pure because of its single tissue source. Based on these advantages, Ti-EVs have attracted increasing attention and have been studied a lot in basic research and clinical applications. Although some progress has been achieved, there are still some challenges and limitations needed to overcome. Firstly, Ti-EVs isolated by existing methods are often contaminated by EVs released from other broken cells. Therefore, it is urgent to develop optimal techniques to isolate and purify Ti-EVs. Furthermore, the tissue microenvironment contains diverse cell types, to elucidate the accurate original cell type of Ti-EVs will provide deep understanding for the intercellular communication within microenvironment. To further investigate the chemical, physical, and biological characteristics of Ti-EVs may help us distinguish Ti-EVs from \u201cimpurities\u201d. Secondly, some researches have demonstrated the potential of Ti-EVs to be diagnostic biomarkers, however, the isolation methods are too complex to apply in clinical practices and the exploration of Ti-EVs in the pathophysiological process of various diseases are still in the initial stage. How to balance the purity and convenience of Ti-EVs extraction is a major challenge in the future. Thirdly, considering the spatiotemporal specificity of tissue development, to illustrate the spatiotemporal profiles of Ti-EVs in all kinds of tissues will help us understand the development process deeply.in vivo (Although the widely clinical application of EVs is still difficult because of the problems in EVs preparation, transmission, targeting and retention, some achievements have been made. A variety of studies have shown diverse biomaterials for delivering EVs efficiently, including implantable scaffolds , injectain vivo .Without a doubt, a deeper comprehension of Ti-EVs biology and the development of standardized techniques for Ti-EVs quantification, extraction and storage, molecular characterization, and potency assays will greatly advance the prospects of Ti-EVs-based diagnostic and therapeutic applications in the future. Challenges do exist at present, but with the improvement of technology and development of research, these difficulties will eventually be overcome. The clinical application of Ti-EVs will gradually come true."}
+{"text": "Extracellular vesicles (EVs) have lately arisen as new metabolic players in energy homeostasis participating in intercellular communication at the local and distant levels. These nanosized lipid bilayer spheres, carrying bioactive molecular cargo, have somehow changed the paradigm of biomedical research not only as a non-classic cell secretion mechanism, but as a rich source of biomarkers and as useful drug-delivery vehicles. Although the research about the role of EVs on metabolism and its deregulation on obesity and associated pathologies lagged slightly behind other diseases, the knowledge about their function under normal and pathological homeostasis is rapidly increasing. In this review, we are focusing on the current research regarding adipose tissue shed extracellular vesicles including their characterization, size profile, and molecular cargo content comprising miRNAs and membrane and intra-vesicular proteins. Finally, we will focus on the functional aspects attributed to vesicles secreted not only by adipocytes, but also by other cells comprising adipose tissue, describing the evidence to date on the deleterious effects of extracellular vesicles released by obese adipose tissue both locally and at the distant level by interacting with other peripheral organs and even at the central level. Obesity has reached pandemic proportions worldwide and continues to increase at an alarming rate; specifically, the World Health Organisation (WHO) estimates that 39% of adults over the age of 18 are overweight or obese . Above aExtracellular vesicles (EVs) are small, rounded membrane spheres that can range in size from 30\u20131000 nm. Depending on their biogenesis and size, EVs can be microvesicles (100 nm\u20131 \u00b5m) derived from plasma membrane blebbing, exosomes (30\u2013100 nm) assembled into multivesicular endosomes (MVE) that are secreted by exocytosis, or large vesicles (50\u20135000 nm) that include apoptotic bodies released by cells preceding apoptosis ,10. InteIn adipose depots, adipocyte derived EVs, together with those liberated by endothelial, immune, and mesenchymal stem cells, play an important role in adipogenesis, targeting tissue formation, modulation of the immune microenvironment, adipokine release, as well as tissue remodeling . FurtherCurrently, the great challenge surrounding EVs research, apart from deciphering the exact functional role and characterizing their dynamic content, is to find a feasible, accurate, and reliable method to isolate these particles in a reproducible manner, allowing to obtain pure EVs, while also maintaining their functional characteristics , thus, aIn the context of obesity, several studies have characterized EVs isolated from primary cell cultures of adipocytes or adipose tissue stem cells by various techniques, ranging from in vitro cell cultures of mesenchymal cells differentiated into adipocytes, to those secreted by whole adipose tissue at different anatomical locations and under different metabolic status. The study by Connolly et al. characterized the EVs secreted during adipocyte differentiation (day 0 to 15 of differentiation) in the 3T3-L1 murine cell line, observing a decrease in the amount of particle secretion measured by NTA from day 0 to 15 of adipocyte differentiation without showing changes in particle size . This re7 particles/mL and measured approximately 115 nm. In contrast, adipocytes from the abdominal fat secreted around 7.60 \u00d7 107 particles/mL with an average size of 125 nm. Notably, both types of EVs were positive for endosomal markers such as TSG101 (tumor susceptibility gene 101) [On the other hand, the study by Akbar et al. isolated EVs from primary cultures of human adipocytes extracted from adipose tissue explants (0.5 g) from the gluteal and abdominal area of healthy donors by ultracentrifugation and filtration . In thisene 101) . In anotene 101) . The lEVene 101) . In thisene 101) .In contrast to previous studies, our group has characterized EVs released from human and murine whole adipose tissue considering the color type (white vs. brown), anatomical location , and metabolic status (obese vs. lean) ,30. The Of interest is the work of Chen and collaborators, in which they demonstrate that murine brown adipocytes also secrete exosomes whose production augments when simulating cold exposure and \u03b2-adrenergic stimulation induced by cAMP treatment ; this efIn summary, studies on the characterization of the size, nature, and concentration of EVs released from adipose tissue shows different profiles depending on the site of origin and on the metabolic statutes (obese and lean), finding a higher concentration of EVs in secretomes isolated from pathological adipocytes or visceral adipose tissue from patients with obesity compared with healthy adipocytes. This suggests that the populations and kinetics of secreted vesicles are highly dynamic being also modulated by the physiology of the cell of origin. Therefore, we can affirm that adipose tissue secretes vesicles of various sizes and different nature, and that their concentration depends on the type of tissue and nutritional status.EVs have been postulated as key factors in various physiological processes, such as development, tissue homeostasis, aging, metabolic regulation, circadian rhythms, lactation, and in pathological processes, such as in various non-infectious diseases  and infectious diseases  ,35. In tThe functionality of EVs is mainly determined by their cargo, whereby their membrane antigens and molecular cargo are crucial to exert a functional effect on a target cell/tissue. Thus, functional role of EVs is related to their ability to interact with target cells and release their content into these cells, which gives EVs an important part in cellular communication and targeted delivery as therapeutic vehicles . In addiMicroRNAs (miRNAs) are a group of short (20\u201324 nucleotides) single-stranded non-coding RNAs with the ability to regulate gene expression at the post-transcriptional level when bound to the mRNA of a target gene . miRNAs,MiRNAs bind to the RNA-induced silencing complex (RISC) and, subsequently, through RISC, they bind to the 3\u2019 untranslated regions (3\u2019 UTR) of the mRNAs leading to translation repression or mRNA degradation . InitialAt the paracrine level, a study by Zhang et al. showed an enhanced population of 45 miRNAs in exosomes secreted from whole rat adipose tissue compared to vesicles that were secreted by adipose tissue-derived stem cells . Of thesFurthermore, under the premise that EVs and their cargo molecules, in this case the miRNAs, play different roles at many levels of intracellular and interorgan crosstalk . ResearcA recent study by Thomou et al. shows that humans with lipodystrophy, as well as a fat-specific knockout of the miRNA processing enzyme Dicer (ADicerKO), have decreased levels of circulating exosomal miRNAs . They haMoreover, it was suggested that adipose tissue secretion of miR-200a loaded in exosomes could be the molecular mechanism underlying the adverse cardiovascular effect of rosiglitazone; thus, establishing a clear crosstalk between adipocytes and cardiomyocytes in mice . In thisRecently, at a different level, Yang Liu et al. have discovered how exosomal miRNAs are expressed in patients with hepatocellular carcinoma (HCC) and an elevated body fat ratio . In thisExosomal microRNAs derived from brown adipose tissue were also shown to be capable of influencing gene expression, specifically in the liver, as demonstrated by Kariba et al. by identifying exosomal miR-132-3p as an endocrine factor regulating hepatic lipogenesis for cold adaptation . FurtherThus, increasing findings are evidence that adipose tissue miRNAs can cause effects by regulating gene expression in different diseases by means of their transport in extracellular vesicles. Precisely, further research regarding the role of miRNAs traveling within EVs in obesity will allow the identification of therapeutic targets for the prevention and treatment of obesity and its comorbidities.As in the case of vesicular miRNAs, there is also great interest in obtaining a better understanding of adipose tissue-specific EVs protein content, and, more precisely, in relation to their anatomical location since there is a clear role of body fat distribution in the metabolic complications of obesity; thus, visceral adipose tissue being considered more deleterious . In addiKranendonk et al. published one of the earliest works describing the protein content of EVs isolated from human adipocytes of Simpson\u2013Golabi\u2013Behmel Syndrome (SGBS), which is a complex congenital overgrowth syndrome . These vIn a research work focused on the relationship of obesity with cancer, Lazar et al. performed a proteome analysis of the EVs released by the murine adipocyte cell line 3T3-F442A differentiated in vitro in which they identified 324 proteins of which the majority were related to lipid metabolism . NotablyOn the other hand, studies of whole adipose tissue explants were performed, such as that of Kranendonk et al., in which they observed characteristic adipose tissue proteins, such as adiponectin and FABP4, and changes in the proteomic composition of EVs isolated from human adipose tissue explants from thin donors according to their anatomical location, subcutaneous and omental, with a higher number of inflammation-related proteins in EVs from the omental depot . In addiIn recent years, our group has performed an extensive qualitative and quantitative proteome analysis of EVs shed by murine pathological adipocyte cell models that included lipid hypertrophy  and insulin resistant adipocytes, by whole explants of subcutaneous and visceral adipose tissues from obese patients, and from whole explants of white and brown tissues of obese and lean rats ,25,29,30In our previous work, common proteins identified in EVs from all adipose tissues or obese adipocytes included structural and cytoskeletal proteins such as annexins A1/A6, which modulate anti-inflammatory processes that may be dysregulated in obesity-associated DM2 (type 2 diabetes) , as wellTGFBI, has been described as a diabetes risk gene in both mice and humans [Furthermore, we have observed that EVs secreted by visceral fat depots of patients with obesity showed a greater variety of proteins compared to subcutaneous, and more interestingly, the functional classification of these proteins revealed that many were previously related to adipose tissue, obesity, and to the obesity-associated inflammation. This is the case for leptin , IL-6 , unlike EVs released from white fat ,96. ThusOn the other hand, other enzymes and proteins related to the insulin signaling pathway, and cytokines, identified in brown adipose tissue, secreted EVs, such as C3/C4, AOC3 (primary membrane amine oxidase), MIF1 (macrophage migration inhibitory factor), REEP5 (receptor enhancer protein 5 expression), PEPCK-C (phosphoenolpyruvate carboxykinase), TARG1 (regulator of GLUT4 trafficking 1), and Gyk (glycerol kinase), which were also identified in the secretome of murine and human brown adipose tissues . ProteinIn addition, EVs from brown deposits of normal-weight animals contained up-regulated proteins such as ACLY (ATP citrate synthetase), Rab 14, ACC (acetyl-CoA carboxylase), and the protein 2-enoyl thioester reductase, compared to EVs from obese brown deposits . ACLY isThe role of EVs, as implicated in the development of metabolic diseases, is still poorly understood . IncreasDifferent functional assays have been performed in which EVs released by adipose tissue interact with metabolic target cells or tissues involved in the dysregulation of obesity disease. Thus, we have described that EVs from adipocytes of the C3H10T1/2 cell line with lipid hypertrophy and insulin resistance induce and stimulate the differentiation and lipid hypertrophy of neighboring healthy adipocytes . This agUnder the above, we have also found that EVs secreted by C3H10T1/2 adipocytes with lipid hypertrophy induced inflammation of non-inflamed healthy macrophages . It is nOn the other hand, it has also been observed that EVs secreted by macrophages from both mouse and human obese adipose tissue suppress insulin secretion and increase \u03b2-cell proliferation . InteresIn the study by Zhao et al., they observed that small EVs (sEVs) secreted by mouse brown adipose tissue after exercise participate in exercise-associated cardio-protection through the delivery of miRNAs into cardiomyocytes, playing a cardioprotective role in the heart . In thisRegarding the association of obesity with a higher risk of developing cancer, several studies have shown that EVs isolated from adipocytes of human obese adipose tissue may be pro-oncogenic, promoting the proliferation of MCF7 tumor breast cells and other breast tumor cell lines within 24 h of treatment ,123. AddFinally, as brown adipose tissue EVs and their functional role is still an emerging field, it should be mentioned that the research by Zhou and collaborators shows that brown adipose tissue secreted exosomes that mitigated the metabolic syndrome in animals fed with a high fat diet . They shCurrently, extracellular vesicles are attracting exponential interest owing to their signaling role and as transporters of adipokines and biomarkers of the cell and tissue of origin, which dynamically reflets pathological deregulation; hence, they also provide new promising therapeutic options. Even though studies on the characterization and functional role of EVs released by adipose tissue took some time to develop, unlike other cellular systems, such as in the immune system or in diseases such as cancer, in recent years, the number of investigations regarding EVs released in the context of metabolic regulation has increased. Consequently, the potential role of EVs in adipose tissue communication at the local and peripheral/central levels has been demonstrated.In this review, we tried to assemble all the research performed to date in relation to the characterization of molecular cargo and the function of adipose tissue EVs, focusing on their modification under metabolic stress and/or in the context of obesity and its comorbidities. Hence, we can conclude that adipose tissue can also secrete adipokines independently of the classical pathway by packaging those into extracellular vesicles. Furthermore, as these vesicles are sophisticated structures, they can target their contents to specific locations thanks to their membrane antigens that may be cell- or tissue-specific. Thus, adipose tissue can send signals both at the autocrine/paracrine and endocrine levels, interacting with distant tissues able to alter the gene expression of target cells due to the microRNAs transported inside. The research performed to date has shown that obesity is characterized by increased secretion of EVs both locally and at the circulating level. In addition, it can be concluded that obese adipocytes release a greater number of EVs whose charge and molecular composition vary according to their anatomical location. Therefore, in relation to the protein load, the visceral deposits secrete a greater number of different vesicular proteins including those related to obesity and its comorbidities, unlike the EVs released by the subcutaneous adipose tissue. Additionally, it must be highlighted that specific exosomal miRNAs liberated by adipose tissue may be implicated in different local and systemic deleterious aspects associated with obesity, such as glucose tolerance, insulin resistance, inflammation, cardiovascular and fatty liver disease, and even participating or fueling tumor growth. Future goals will need to consider the possibility of using the functional effects of adipose tissue-EVs for therapeutical purposes, which may represent important curative options to treat obesity."}
+{"text": "Strong, trusted partnerships within a social capital framework are core to Peace Corps\u2019 successful international, national, and community interactions and outcomes. The Peace Corps\u2019 integrated three level model has thrived since its beginning in 1961. During this time, about 250 000 two-year Volunteers have lived and worked in 142 countries. In March 2020, the Peace Corps had to evacuate all 7 000 currently serving Volunteers because of the world-wide Covid-19 pandemic, a task organized and completed in nine days. The evacuation\u2019s success depended on the resiliency of these honored long-term partnerships, and specifically:a partnership model with three intertwining layers of collaboration between national and host country staff and Volunteers;years of respectful integration of Volunteers in local communities building mutual trust;in-country and cross-nation preparedness for health, safety, and security emergencies;transparent communication during the evacuation among all involved parties in every nation with Volunteers; andin-country and host country staff support across countries during the evacuation.This case study illustrates elements of effective and sustainable partnerships that ensure their effectiveness during a crisis and survival beyond the crisis.I write this at the anniversary of one year from my stepping down as the twentieth Director of the Peace Corps. During my tenure, I brought home all 7 000 currently serving Volunteers in nine days as the Covid-19 pandemic spread across countries. This massive \u2013 and successful \u2013 undertaking was possible because of long-standing and trusted partnerships between the Peace Corps and the communities and countries hosting Peace Corps Volunteers. This is my personal reflection and a reminder to build, honor, and tend relationships with international partners who are collaborators in learning, service, and research in good times and our friends, protectors, and allies in times of crisis. Sustainable partnerships between 142 countries and the United States over the 60-year history of the Peace Corps have created relationships resilient enough to withstand the total (temporary) withdrawal of all serving Volunteers that happened in March 2020. These partnerships supported the Volunteers returning to the United States, and are now facilitating host country staffs\u2019 continued in-country activities and will support the Volunteers\u2019 safe return to service beginning in 2022.Sustainable international partnerships are well-defined and include elements of reciprocal relationships and trust. For example, Davy says:The characteristics that are common to partnerships include voluntary engagement, mutually agreed objectives, distinct accountabilities and reciprocal obligations, and \u2018added value\u2019 to what each partner could achieve alone .Another definition suggests five key components of partnership implementation built and maintained over time:\u2026 (i) shared goals, (ii) relations with partners, (iii) capacity for partnership work, (iv) governance and leadership, and (v) trust and trustworthiness .In addition, commitment and conflict resolution are also important characteristics. Mohr and Spekman note:\u2026 [p]artnership characteristics include \u2026 attributes of commitment, coordination, and trust; communication quality and participation; and the conflict resolution technique of joint problem solving .Trust, in the case of successful international partnerships, is developed through intentional practices.Trust accrues from behaving in a trustworthy manner toward others but also requires consistently exuding trust in others. \u2026 ensuring that others have the freedom to present their concerns and that these concerns are heard and honored. \u2026 Trust builds when members of a network acknowledge the legitimacy of each other\u2019s goals even if they differ from one\u2019s own .Proposed structures for equitable partnerships in global health research provide insight into partnerships designed to be mutually beneficial on a number of levels. The elements include co-creation, understanding and compensating for inherent inequalities to enable all partners to fully participate and benefit from interactions; communication, mutual understanding and respect for cultural norms, including religious, cultural and societal boundaries; commitment, patience, building trust and long-term commitment; and continuous review process of review and consultation to develop and refine partnership research model .The above noted principles and frameworks for international partnerships are consistent with the founding goals of the Peace Corps that have been the basis of partnerships since the organization\u2019s inception. The three goals were written into the Peace Corps\u2019 original legislation, signed by President Kennedy in September 1961. These intertwining and equal goals areto help the people of interested countries in meeting their need for trained men and women;to help promote a better understanding of Americans on the part of the peoples served; andhttps://www.peacecorps.gov/about/).to help promote a better understanding of other peoples on the part of Americans  and national leaders of each country of service; between Peace Corps host country staff and their ministry counter-parts, local officials, and Peace Corps Volunteers; and between Peace Corps Volunteers and communities in which they live and work.First, country to country partnerships. Peace Corps has official government to government partnerships between the US and host governments, ensuring \u201cvoluntary engagement, mutually agreed objectives, distinct accountabilities and reciprocal obligations.\u201d [ations.\u201d  NegotiatOngoing informal discussions, as recommended by Zaman et al., ensure that each entity is meeting its interests and obligations, engendering trust, and that Volunteers are continuing to meet individual host country national interests . The couSecond, host country staff partnerships with their ministry counter-parts, local officials, and Volunteers. Peace Corps host country staff  are the middle layer of country partnerships. They negotiate with Ministry personnel to ensure relevant locally-implemented program development and implementation; manage Volunteer training; work with communities preparing projects for incoming Volunteers; and support Volunteers throughout their two-year tours. Host country staff work in both local and national languages when discussing Volunteer projects. Volunteers turn to, and trust, host staff for health and safety emergencies, personal issues, and project technical guidance demonstrating that these are partnerships built on mutual respect and trust.Third, Volunteer partnerships with local communities. Each Volunteer and community is a mini-partnership based on the trust and respect embodied in the national agreements. Individual Volunteers\u2019 living with host families, sharing meals, engaging in family activities, and developing language and detailed cultural knowledge establish this core element of sustainable partnerships. Volunteers\u2019 project goals, whether in health, education, or other technical areas of service, depend on community involvement in order to build, as noted by Mohr above, \u201cattributes of commitment, coordination, and trust.\u201d [ trust.\u201d  As an exThese personal and professional relationships continue after Volunteers return to the US and share what they learned from the families and communities in which they lived and worked. I have heard host country Ambassadors to the US say at Peace Corps Embassy events, \u201creturned Volunteers are better ambassadors to the US for my country than I am.\u201dPeace Corps has worked across the three-layer partnership model described above to strengthen a sustainable framework for Volunteer service. To do this, Peace Corps has developed two community integrative models and a safety and security acceptance model. The first community integrative model, Participatory Analysis for Community Action (PACA) , guides PACA. The PACA model has been tested and revised by Peace Corps for over 20 years with full participation at each country\u2019s national, regional, and local level. Volunteers use the PACA approach to engage in capacity building initiatives driven by community needs. The approach empowers community members to be their own decision makers, develop the skills needed to carry out those decisions, and take the lead in improving their own lives [wn lives . Using twn lives .Through the use of PACA, Volunteers develop relationships through trust, integrating into the community, and bringing different community members together (co-creation). They gather information and gain key insights by observing, learning from, and engaging community members (communication). Together they make sense of observations, insights, and discoveries to generate or improve project ideas (commitment), and they test, refine, and continue to improve project ideas (continuous review).Logic Project Framework or LPF. As noted earlier, partnership roles need to be clarified, boundaries drawn, added value defined, and outcomes measured. Co-created with community members at the start of a Volunteer\u2019s service, the LPF outlines the project situation, inputs, outputs, outcomes and impact. These components are based on the local community counterpart(s) and Volunteer jointly gathering data which is uploaded quarterly, reviewed by both Volunteers and their community based local counterpart who help design the project, the in-country Peace Corps staff, and Peace Corps Headquarters in Washington, DC. The LPF enables collaborative discussions for clarifying roles, boundaries, value, and outcomes.Core to sustainable PACA integration and LPF project development and measurement is Volunteers\u2019 living with host families and sharing meals, family activities, language, and daily cultural knowledge. As suggested by Mohr above, important partnership characteristics include \u201cattributes of commitment, coordination, and trust.\u201d  Peace CoWhat Volunteers bring home\u2014new skills and insights into community public health, literacy, education, the environment, public service\u2014are learned through their interactions in communities culturally, linguistically, and traditionally different from their pre-Peace Corps experiences. Even as PACA and the Logic Framework measure impacts of the partnerships, individual stories remain strong indicators of success.The Acceptance Model for Safety and Security. To ensure Volunteer safety and security, Volunteers are trained in emergency procedures during pre-service training and supported during their two years in-country. The Peace Corps\u2019 integrated three-layer partnership approach is essential in assuring Volunteers, host families, and community counter-parts are integrated into these safety and security systems. The Peace Corps model is based on building trusting close relationships to keep Volunteers safe.For example, Volunteers are provided a phone pre-populated with emergency information, and then tested in emergency safety and health procedures every few months. As community and national needs and assets change, Peace Corps\u2019 emergency plans change accordingly. Plans are adapted to local environments such as Volunteer distance from the capital city, modes of transportation, and village isolation. These plans are shared and discussed  regularly with local and national host officials to ensure full partner participation. Even the local police chief and the health clinic health official are integral to Volunteers\u2019 health and safety .Peace Corps\u2019 key messages for Volunteer recruitment, host-country and American staff training, and host-country program and project development embody model practices for effective international partnership and are consistent with Peace Corps\u2019 own 60-year partnership framework. The importance of this approach was shared with me when the Acting Peace Corps Director addressed all living former Peace Corps Directors (myself included) in 2021:The Peace Corps is an international network of Volunteers, community members, host country partners, and staff who each have a unique service journey, defined by an enduring desire to learn and grow.The Peace Corps\u2019 approach, grounded in mutual respect and collaboration, is a catalyst for grassroots development.At the invitation of governments around the world, Peace Corps Volunteers work alongside community members on locally- prioritized projects that build relationships, promote knowledge exchange, and make a lasting and measurable impact .In early spring 2020, the Covid-19 pandemic\u2019s meteoric spread across countries required global agencies and organizations to immediately evacuate staff and participants or take other health safety measures to protect citizens while reducing the disease\u2019s further spread. The Peace Corps had 7 000 Volunteers in communities in 61 countries in March 2020, when it ordered all Volunteers to return to the United States to protect both Volunteer health and the health of citizens in countries in which they were serving. A full Peace Corps evacuation had never happened before in its 60-year history.At midnight on March 15, 2020, as Peace Corps Director, I sent out a communication to all Volunteers and host country staff. Included in the communication were the following statements:I have made the difficult decision to evacuate all of our Volunteers globally. As Covid-19 continues to spread and international travel becomes more challenging by the day, we are acting now to safeguard your well-being and prevent a situation where Volunteers are unable to leave their host countries.I also want to assure you and our host country partners that these evacuations represent the temporary suspension of Volunteer activities. We are not closing posts, and we will be ready to return to normal operations when conditions permit .Undergirding my communique was Mohr et al.\u2019s notion that \u201ccommitment, coordination, and trust; communication quality and participation; and conflict resolution technique of joint problem solving\u201d . would eWhen the March 15 email was sent, everyone knew what to do based on extensive practiced procedures. They followed protocols locally prepared, regularly updated, and successfully improvised based on that country\u2019s assets at the time.Within three hours of the initial communication, the staff teams on the ground were in touch with country national, state, and local officials. Similar communications were sent to Embassy officials. Host country staff personally explained the decision to counterparts at all levels of government. Foreign affairs officials, national and regional safety and security officials, and community leaders were critical to the support for Volunteers as they said goodbye and moved across the country to their international flights. National leaders and ministry officials had trust that leaving was related to a global health threat and that their leadership support would ensure long term partnership in the future. As stated by Pennink, \u201cTrust is increasingly seen as important for long-term interactions of high interdependence \u2026 trust can act as a governing mechanism in situations of high risk.\u201d To move seven thousand Volunteers from small villages, to capital cities, airports, international and domestic flights and quarantine rooms required support and collaboration from those in communities everywhere in the world. Individuals offered help\u2014quickly organized goodbye ceremonies for students and families, seats on buses, extra packets of food, free hostel rooms for the night, rides to train stations, airlines scheduled adjustments for Volunteers, and safety support by ministry officials. The core of the evacuation\u2019s success was thousands of willing hands given with care and trust. With country level support and care, all Volunteers returned to the US within nine days with no one getting lost, injured, or sick.Volunteers, host country staff, and national officials knew what to do in an emergency. For example, host staff members organized hundreds of tasks to make the evacuation logistically successful and personally heart felt. They cared about Volunteers and communities with each logistical step they took, even as they coped with their own family Covid-19 emergencies. Peace Corps headquarters assured host country staff and national leaders that Peace Corps in-country offices and all staff would continue until Volunteers returned. Peace Corps continued to honor the country agreements.The evacuation success came directly from the Peace Corps\u2019 years of sustainable international partnerships across all regions of the world. Nearly two years later, these commitments have continued even as Volunteers have not been able to return.During the evacuation and immediately afterward, many staff and Volunteers wrote about their experiences during the evacuation illustrating the themes of trust. Below are three short examples, one from a Volunteer, and two from Peace Corps Country Directors supervising the in-country efforts.I firmly believe that one-to-one relationships built at a grassroots level between people who are fundamentally different is the best pathway to world peace. But I forgot how much it hurts to leave your friends.\u201d The farthest part of the country is normally a 40-hour bus ride \u2026 And of course, because it\u2019s Mongolia, there was a blizzard. \u2026 My communities said as Volunteers left, \u2018We want the Volunteers back as soon as possible\u2019. (CD in Mongolia)WorldView, NPCA, 2020)We move PCVs in six different buses, with police and regional security officer escort since it is now illegal to gather more than 10 people because of Covid-19. At Kyiv airport, there\u2019s a technical issue: They can\u2019t issue or print boarding passes. So, airport staff write them all out by hand.\u201d (CD in Ukraine)  make possible the revised training and programming requests countries are making and will make in the future to address current community and country needs.Even without Volunteers in the field, Peace Corps\u2019 strong international partnerships continue. All in-country Peace Corps offices have remained open and fully staffed, with staff working at home or in offices depending on country specific Covid-19 situations. Host country staff, local counterparts, and national officials facilitate daily and weekly in-country communication with national ministries and communities. These are matched with the communication to Peace Corps headquarters. This ongoing activity in the face of uncertainty depends on trust that Volunteers will return and trust that when Volunteers return, they will be ready to meet the new challenges created by Covid-19 in the health, economic, and social sectors of the countries and communities where they will work.I have been shaped by my own Peace Corps service 55 years ago which included time with a Tunisian host family who became my immediate family and students who became my extended family. My personal experiences have been replicated thousands of times over the years by other Peace Corps Volunteers bound together as host family and a Volunteer, head master and Volunteer, or health clinic nurse and Volunteer \u2013 all sharing humility and respect for each other as they work to achieve common goals. The Volunteer\u2019s statement earlier, \u201cbut I forgot how much it hurts to leave your friends,\u201d reflects the depth of personal trust developed over long-term, intentional immersion experiences. The example of airport staffers writing all the tickets out by hand shows the breadth of partnership commitment, even in one small unanticipated action. It reflects years of commitment, patience, and trust. As Peace Corps continues its planned return, it will rely on the collaboration within the three-layered partnership model. This will enable Volunteers to respond to community-defined needs and interests resulting from Covid-19\u2019s world-wide impact on health, education, economic, and culture changes."}
+{"text": "Metallic deposition is a commonly observed phenomenon on the surface of revised femoral heads in total hip arthroplasty and can lead to increased wear due to third bodies. In order to find out the origin and composition of the transfer material, 98 retrieved femoral heads of different materials were examined with regard to the cause of revision, localization, pattern and composition of the transfer material by energy dispersive X-ray spectroscopy. We found that in 53.1%, the deposition was mostly in the region of the equator and the adjacent pole of the femoral heads. The most common cause for revision of heads with metallic deposition was polyethylene wear (43.9%). Random stripes (44.9%), random patches (41.8%) and solid patches (35.7%) were most prevalent on retrieved femoral heads. Random patches were a typical pattern in ceramic-on-ceramic bearing couples. The solid patch frequently occurred in association with dislocation of the femoral head (55%). The elemental analysis of the depositions showed a variety of different materials. In most cases, titanium was an element of the transferred material (76.5%). In addition to metallic components, several non-metallic components were also detected, such as carbon (49%) or sulfur (4.1%). Many of the determined elements could be assigned with regard to their origin with the help of the associated revision cause. Since the depositions lead to an introduction of third-body particles and thus to increased wear, the depositions on the bearing surfaces should be avoided in any case. Nowadays, aseptic loosening due to wear particles is still the main cause for total hip revisions ,2. NumerSubsequently, the metallic deposit on the surfaces of the bearing couples leads to a change in the wetting behavior  and an iDifferent motion sequences result in different stress distributions in the hip joint . This ca2 up to 850 mm2 of the transfer with a maximum height of 30 \u00b5m were determined. In some studies, energy dispersive X-ray (EDX) analysis in combination with scanning electron microscopy (SEM) was used to determine the composition of the metallic deposit on the femoral heads. In most cases, CoCr or titanium alloys were identified as transferred material. However, EDX has been performed only on small numbers of samples and only on ceramic heads, often searching for explicit materials [The amount of total wear correlates with the extent of the deposited area ,9,26,27.In numerous studies, retrieved femoral heads are examined with regard to their abrasion and wear behavior. Thereby, the metallic deposition was often documented as well. However, a detailed examination of the transferred material mostly did not follow ,29,30. SThe workflow and methods used are summarized in For this study, retrieved femoral heads of various designs with metallic deposition localized on the surface were selected from the retrieval archive of our hospital. Patients signed a consent statement for the safekeeping and examination of the explants. The data protection regulations were observed. The study was approved by the local Ethics Committee (registration number A 2019-0103). The selected 98 heads consisted of five different materials. The numbers of heads per material group are listed in The patient data collected in reference to the selected explants were anonymized. Since many of the collected samples were not implanted at our Orthopedic Hospital, the implantation period is mostly unknown and was therefore not investigated in this study. The type of bearing materials as well as the cause for revision were considered for the evaluation of the results. After revision, all implant systems were sanitized at 95 \u00b0C. Furthermore, all femoral heads were cleaned in deionized water in an ultrasonic bath  and dried with nitrogen to remove loose wear debris before examination.An experienced and board-certified orthopedic surgeon, specially trained and approved in total joint arthroplasty at a center of excellence (Endocert-Certification Germany), was consulted for all subsequent examinations.The deposition patterns were classified according to the study of Fredette et al. . The patIn order to standardize the mapping of the localization of the metallic contamination, a grid was drawn on the acquired images of the analyzed femoral heads. The grid was used to divide the head surface into the areas pole, equator and near taper. An example image of the adopted segmentation is shown in During a revision, the implants are removed, cleaned and stored in single parts (unassembled) in the retrieval archive. The position in which the femoral head was mounted on the femoral stem was not documented. Therefore, an exact determination of the contact surface cannot be made for most heads.\u00ae VP Compact, Co., Zeiss, Oberkochen, Germany) equipped with a detector (XFlash 6/30) for EDX spectroscopy and analysis software . Representative areas of the samples were analyzed and mapped to determine the elemental distribution on basis of the EDX-spectra data by the QUANTAX ESPRIT Microanalysis software (version 2.0).To analyze the metallic coating, all femoral heads were examined a by field emission scanning electron microscope . For the selected femoral heads, the absolute frequencies of the transfer materials, the deposition patterns and the cause for revision were analyzed. Cross tables with Chi-square test and Fisher\u2019s exact test (two-sided) were used to determine associations in deposited material and deposition pattern due to the cause of revision or the localization of the depositions on the femoral head. p-values of <0.05 were considered significant.Statistical analysis was performed using the IBMIn order to determine the cause of the revision, revised total hip implant systems (if available) were also examined in addition to the retrieved heads see .The metallic deposition can also be found in part in the associated metallic or ceramic bearing surfaces see . HoweverThe frequencies of the cause for revision of the retrieved heads are listed in p = 0.022). A further significant correlation of the pattern or the localization of the deposition with the different bearing partners could not be found. In total, 11.2% of the heads showed global patterns distributed over the entire head.Random stripes, random patches and solid patches were most prevalent on retrieved femoral heads see . The ranThe metallic deposition occurred most frequently in the area of the equator. In 53.1% of the heads, metallic deposits were found in the equator and pole regions without contamination of the taper-near area. However, there was no case in this study in which metallic contamination occurred exclusively in the pole area. A total of 40.8% of the heads had no metallic depositions in the pole area at all. The most prevalent deposition patterns on the pole area were random stripes (12.2%) and longitudinal stripes (10.2%). In the equatorial area, the three most recurrent deposition patterns were solid patches (14.3%), random stripes (13.3%) and a combination of solid patches and longitudinal stripes (8.2%). In the near taper area, the majority of heads also showed no deposition (44.9%). The most prevalent patterns of deposition were random patches and a combination of random stripes and random patches .In a statistical comparison, Fisher\u2019s exact test (two-sided) was used to analyze whether there was an association between the metallic patterns and the cause for revision. It was found that a solid patch was detected in more than half (55%) of the femoral heads that were retrieved due to dislocation and thus a direct association cannot be excluded (p = 0.028). It was also shown that in case of dislocation, there were no random patches in 80% of the cases (p = 0.041) and no patterned coverage in 70% of the cases (p = 0.008).When solid patches were present, they were always localized in the equator area (11.4% of heads with solid patches) or in the equator and adjacent area (88.6% of heads with solid patches). In nearly half of the femoral heads with solid patches, it occurred in the equator and pole region (p = 0.087 in each case). While in both cases all heads showed random stripes, the total number of heads for these specific causes was limited with only three retrieved heads each. This makes it difficult to draw a conclusion from the results.A possible association exists for the occurrence of random stripes in gluteal insufficiency as well as implant fracture, which may include a fracture of the acetabular cup as well as a fracture of the surrounding bone cement .Many elements, often including carbon and silicon were only detected as background noise and could not be directly defined as a deposition material. It was hypothesized that this was background noise caused by remaining material from previous revision surgeries. However, this hypothesis could not be confirmed statistically (p = 0.035). All heads (n = 3) which had to be revised due to implant fracture had depositions of Fe on the head surface (p = 0.039). Five out of seven heads with deposition involving oxygen were retrieved due to particle disease (p = 0.002). Out of four heads with sulfurous deposit, three had septic loosening as the cause for revision (p = 0.004). It should be mentioned here that all three cases were bearing couples of alumina-on-PE. In the first case, the bacterial strains Finegoldia magna, Bacillus cereus, Corynebacterium species, Staphylococcus aureus and Neisseria species were detected. The second case infection was again caused by Staphylococcus aureus, while in the third case Enterococcus faecalis was the causative pathogen. For the revision cause septic (without loosening), eight out of nine heads showed carbon deposition (p = 0.015). One out of three heads with revision due to bone fracture (including trochanteric major contact) showed deposition of calcium (p = 0.031).In order to determine the origin of the transferred and deposited materials, it was verified whether there were statistical associations between the transfer materials and the cause for revision of the retrievals. The detailed analyses showed that some of the elements that were detected by EDX occurred more often after specific causes of revision. Only the significant associations in Chi-square test between certain elements and specific causes are described in detail. Of 34 femoral heads with depositions including Fe, 24 heads were retrieved due to PE wear , but may also contain components of ZrO2, BaSO4 (barium sulfate), Na, P, Ca, Si and Cl. The elements Na, P, Ca and Cl are also components of the blood plasma and can diffuse into the bone cement over time [Since the articulating joint is surrounded by a joint capsule, it is likely that the transferred material originates from wear particles of the implant as well as the bone and the surrounding tissue. The main components of metallic acetabular cups and stems are Ti alloys (mainly Ti-6Al-4V) and CoCrMo alloys. Thereby, Ti alloys are the softer material and are often associated with tissue discoloration and the release of wear particles . Additioten used . Wear paver time . Wear paver time ,39,40. OStaphylococcus aureus and Enterococcus faecalis are among the most common pathogens in septic revisions [S. aureus can use hydrogen sulfide to protect against cationizing molecules from antibiotics [E. faecalis is also known to produce sulfur-containing radicals to reduce oxidative stress [In the case of septic loosening, a significant occurrence of sulfur was found in the transferred materials on the femoral heads. A possible source could be in the biofilm formed by colonizing bacteria . The bacevisions . Some stibiotics ,44,45. Ee stress . Alternae stress . In septe stress . Carbon n = 28), but only comparatively few coated metallic heads (n = 6). A critical limiting factor is the patient data. Because the primary surgeries often took place in other clinics, the implantation period and some other data could not be collected and thus could not be included in the correlation. After revision, specimens must be initially stored for one year before examination, as the patient is allowed to recall the retrieval within this time. During storage, additional metallic deposition by the enclosed implant system cannot be excluded. Therefore, heads with very slight deposition were excluded in the sample acquisition. In addition, only single elements could be detected by EDX analysis. The advantage of the EDX analysis is the non-destructive measurement. Nevertheless, a detailed investigation of the chemical composition is desirable. Furthermore, it is assumed that the metallic deposit is formed layer by layer on the surface. Therefore, an erosive analysis to investigate the individual layers of the metallic deposition would be of interest.The limitations of the study are summarized below. Due to the high variability of head materials and deposition forms, the group sizes differ remarkably. For example, a very large number of heads made of alumina were examined (Material deposits are not only found on femoral ceramic heads. They can also occur on metallic heads and on the surface of the rest of the revised implants parts, such as acetabular cups or hip tapers. Our study showed that titanium is the most commonly transferred material. However, the deposition materials were not limited to metallic elements, and non-metallic materials such as carbon or sulfur were also transferred. Some possible sources of the deposition materials could be identified; for example, random patterns due to implant system contact in unstable joint situations. It was further shown that the deposition pattern solid patch results from dislocations of the femoral head. Random patches were identified as a typical pattern on ceramic-on-ceramic bearing couples. In general, depositions on bearing surfaces should be avoided, as they lead to an introduction of third-body particles and increased wear. Further long-term studies, with complete information on the implantation period and photo documentation during revision, are recommended. An examination of the metallic deposition with XPS to determine the complete elemental composition can provide more precise information on the origin of the deposition. In order to better comprehend the origin and formation of the metallic deposition and to better assess its influence on abrasive wear, the use of simulation models would be a promising option . Investi"}
+{"text": "Previous studies claimed that, apart from other side effects, these anti-ulcerant therapies significantly altered bone mineral density by interfering with intestinal reabsorption of minerals and vitamin B12, and the most widely prescribed PPIs were significantly associated with increased risks of hip and spine fractures. However, the potential skeletal side effects of these antiulcerants are unknown in Bangladesh.Proton pump inhibitors (PPIs) and HTo examine safety concerns of anti-ulcer therapies and their impact on musculoskeletal health among patients in Bangladesh, the present work surveyed 200 patients in five different hospitals from December 2019 to February 2020.The current study revealed that most respondents (95\u00a0%) received PPIs for gastrointestinal indications while the rest were taking H2 receptor antagonists for their gastric ailments. Most patients taking PPIs alone  claimed some unusual musculoskeletal side effects, such as weakness, flank pain, spasm of hands and feet, muscle aches, numbness, and tremor. About 61\u00a0% of patients taking PPIs experienced low back pain whereas the respondents with neck pain and knee joint pain were 10\u00a0% and 7\u00a0%, respectively. However, few osteopenia and osteoporotic incidences have been also recorded. Although further studies are required to confirm the impact of these antiulcerants on the bone, these patient responses suggest that these musculoskeletal side effects might have some links with altered bone metabolism.It is possible that anti-ulcerant therapies may worsen the bone metabolism of patients suffering from osteoporosis or other bone disorders, and awareness and precautions should be raised among the patients and clinicians for the careful administration of PPIs to patients suffering from bone disorders.  \u2022Anti-ulcerant therapies can interfere with intestinal reabsorption of minerals and vitamin B12.\u2022PPIs are associated with increased risks of hip and spine fractures.\u2022Patients taking PPIs  claimed unusual musculoskeletal side effects.\u2022Anti-ulcerant therapies may worsen the bone metabolism of patients suffering from osteoporosis or other bone disorders.\u2022Awareness should be raised among the patients and clinicians for the careful administration of anti-ulcerant therapies. This study also evaluated some factors such as the family history of fracture incidence, some physical conditions as well as risk factors, diseases and medications that may have undesirable repercussions on bone health.22.1This survey was carried out among Bangladesh patients taking two common anti-ulcerant therapies for a long term (at least three years) so to investigate the present consumption, health-hazardous patterns and their potential musculoskeletal side effects. Depending on the easy accessibility and easy availability of participants, the respondents were chosen from five different hospitals in Chattogram, Bangladesh  between December 2019 and February 2020. A simple random sampling technique was used for the selection of study participants. Besides, to ensure the respondent's least knowledge about the medications and their effects, this current study only considered respondents who have completed at least their higher secondary education (HSC). A total of 200 participants were selected, including 130 male participants and 70 female participants. After having their consent with the proper explanation of the research objectives and assurance of the confidentiality of their participation, the pre-designed questionnaires were explained to participants. The study protocol was approved by Noakhali Science and Technology University, Bangladesh.2.2A face-to-face interview was conducted by the field researchers. During the interview in person, the interviewers translated the questionnaire from English to Bengali to make the questions easily understandable to the respondents. Interviewers clarified any doubts that participants had and explained the symptoms and medical terms in their familiar names and terms. The interview lasted 45\u201360\u00a0min and included a range of questions about age, education, income and health status of respondents, history of diagnosis and treatment protocols for gastrointestinal disease, other drug information and complications  they experienced during the therapy.2.3All data obtained were coded into a Microsoft Excel spreadsheet and categorized into groups according to their background. The data were summarized as counts (or percentages) and represented as percentage\u00a0\u00b1\u00a0SEM (standard error of the mean), where possible descriptive statistics  were computed by using SPSS version 26 . P\u00a0<\u00a00.05 was considered statistically significant.33.1The current survey was conducted among individuals who had been receiving frequent antiulcerants at least for the last 3 years from the date they were surveyed. As 3.23.3The methods of diagnosis for different gastrointestinal (GI) problems in respondents are shown in 3.4As shown in It is evident from the survey that, among all PPI users (190), most respondents (67\u00a0%) received omeprazole, with the second major response being recorded for esomeprazole, and very few respondents were taking rabeprazole and pantoprazole-based treatments . This st3.5s are statistically significant (p\u00a0<\u00a00.05) during their subgroup comparison except for ranitidine and omeprazole groups.3.6During this survey, ranitidine (only H2-blocker recorded) users were not reported with any musculoskeletal side effects. Besides these musculoskeletal side effects, PPI users also had some localized bone pains. 3.73.8Besides, the GI disorders, respondents also had some skeletal and other problems within the period of taking anti-ulcerants for GI problems. About 15\u00a0% of the respondents were suffering from osteopenia while only six respondents were diagnosed with serious problems in bone mineral density (osteoporosis) (within the last three years of taking PPIs treatment) . Meanwhi3.9Furthermore, while comparing the responses about the paternal history of fracture risk, the highest number of respondents said about femur fractures while the history of shoulder and hip fractures have taken 21\u00a0% and 15\u00a0% scores respectively.3.104This prospective study examined the relationships between acid-suppressive medication use and the incidence of musculoskeletal side effects among 200 respondents from five different hospitals in Bangladesh. In a country like Bangladesh, people are still suffering from mineral and vitamin deficiencies During this survey, respondents have chosen between PPIs and H2-blockers users for several GI problems where most of them were PPIs users (95\u00a0%). This large usage difference between these two antiulcerants might be due to the good treatment success with greater inhibition of gastric acid secretion properties of PPIs at low doses while safety concerns for the H2-blocker use in some cases The study computed several influencing factors such as age, gender, BMI, dietary intake, cigarette smoking and alcohol status and a few other parameters, but we didn\u2019t observe any significant differences between these parameters that can affect the study outcomes . After c12 deficiencies  ion exchanger and lead to a profound inhibition of gastric acid secretion, thus, impairing the number of minerals and vitamins absorption while also can dysregulate the hormonal functions Numerous epidemiological and cohort studies have provided evidence of an association between long-term PPI use and increased musculoskeletal abnormalities among users 2+, Ca2+ and other minerals absorption The possible mechanism is PPIs administration can reduce the epithelial acidification and thereby increase the colonial pH and fate to the reduction of MgNowadays, proton-pump inhibitor-induced hypomagnesemia is one of the most recognized side effects of PPIs and the situation becomes worst when these drugs are given to the patients for a longer duration with frequent dosing. A list of previous cases demonstrated that long-term PPIs medications were linked to hypomagnesemia whereas the situation was resolved by stopping PPIs therapy and repeated by reintroducing PPI therapy 12 but have no action on crystalline VB12 absorption in-vitro and in-vivo studies validate the importance of normal regulation and function of vitamin B-12 in bone health in-vitro study showed that vitamin B-12 is important for the expression and function of alkaline phosphatase (ALP) in osteoblast proliferation and bone mineralization 12 supplementation In many previous studies, proton pump inhibitors (PPIs) have proven their negative role in vitamin B-12 absorption Furthermore, several previous publications revealed that individuals with a familiar history of bone fractures are considered the risk group for experiencing fractures in future In summary, the current study data suggest that long-term PPI treatment has some linkage to musculoskeletal problems. With the current findings, it is evident that anti-ulcerant therapies might develop skeletal problems in long-term usage while may also delaying the healing of osteoporosis or fractures in patients. Thus, awareness and precautions should be raised among the patients and clinicians for the careful administration of PPIs to individuals, especially to patients already suffering from bone disorders and family history of fractures. It is difficult to find a treatment/medication that would bypass the musculoskeletal side effects of PPIs, as there are no such guidelines in the literature. However, patients should avoid long-term or unprescribed use of PPIs, and should monitor their bone health if they need to take it for longer than usual. If a patient needs additional medication or supplements to maintain their musculoskeletal health, they should contact a doctor. However, the dietary uptake rich in essential minerals, phosphates and vitamins could be helpful in this aspect.5The present data only covered 200 respondents from five different hospitals in Bangladesh. So, the current data seems not enough to give the whole scenario of musculoskeletal problems of all antiulcerants users in Bangladesh. So, a cohort study in a large population size with a more descriptive questionnaire should be conducted to have a complete scenario of the risk between the antiulcerants users and probable complications to musculoskeletal systems.6PPIs are relatively safe and less toxic in comparison to other antiulcerants but the risk of osteoporosis and fracture in long-term administration should not be overlooked. Thus, suggestions from the current study are consciousness should be created among the participants and clinicians in the hospital so that the appropriate prescription of PPIs will improve patient satisfaction while the incidence of osteoporosis and fractures can be avoided. The current study represents the outcomes from a small group population hence a multi-centered investigation and clinical studies are recommended to obtain more conclusive and valid results that would better reflect the usage and health-hazardous patterns of anti-ulcerant therapy in Bangladesh.All authors meticulously reviewed the draft and gave their consent to publish this article.Consent was taken from all the study participants before the interview. Information taken from the participants was preserved confidentially.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."}
+{"text": "ACE2 exhibits a high degree of genetic polymorphism that may affect its structure and stability, interfering with these cardioprotective actions. The aim of this study was to analyse the relationship of ACE2 polymorphisms with cardiovascular risk factors in children.In the cardiovascular (CV) system, overactivation of the angiotensin converting enzyme (ACE) may trigger deleterious responses derived from angiotensin (Ang)-II, which can be attenuated by stimulation of ACE2 and subsequent Ang-(1-7) metabolite. However, ACE2-single nucleotide polymorphisms (SNP), rs4646188, rs2158083, rs233575, rs879922, and rs2074192, previously related to CV risk factors, were analyzed in a representative sample of 12\u201316-year-old children and tested for their potential association with anthropometric parameters, insulin levels and the lipid profile.Five N = 461) exhibited lower rates of overweight, obesity, blood pressure, and glycemia than boys (N = 412), though increased plasma lipids. The triglycerides (TG)/HDL-C ratio was, however, lower in females. Interestingly, only in girls, the occurrence of overweight/obesity was associated with the SNPs rs879922 [OR 1.67 (1.02\u20132.75)], rs233575 [OR 1.98 (1.21- 3.22)] and rs2158083 [OR 1.67 (1.04\u20132.68)]. Also, TG levels were linked to the rs879922, rs233575, and rs2158083 SNPs, and the TG/HDL-C ratio was associated with rs879922 and rs233575. Levels of TC and LDL-C were associated with rs2074192 and rs2158083. Furthermore, the established cut-off level for TG \u2265 90 mg/dL was related to rs879922 [OR 1.78 (1.06\u20132.96)], rs2158083 [OR 1.75 (1.08\u20132.82)], and rs233575 [OR 1.62 (1.00\u20132.61)]. The cut-off level for TC \u2265 170 mg/dL was associated with rs2074192 OR 1.54 (1.04\u20132.28) and rs2158083 [OR 1.53 (1.04\u20132.25)]. Additionally, the haplotype (C-G-C) derived from rs879922-rs2158083-rs233575 was related to higher prevalence of overweight/obesity and TG elevation.Girls  of the three former could induce vulnerability to obesity and hyperlipidemia in women. Thus, these SNPs might be used as predictive biomarkers for CV diseases and as molecular targets for CV therapy.The expression and activity of ACE2 may be essential for CV homeostasis. Interestingly, the  The Angiotensin converting enzyme-2 (ACE2) is a significant regulator of the Renin-Angiotensin-Aldosterone system (RAAS), which plays key roles in the control of cardiovascular (CV) system . In the ACE2 gene is highly conserved in mammals and expressed in testes, renal and CV system, and especially in gastrointestinal tissues    have beeregions) . In chilregions) , 13. Nowregions) , 14. ThuACE2 SNPs in relationship with the presence of overweight and obesity in a representative sample of 12-to 16-year-old children, and we assess the potential association of the polymorphisms with the lipid profile and insulin levels.In our study, we examine five The present study comprised 873 adolescents (11\u201317 years old) recruited in the \u201cFour Provinces study\u201d during 2004\u201308 in whom information on biochemical variables and DNA samples were available . This cr2). The age- and sex-specific cut-off points of BMI for overweight and obesity were established according to Cole et al.  and were used to estimate the body mass index (BMI), as kilograms divided by height in squared meters . LDL-C levels were calculated according to Friedewald's formula. Also, the glucose concentration was evaluated by the glucose oxidase method, and insulin levels were achieved by using a RIA commercial kit . To estimate insulin resistance, the homeostatic model assessment of insulin resistance (HOMA-IR) index was calculated following formula: fasting insulin (\u03bcU/mL) x fasting glucose (mmol/liter)/22.5.Blood samples were obtained from subjects in the morning after a 12-h fasting period by using EDTA-collection tubes . Plasma was separated by centrifuging blood at 1,500 g at 4\u00b0C for 20 min. The upper fraction was stored at \u221280\u00b0C until use. Total cholesterol and triglycerides were enzymatically quantified in a RA-1000 Autoanalyzer . HDL-C concentrations were measured after precipitation of apo B-containing lipoproteins with phosphotungstic acid and MgACE2 gene  were genotyped by using predesigned TaqMan\u00ae SNP Genotyping Assays from Applied Biosystems . A StepOnePlus\u2122 Real-Time PCR System (Applied Biosystems) was used for allelic discrimination. PCR was performed with a mixture of 10 ng of genomic DNA, TaqMan\u00ae SNP Genotyping Assay (20X), and TaqPath\u2122 ProAmp\u2122 Master Mix (Applied Biosystems). Samples were cycled under the recommended conditions: 95 \u00b0C for 10 min, 95 \u00b0C for 15 s and 60 \u00b0C for 1 min, repeated over 40 cycles. The data presented in the study are deposited in the dbSNP repository, submission batch ID 1063361 (dbSNP Build ID: B156). The genotyping data were upload in a public repository (accession number).Genomic DNA was also obtained from blood samples. After plasma separation, the interphasic fraction (leukocytes) was used to isolate total DNA by adaptation of a classic procedure, which involves salting out of the cellular proteins by dehydration and precipitation with a saturated NaCl solution . The quaThe five SNPs  were selected to construct haplotypes. Linear generalized models were used to compare outcomes levels between haplotypes. The comparisons were summarized by coefficients and odds ratios for continuous and categorical outcomes respectively, and with 95% confidence intervals and p-values. These analyses were performed using the haplo.glm function from the haplo.stats R package. Haplotypes with frequencies less than 5% were included in the category \u201cother\u201d.post-hoc test. Also, they were summarized as mean values and 95% confidence interval (CI). Some variables with non-normal distribution were normalized by log base-10 transformation  and were expressed as geometric mean values and 95% confidence interval. Other variables with non-normal distribution  were compared using the Mann\u2013Whitney U test and Kruskal-Wallis test followed by Dunn's (post-hoc). These variables were summarized by median and interquartile range. The associations of haplotypes with anthropometric, blood pressure and plasma parameters were analyzed by regression models. For continuous variables, we used linear models, whereas dichotomous coded variables were examined by logistic models. The former were summarized by coefficients (coef.) while the later were summarized by odds ratio (OR). Also, a 95% CI and p-values were reported. Statistical analyses were performed using the statistical language R version 4.0.5 .Qualitative variables were included as absolute and relative frequencies. Associations between qualitative variables were studied by the chi-square test and odds ratio. On the other hand, normality of quantitative variables was analyzed by the Kolmogorov\u2013Smirnov test. On one hand, variables with normal distribution (age and HDL-C) were compared using a Student's t-test and ANOVA one way, followed by Tukey  or Games-Howell  2. In the study, we found that 71.8 percent of children were normo-weight, whereas 28.2 percent presented an excessive weight (p = 0.569). Also, according to the latest clinical guides of the American College of Cardiology/American Heart Association/American Diabetes Association for pediatric population  of the injuries , 10, 21 injuries .ACE2 SNPs  might associate with the anthropomorphic variables of the population. Remarkably, none of the SNPs were significantly related to the SBP or DBP, either in girls or boys (not shown). However, in females, the occurrence of overweight or obesity was found to be significantly associated with three of the studied SNPs , p = 0.006] and 27.0%  of overweight or obese girls, respectively , rs233575 (p = 0.017) and rs2158083 (p = 0.036)  and rs2158083  . Lipid parameters did not show any relationship with any SNPs in male adolescents. However, again in females, four out of five SNPs were related to the lipid profile. The highest levels of TG were significantly associated with the presence of heterozygous genotypes of rs879922 (= 0.036) . The TG/= 0.036)  were rel= 0.036) . Furtherctively) . Similarctively)  were assctively) . The HDLp = 0.038], in comparison with the haplotype composed by the major alleles (G-A-T) , p = 0.031] and TG/HDL ratio . Interestingly, after adjusting by BMI, the C-G-C haplotype maintained its association with higher TG levels   was overrepresented in girls (34%) and linked to significantly higher BMI  . On the ACE2 gene in a general population of apparently healthy adolescents from Spain. The prevalence of these polymorphisms in females and males was comparable to previous findings  and its activity can be regulated by hormone release , and an estimator of LDL-C particle size . Thus, t release . Neverth release . Moreove release . Also, A release , 31. In  release .ACE2 was associated with overweight/obesity and elevated plasma TG in Spanish girls. The rs879922 and rs233575 polymorphisms were also related to upper TG/HDL-C ratio, and the rs2074192 and rs2158083 with higher TC levels. Reinforcing our findings, we observed that the C-G-C haplotype of rs879922, rs233575, and rs2158083 was associated with excessive weight and elevated TG levels. Previous data on ACE2 haplotypes linked higher risk of cardiac hypertrophy with minor alleles of rs2074192 and rs2106809 in females -MasR signaling can promote CV failures . ACE2 kn females .ACE2's intron 4 and was previously associated with higher blood pressure . Furthermore, these ACE2-mutations might be generated in rodent models with/without obesity and cardiovascular failures in order to characterize atherogenic responses under ACE2 modulation.This study lays the groundwork for subsequent trials in patients and experimental animals. The ACE2 gene with overweight/obesity or plasma lipids elevation remain to be clarified. Finally, functional investigations of the association between C-G-C haplotype with overweight/obesity or TG elevation could be required.At these ages, girls and boys may produce different sexual hormones and factors, which can influence on the lipid profile. Thus, their puberal stage may add important information to explain differences between sex. Also, the precise biological mechanism and other possible factors underlying the association of the ACE2 gene may be somehow protected from increased adiposity and hyperlipidemia. However, Spanish girls carrying the rare alleles of the ACE2-SNPs rs879922, rs233575, rs2158083, or rs2074192 may be vulnerable to future obesity and CV injuries. The haplotype rs879922-rs233575-rs2158083 may be considered a valid biomarker for both pathologies. Therefore, these ACE2-SNPs might be addressed for therapeutic and prognostic purposes against CV diseases. In this sense, new studies might evaluate their role as predictors of worse evolution in COVID-19 subjects, where CV homeostasis may be seriously damaged.The ACE2 gene may become a double-sword factor for CV protection, at least in women. Those female adolescents who carry the wild-type The data presented in the study are deposited in the dbSNP repository, submission batch ID 1063361 (dbSNP Build ID: B156).The study protocol was approved by the Ethics Committee of Clinical Investigation of the Fundaci\u00f3n Jim\u00e9nez D\u00edaz (Code number: PIC016-2019 FJD). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.ACE2-SNPs and analyze its associations with clinical variables. IM-F performed the statistic studies. LS-G, OL, and CG designed and wrote the manuscript. All authors participate in the discussion of the work.JL-C, CV-V, and IP-N quantified the gene expression of This research was funded by SPACE2-CV-COVID-CM grant from REACT-EU, Comunidad de Madrid and European Regional Development Fund, and by the Fondo de Investigaci\u00f3n Sanitaria-IS. Carlos III (ref.: PI18/01016 and PI20/00923), Biobank (ref.: FEDERRD09/0076/00101), and Ciberdem (ref.: CB07/08/2007). JL-C was granted by FPI contract from Universidad Aut\u00f3noma, Madrid.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": "We aimed to investigate the association between the GNRI and the risk of stroke in elderly patients with hypertension.A total of 5312 elderly hypertensive patients free of history of stroke were included. Multivariate Cox models were used to calculate hazard ratios (HRs) and their 95% confidence intervals (CIs) for stroke and its subtypes.The average time of follow-up was 3.8 years, and the median time was 3.2 years. We identified 640 individuals with stroke, of whom 526 had an ischemic stroke (IS) and 114 had a hemorrhagic stroke (HS). After adjusting for confounding variables, compared with participants in the lowest quartile of the GNRI, those in the third and fourth quartiles were associated with a decreased risk of stroke . Similar results were found for IS and HS. Moreover, there were L-shaped associations of GNRI with new-onset HS (P for non-linearity = 0.034). Multiple sensitivity analyses and stratified analyses did not materially change the results.In summary, we found that a lower GNRI was associated with a higher risk of incident stroke in elderly hypertensive patients. Additional prospective data collection is required to confirm our findings. Stroke, including ischemic stroke (IS) and hemorrhagic stroke (HS), is the leading cause of the global burden of disease . China hMalnutrition is associated with a poor clinical prognosis in patients with various diseases . AccordiTherefore, the present study is based on a cohort study aiming to investigate the association between GNRI and the risk of stroke and its subtypes in elderly hypertensive patients and to characterize the nature of the dose-response relationship.We conducted a cohort study at the People's Hospital of Xinjiang Uygur Autonomous Region, Xinjiang, China. All patients were either older than 60 years of age and were recruited between January 1, 2010, and December 31, 2021. First, we excluded patients who had < 6 months of follow-up or had the outcome at baseline. Second, we further excluded individuals with missing data on body height, body weight, or serum albumin level. Third, we excluded participants with severe wasting diseases . Finally, this left a final study population of 5,312 patients. Participant flow is shown in 2) was computed from the measured weight and height. Smoking status included categories of current smokers and non-smokers. Participants are classified as current drinkers and non-smokers. Blood samples were drawn after an overnight fast. The participants' prior medical histories were evaluated using ICD-10 codes. To ensure the accuracy of diagnoses, coronary heart disease (CHD) (I24 and I25), diabetes (E10-E14), atrial fibrillation (I48), and dyslipidemia (E78) were regarded as present if a participant was treated \u2265 2 times. To quantify the burden of comorbidities, the Charlson Comorbidity Index (CCI) was calculated as described previously , prescribed medications, and laboratory reports. Weight, height, heart rate, and blood pressure (BP) were measured using standard protocols. The BMI , either nonfatal or fatal. Secondary outcomes included the first ischemic stroke and the first hemorrhagic stroke. Methods of determination of incident stroke are described in the P-values < 0.05 were considered statistically significant.Details of the missing covariates are shown in As illustrated in the flow chart , a totalP < 0.001, P = 0.001, P = 0.001, The average time of follow-up was 3.8 years, and the median time was 3.2 years. We identified 640 individuals with stroke, of which 526 had IS and 114 had HS. The Kaplan-Meier curve showed that participants in the quartile 1 group had a higher risk of total stroke, IS instead of HS than those in other groups  with total stroke and its subtypes, as provided in P < 0.001). Moreover, according to continuous NRI and IDI, the GNRI significantly improved risk discrimination for total stroke . Furthermore, the DCA for the different models is shown in As illustrated in This study investigated the relationship between GNRI and incident stroke in elderly hypertensive patients. The findings revealed that the risk of stroke was significantly associated with baseline GNRI after adjusting for multiple confounders. In addition, a significant L-shaped dose-response relationship between GNRI and the risk of incident HS was observed, indicating a rapid increase in the risk of HS when GNRI was below 103. These findings were reliable in subgroup and multiple sensitivity analyses. Overall, the present study revealed that low CNRI was associated with a higher risk of incident stroke. To our knowledge, this is the first study to show an association between GNRI and the risk of incident stroke in a large retrospective cohort.Aging is a condition that affects all people. One of the most susceptible demographics and one that is more likely to experience nutritional issues is the elderly . SimilarAlthough the underlying mechanisms remain unclear, there are some possible explanations. Oxidative stress and inflammation play key roles in the pathogenesis of stroke . First, The strengths of this study lie in the novelty, the long observational period, and the well-characterized participants. Despite the aforementioned merits, several limitations of the present study merit discussion. First, it was observational and cannot establish causation. Second, the time-dependent changes of GNRI during the follow-up period were not assessed. Third, we didn't examine the predictive value of GNRI against more thorough nutrition evaluations. Despite adjustment for major confounding factors, the risk of residual unmeasured confounding remains possible. Finally, this study is limited to China and needs to be replicated in other different populations. Given the limitations inherent in this study, these results should be interpreted with caution but warrant further investigation in subsequent studies.In summary, we demonstrated that a lower GNRI was associated with a higher risk of incident stroke in elderly hypertensive patients. In addition, a significant L-shaped dose-response relationship between GNRI and the risk of incident HS was observed. Additional prospective data collection is required to confirm our findings.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by People's Hospital of Xinjiang Uygur Autonomous Region. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.XC and JHu analyzed the data and wrote the manuscript. JHo, QZ, MW, YD, and WY helped with copyediting. XC and NL audited the data. SL, JHu, and XC conducted research. NL had primary responsibility for the final content of the manuscript. All authors read and approved the final manuscript.This research was supported by the Chinese Academy of Medical Sciences (2020-RW330-002).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": "The motivation of this research is to review all methods used in data compression of collected data in monitoring the condition of equipment based on the framework of edge computing. Since a large amount of signal data is collected when monitoring conditions of mechanical equipment, namely, signals of running machines are continuously transmitted to be crunched, compressed data should be handled effectively. However, this process occupies resources since data transmission requires the allocation of a large capacity. To resolve this problem, this article examines the monitoring conditions of equipment based on edge computing. First, the signal is pre-processed by edge computing, so that the fault characteristics can be identified quickly. Second, signals with difficult-to-identify fault characteristics need to be compressed to save transmission resources. Then, different types of signal data collected in mechanical equipment conditions are compressed by various compression methods and uploaded to the cloud. Finally, the cloud platform, which has powerful processing capability, is processed to improve the volume of the data transmission. By examining and analyzing the monitoring conditions and signal compression methods of mechanical equipment, the future development trend is elaborated to provide references and ideas for the contemporary research of data monitoring and data compression algorithms. Consequently, the manuscript presents different compression methods in detail and clarifies the data compression methods used for the signal compression of equipment based on edge computing. Mechanical equipment was monitored to detect whether a mechanical failure occurred or not in the previous investigations, which is fairly a simple method to deal with monitoring mechanical failures. However, before a failure occurred, the machinery could have signaled to have stepped into serious failure mode. To resolve this issue, the first implemented attempt was the signal processing technology  that wasData compression is a generic name that encodes data to squeeze , mainly Even though data can be compressed well through compression methods, powerful computing ability is required in this process when an increase in the complexity of algorithms is encountered. So, hardware becomes the key component to improve the capability of the algorithm. The development of data processing technology based on edge computing , 13 makeTo monitor the signal condition of mechanical equipment, the first step is to classify the status signal, which currently contains signals changing slowly and changing rapidly . While aThe rest of the article is organized as follows: Section 2 introduces the investigation of data pre-processing based on edge computing, then the available compression methods according to the monitoring condition characteristics of signals employing different mechanical equipment are introduced. The assessment indexes used by various compression methods in data compression methods are presented in Section 3. Section 4 discusses the future directions of these methods. Section 5 concludes the review research.Monitoring the status of equipment based on edge computing first contains the acquisition of signals, including slowly changing signals, such as water temperature and oil temperature, as well as fast-changing signals, such as engine vibration signals and transmission vibration signals. Then, the collected signals, and data, are pre-processed by edge computing, and the signals of the fault characteristics not easily identified are compressed and transmitted to the cloud for signal reconstruction and fault diagnosis. The whole signal analysis process is shown in To avoid occupying a large number of transmission resources and energy loss, it is necessary to pre-process the data. In this article, we apply a pre-processing method based on edge computing to monitor the signal process of equipment status and extract the information of fault features through a short-time Fourier transform or wavelet transform , such asWhen signal data needs complex calculation, such as pulse signal, decay signal, noise signal, and other combined signal components, the data is transmitted to the cloud for analysis and calculation, therefore, we need to compress and transmit them, and decompose the signal and identify the fault characteristics in the cloud for such data types.Different processing methods are required for compression based on the signal type. If the sampling frequency of fast-varying signals is too high, the amount of data collected will be very large, resulting in a large amount of transmitted data. A lossless compression algorithm can compress them, but it cannot meet the CR required for the fast-varying signal, so lossy compression is applied to the fast-varying signal compression since a lossless compression algorithm is used for slow-varying signals.Due to the small variability occurring in slow-varying signals over a short period, the amplitude of the remaining numbers is significantly reduced by the differential transformation of the data, which results in a lower number of binary bits required for representation. This implies that lossless compression is more effective. Lossless compression was initially used in data monitoring algorithms based on a single compression algorithm, which is more effective regarding the computational complexity, but its CR is less than expected. To improve its compression effect, multiple compression algorithms need to be utilized in combination, so numerous hybrid algorithm compression models have been suggested, which have the advantage of improving further the CR of the data. However, the complexity of the algorithm increases.The single-algorithm model mainly includes statistical-based and dictionary-based compression algorithms. While statistical-based compression algorithms compress data by counting the frequency of characters and providing fewer bits of encoding for characters with high frequency, the dictionary-based compression algorithm compresses data by constructing a dictionary and outputting its dictionary index value, which can be adapted to various types of data.When the hybrid algorithm compression models are under consideration, one type is based on the improved compression algorithm, such as moving to front (MTF) coding , BurrowsThe other type is a compression method that combines different single-algorithm models, mainly through a mixture of statistical-based compression algorithms and dictionary-based compression algorithms, which outputs its index value through the dictionary coding and compress data through a statistical-based encoding algorithm. Through the analysis of the various lossless compression methods in When monitoring equipment is conducted, the most applied mechanical vibration signal is analyzed to understand the operation status of the equipment. In this article, vibration signal analysis is employed as an example to investigate and review the compression algorithms. After the vibration signal is collected, the fault signal is detected by edge computing to extract more effective signal information. Then, the research on the lossy compression algorithm is carried out on this basis. At present, there have been many studies dealing with lossy compression algorithms for vibration signals, but they have not been classified yet.In this research, lossy compression methods for vibration signals are studied and classified to present their research directions. In the early stage, the quantization compression algorithm has higher efficiency, but the CR is not high. To improve its compression effect, one method applied is based on decomposing signals to extract its effective components and remove invalid components. Therefore, the removal methods include threshold processing and correlation analysis, such as empirical mode decomposition (EMD)  and imprTherefore, the super-complete dictionary is studied , which iWith the development of deep learning technology, compression algorithms based on deep learning have also been studied in more detail, such as recurrent neural network-based recurrent neural networks (RNN)  and longAlthough the single-algorithm compression model can effectively reduce the redundancy of data and improve the CR, there exists still room for the improvement of the CR. By studying the compression model based on a hybrid algorithm utilizing the single algorithm, the signal is first decomposed or sparsely transformed and then compressed using lossless compression methods. So, its algorithm can further improve the CR, but its computational complexity increases. Lossy compression methods and their advantages and disadvantages are described in By comparing the advantages and disadvantages of the data compression methods in 0 denotes the bit number of original data and Data1 denotes the bit number of compressed data.The performance of the data compression algorithm needs to be evaluated by establishing a corresponding evaluation index. The number of the current compression algorithm evaluations related to the lossless method is relatively few, which is mainly due to the original signal and reconstruction signals remaining the same, so there occurs no data loss. Therefore, the lossless compression algorithm is mainly evaluated through the CR and time. The greater the CR is, the more effective the compression would be, the shorter the compression time is, the lower the complexity of the algorithm would be, and the better the processing of data would be. The formula for its CR is defined bySince lossless compression needs to ensure the consistency between the original data and the reconstructed data, it limits the increase of the CR, and many optimization algorithms have still been struggling to improve the performance of the CR.Unlike lossless compression, the reconstructed signal in lossy compression methods does not need to be consistent with the original signal, so in addition to the CR and time, the fidelity of its signal needs to be evaluated. The corresponding evaluation indexes are presented as follows .xi and xir are the original signal and the reconstructed signal, respectively and N is the amount of signal data.The RMSE method quantifies the degree of data distortion caused by the compression algorithm. When the RMSE value nears zero, the original signal is consistent with the reconstructed signal and there is no compression effect. To reduce the influence of the signal mean on the RMSE, a normalized PRD is established, thus disregarding the influence of its mean, which is given byIt is used to evaluate the similarity between the original signal and the reconstructed signal byTo understand whether there is a loss of key features when performing fault feature extraction before and after compression, it is necessary to compare the fault feature information before and after compression to ensure that useful fault features can be extracted from the reconstructed signal even though the evaluation metrics above for lossy compression are employed. The current study of fault features requires the examination of fault information in the time domain, frequency domain, and time\u2013frequency domain . Lei andWith the development of edge computing and data compression methods, monitoring mechanical equipment has become more practical and useful than ever. In this section, the progression trend of monitoring mechanical equipment and data compression algorithms will be presented in detail.With the development of automated equipment, the inspection of offline mechanical equipment using automated equipment was realized. However, this method has still a certain deficiency in the inspection process. Due to the development of signal processing technology and the emergence of intelligent technology, online remote monitoring of mechanical equipment in real time has become more possible and reliable. However, data collected in this process is all uplinked to the cloud for crunching. Although this processing satisfies the functional requirements to a certain extent, there has still been a certain delay.The edge node is utilized by transferring the data processing from the cloud to the edge node, thus speeding up its downlink computational process. Moreover, the real-time capability of data monitoring is effectively improved by filtering out data, processing it, and uploading it to the cloud for analysis. So, the amount of signal data that needs to be compressed is effectively reduced, and so does the amount of data transmission.Based on the development trend of smart methods, monitoring of mechanical equipment also contribute to the improvement of the miniaturization of its equipment. So, the development of microelectronics technology, such as the application of digital signal processing and field programmable gate array-based devices, has effectively improved its micro-processing capability. On the other hand, the development of communication technologies, such as Internet of Things  and 5G , 68 tranWhen equipment is monitored, the signal data is compressed with the help of different compression methods due to the different characteristics of the signal data.To improve the CR, the CR of the single algorithm is enhanced on the one hand, and the complexity of the hybrid compression algorithm is reduced on the other hand by employing the characteristics of slowly varying data.In the single algorithm, the traditional compression method is algorithmically optimized to improve its compression performance. The algorithm proposed by Huffman is improved based on the adaptive Huffman coding  and weigZ-standard combined with LZ77 [Based on the ANS algorithm, a finite state entropy algorithm was developed. To further improve the compression and decompression speed, an optimization algorithm called ith LZ77  was deveith LZ77 . AlthougIn the hybrid compression algorithm, to meet the impact of real-time transmission and reduce the time consumed by compression, it is necessary to reduce the complexity of the algorithm. Many of the current algorithms , 79 are On the other hand, fast-varying signals are researched regarding lossy compression methods. Since it allows a certain information loss in reconstructed signals, the CR is much higher when compared to a lossless one. When signal processing technology is under consideration, compression methods based on signal processing have become the mainstream research direction, such as compression methods run by sparse processing or threshold processing algorithms based on signal decomposition.In the compression method of sparse processing, the energy distribution of the data is more concentrated by employing entropy transformation, and the low-energy data are removed, and then quantization is performed. Then, the quantized data are encoded , 34. TheThe motivation of this research is to review all methods used in data compression of collected data in monitoring the condition of equipment based on the framework of edge computing. This article introduces a literature review of various data compression methods based on edge computing, which pre-processes the collected signal through edge computing to obtain easily identifiable fault signals and then performs data compression that is not generally easy to conduct. Two key indicators, algorithm complexity and CR are paid attention to when the literature is reviewed.Concluded that when signal processing technology is under consideration, compression methods based on signal processing have become the mainstream research direction, such as compression methods run by sparse processing or threshold processing algorithms based on signal decomposition equipped with deep learning technology.The manuscript presents different compression methods in detail and clarifies the data compression methods used for the signal compression of equipment. Then, comprehensive classification is presented based on various assessment methods to determine the fidelity of the compression methods.The future development trend of monitoring equipment and data compression is comprehensively provided by presenting several research findings in the literature. Consequently, more detailed references and ideas for data compression of equipment are summarized for interested readers."}
+{"text": "Zika virus and dengue virus are co-circulating flaviviruses with a widespread endemic range. Eliciting broad and potent neutralizing antibodies is an attractive goal for developing a vaccine to simultaneously protect against these viruses. However, the capacity of viral mutations to confer escape from broadly neutralizing antibodies remains undescribed, due in part to limited throughput and scope of traditional approaches. Here, we use deep mutational scanning to map how all possible single amino acid mutations in Zika virus envelope protein affect neutralization by antibodies of varying breadth and potency. While all antibodies selected viral escape mutations, the mutations selected by broadly neutralizing antibodies conferred less escape relative to those selected by narrow, virus-specific antibodies. Surprisingly, even for broadly neutralizing antibodies with similar binding footprints, different single mutations led to escape, indicating distinct functional requirements for neutralization not captured by existing structures. Additionally, the antigenic effects of mutations selected by broadly neutralizing antibodies were conserved across divergent, albeit related, flaviviruses. Our approach identifies residues critical for antibody neutralization, thus comprehensively defining the as-yet-unknown functional epitopes of antibodies with clinical potential.The wide endemic range of mosquito-vectored flaviviruses\u2014such as Zika virus and dengue virus serotypes 1\u20134\u2014places hundreds of millions of people at risk of infection every year. Despite this, there are no widely available vaccines, and treatment of severe cases is limited to supportive care. An avenue toward development of more widely applicable vaccines and targeted therapies is the characterization of monoclonal antibodies that broadly neutralize all these viruses. Here, we measure how single amino acid mutations in viral envelope protein affect neutralizing antibodies with both broad and narrow specificities. We find that broadly neutralizing antibodies with potential as vaccine prototypes or biological therapeutics are quantifiably more difficult to escape than narrow, virus-specific neutralizing antibodies. Viruses can evade immune pressures by antigenic variation and adaptation. For co-circulating, antigenically similar viruses, this can mean that population immunity to one virus might determine the fitness of another virus strain or serotype. Zika and dengue are two such antigenically related, co-circulating flaviviruses, with dengue virus clustering into four unique serotypes and Zika virus occupying a single serotype . An initFurthermore, primary exposure to Zika or dengue virus has also been shown to increase risk of severe disease upon a second infection with a heterotypic dengue virus \u201312. ThWhile proposing these broadly neutralizing anti-E antibodies as therapeutics or as a template for vaccine design , 30 is pHere, we use deep mutational scanning to identify functional epitopes by measuring the effect of all possible single amino acid mutations in Zika virus E protein on neutralization by both broadly neutralizing and narrow virus-specific antibodies. We find that while all antibodies are affected by single amino acid mutations, the magnitudes of these effects vary widely across antibodies. Specifically, single mutations only modestly increase neutralization resistance against broad antibodies, while single mutations completely ablate neutralization by antibodies with narrow specificities. We extrapolate these results to other flavivirus genetic contexts by engineering neutralization escape mutations in other Zika virus and dengue virus E proteins. Interestingly, we show that some antigenic effects are conserved across these overall divergent viral surface proteins.We first assembled a panel of neutralizing antibodies of varying breadth and potency against Zika and dengue viruses. The neutralization profiles of these antibodies and their structures in complex with E protein homodimer are shown in To perform deep mutational scanning, we regenerated previously described mutant E virus libraries from Zika virus \u201cAfrican\u201d lineage strain MR766 . DespiteWe next plotted the site-level effects of all mutations by summing all individual amino acid antibody escape measurements at a given site in The effects of individual amino acid mutations at each site are shown in Fig. S2 to S6. For ZV-67 and MZ4, many different amino acid mutations tended to cause antibody escape at the key sites. However, for the broad antibodies, typically, only one or a handful of mutations at a given site led to viral escape, and the magnitude of escape tended to be lower. A likely explanation is that our approach only identifies escape mutations that are functionally tolerated in live replicative virus, and the broad antibodies tend to target sites in E that are relatively intolerant of mutations.We next compared the sites of antibody escape from the deep mutational scanning to the structural epitopes of the antibodies as determined by prior structural studies . The ZikIn contrast, broadly neutralizing antibodies were escaped by fewer mutations of weaker effects both within and outside structural epitopes. Despite similar structural epitopes \u201326, 28Another broad antibody, SIgN-3C, had escape mutations distributed across E protein domains. We identified sites S72, M140, and T315 as those comprising the functional epitope, where few mutations had large individual effects . These fTo validate the findings from our deep mutational scanning, we made single mutations in Zika virus E protein and tested them in dose-response neutralization assays . These iWe first assessed the effects of mutations identified in deep mutational scanning of narrow and pseudo-broad antibodies ZV-67 and MZ4 . We saw For broadly neutralizing antibodies, single mutations had far more modest effects. As above, we generated mutant Zika virus MR766 pseudovirus particles and assayed their effects in neutralization assays. Antibody selection with EDE1-C10 had identified the mutations V33I at the intra-dimer interface and T49D within the structural epitope . Our neuDeep mutational scanning of another broad antibody, EDE1-C8, selected mutations S7P and N439K outside its structural epitope . When weFinally, we tested the broad antibody SIgN-3C against a panel of predicted escape mutations, N8T, S72G, and T315Y. To a certain extent, the effect size of these mutations identified from our deep mutational scanning data  correlatOverall, these neutralization assays validate the finding from the deep mutational scanning that narrow and pseudo-broad antibodies have very large-magnitude escape mutations, while no mutations more than modestly affect neutralization by broad antibodies . For theTogether, our deep mutational scanning and traditional neutralization assays showed that many single mutations across a few sites had large effects on neutralization by antibodies with narrow specificities. However, for broadly neutralizing antibodies, we identified only a few mutations across many E protein sites with small effects. Was this because broad antibodies tended to be escaped by mutations that were not well tolerated in the replicative viruses used in our libraries? Or because broadly neutralizing antibodies distribute interaction requirements over a wide range of sites?in vitro. We compared these mutational tolerance values against mutation-level antibody escape in To address our first question, we leveraged data published in Sourisseau et al.  assessinNext, we examined site-level data to see if interaction requirements were distributed differently between antibodies with broad and narrow specificities. To estimate site-level mutational tolerance, we used two metrics: number of effective amino acids and site entropy. These values were plotted against site-wise summed antibody escape values for all antibodies in We next tested if the antigenic effects of mutations that we had identified and validated in Zika virus MR766 were similar in other flaviviruses. Zika virus MR766 is closely related to a widely utilized contemporary strain of Zika virus, H/PF/2013, sharing ~96% amino acid E sequence identity. It is more distantly related to dengue viruses, sharing only ~54% identity with dengue virus serotype 2 (strain 16681). Nevertheless, as the broad and pseudo-broad antibodies within our panel neutralized this wide variety of viruses, we hypothesized that some degree of antigenicity might be conserved.To test this hypothesis, we generated single-mutant pseudovirus particles of Zika virus strain H/PF/2013 and dengue virus serotype 2 strain 16681. For each antibody, we selected a single viral E mutation that escaped Zika virus strain MR766  and Vero-C1008 cells  were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM)  supplemented with 7% fetal bovine serum (FBS)  and 100 U/mL penicillin-streptomycin . Raji cells stably expressing dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin-related (Raji/DC-SIGNR)  .We selected a single representative strain of each dengue serotypes 1\u20134, two strains of Zika virus, and a strain of West Nile virus as an outgroup. We downloaded both protein and DNA sequences for each of these strains from NCBI and used mafft  to alignHQ234498)   in full Q234498) . Using aQ234498) , these mDeep mutational scanning was performed as described , 36, 54.To determine the percent infectivity reported in Table S1, we used reverse transcription-quantitative PCR (qRT-PCR) against Zika virus E gene [primers reported in Sourisseau et al. ] and GAPhttps://jbloomlab.github.io/dms_tools2/) (https://github.com/jbloomlab/ZIKV_MAP_GooLab). Methods used in this paper include calculation of differential selection (dms2_batch_diffsel), which was used as our metric of antibody escape in https://jbloomlab.github.io/dmslogo/). For estimation of mutation-level tolerance in The deep sequencing data were analyzed and visualized using dms_tools2 (tools2/) , 58. Allhttps://jbloomlab.github.io/polyclonal/)  . These m2. Subsequently, reporter virus particle-containing supernatant was passed through a 0.22-\u00b5m Millex SteriFlip Vacuum-driven Filtration System  before aliquoting and storage at \u221280\u00b0C. Infectious units per milliliter were determined by titration of reporter virus particles onto Raji/DC-SIGNR cells and then incubated at 37\u00b0C for 48 hours. Cells were fixed in 2% paraformaldehyde  before percentage of GFP-positive cells was determined by high-throughput flow cytometry .Reporter virus particles were generated as previously described . BrieflySingle-mutant reporter virus particles were generated by site-directed mutagenesis of the CprME plasmid. As in previous work , PCRs weFor dose-dependent neutralization assays with reporter virus particles, antibodies of interest were diluted to an initial concentration of 10\u201350 \u00b5g/mL in RPMI supplemented with 7% FBS and then fivefold serially diluted across a 384-well v-shape microplate . Reporter virus particles were diluted to 5%\u201310% expected infectivity and added to diluted antibodies. Antibody-virus mixtures were incubated at 37\u00b0C for 4\u20136 hours. Next, 20 \u00b5L of 1e6/mL Raji/DC-SIGNR cells was added, and the mixture was incubated at 37\u00b0C for 48 hours. As above, cells were fixed in 2% paraformaldehyde prior to measurement of %GFP by high-throughput flow cytometry. Neutralization assays were performed in technical triplicate on the same day to minimize batch effects.https://github.com/jbloomlab/ZIKV_MAP_GooLab/tree/main/paper_figures/neutralizations_escape_mutants) and wild-type viruses . Fraction infectivity was calculated by dividing samples containing antibodies by the average of those containing no antibody. Plotting of neutralization curves and interpolation of Hill curves and IC50s were performed using the Python package neutcurve (https://jbloomlab.github.io/neutcurve/). Tables of percent infectivity, replicate IC50s, and statistical tests in Tables S1 to S6 were rendered with custom code  using the Python package pyMSAviz (https://moshi4.github.io/pyMSAviz/). Statistical tests used and more details can be found in the above linked directories and corresponding Jupyter notebooks.Downstream analysis was performed using custom Python code with mutant viruses ("}
+{"text": "Influenza outbreaks pose a significant threat to global public health. Traditional surveillance systems and simple algorithms often struggle to predict influenza outbreaks in an accurate and timely manner. Big data and modern technology have offered new modalities for disease surveillance and prediction. Influenza-like illness can serve as a valuable surveillance tool for emerging respiratory infectious diseases like influenza and COVID-19, especially when reported case data may not fully reflect the actual epidemic curve.This study aimed to develop a predictive model for influenza outbreaks by combining Baidu search query data with traditional virological surveillance data. The goal was to improve early detection and preparedness for influenza outbreaks in both northern and southern China, providing evidence for supplementing modern intelligence epidemic surveillance methods.We collected virological data from the National Influenza Surveillance Network and Baidu search query data from January 2011 to July 2018, totaling 3,691,865 and 1,563,361 respective samples. Relevant search terms related to influenza were identified and analyzed for their correlation with influenza-positive rates using Pearson correlation analysis. A distributed lag nonlinear model was used to assess the lag correlation of the search terms with influenza activity. Subsequently, a predictive model based on the gated recurrent unit and multiple attention mechanisms was developed to forecast the influenza-positive trend.This study revealed a high correlation between specific Baidu search terms and influenza-positive rates in both northern and southern China, except for 1 term. The search terms were categorized into 4 groups: essential facts on influenza, influenza symptoms, influenza treatment and medicine, and influenza prevention, all of which showed correlation with the influenza-positive rate. The influenza prevention and influenza symptom groups had a lag correlation of 1.4-3.2 and 5.0-8.0 days, respectively. The Baidu search terms could help predict the influenza-positive rate 14-22 days in advance in southern China but interfered with influenza surveillance in northern China.Complementing traditional disease surveillance systems with information from web-based data sources can aid in detecting warning signs of influenza outbreaks earlier. However, supplementation of modern surveillance with search engine information should be approached cautiously. This approach provides valuable insights for digital epidemiology and has the potential for broader application in respiratory infectious disease surveillance. Further research should explore the optimization and customization of search terms for different regions and languages to improve the accuracy of influenza prediction models. The seasonal influenza epidemic poses a persistent and severe threat to global public health . Over thThe rapid spread of these infectious diseases not only endangers the public health of the population but also leads to a surge in medical demand, straining the health care infrastructure. Effective disease prevention and control measures can help significantly reduce or prevent further damage to population health, society, and the economy. Hence, early detection of infectious disease outbreaks is essential for specific regions affected by the outbreak as well as for the development of global seasonal influenza prevention and control.The growing consensus that traditional surveillance systems and early warning data are inadequate for tackling current pandemics indicates the emergence of a complex public health concern. Conventional syndromic surveillance relying on case reporting suffers from delays due to tendencies to seek medical attention, laboratory confirmation, and reporting procedures, hindering the timely detection of outbreaks of influenza ,7. As a Modern pathogen surveillance offers a broader application scope beyond national boundaries with robust surveillance systems and can be extended to developing countries with limited surveillance capabilities. As of the second quarter of 2022, Baidu, the most widely used search engine in China, had more than 600 million active users worldwide. The first effort to use internet search engines for biosurveillance was reported in 2008 when the Google Flu Trend (GFT) was launched, which displayed real-time updates of flu-related events in the United States 1 week before the publication of government-led flu reports . HoweverExisting studies have largely been based on a single city or a crude national analysis based on monthly data. However, whether modern data are available in both north and south climates remains to be explored. Therefore, we developed a predictive model for influenza supplied by Baidu search query data to combine traditional surveillance with modern big data. This study aimed to detect new infectious disease outbreaks earlier and better prepare for such outbreaks.In accordance with ethical guidelines, this research received approval from the Ethics Review Committee of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (CAMS&PUMC-IEC-2021-032).The virological data were retrieved from our previously published study , which uThe Baidu query data were retrieved from the public Baidu index website , which cPotentially relevant search terms were determined using the procedure illustrated in P<.05. According to the intensity of the correlation, we classified the correlation as high (PPC>0.6-0.7), medium (PPC 0.4-0.6), and low (PPC<0.4).Pearson correlation coefficients (PCCs) between the included Baidu search terms and the influenza-positive rate were calculated. First, we merged the synonyms into 1 combined search term. Subsequently, the PCC between each Baidu search term and the influenza-positive rate was calculated using equation 1, and statistical significance was set as PCC refers to the Pearson correlation coefficient, x refers to the values of the Baidu search index data, y refers to the values of the influenza-positive rate data, and n refers to the total number of values.Here, The lag correlation of the Baidu search index was analyzed using a distributed lag nonlinear model (DLNM), that is, a regression model in a nonlinear process , as descE[Yt] refers to the time series of the daily influenza-positive rate in northern and southern China, and cb(BI) refers to the cross-basis matrix of the time series of daily Baidu search terms, respectively. The natural cubic spline function was adopted in all the element spaces, and a lag period of 1-20 days was selected when establishing a cross-basis matrix. The quasi-Poisson function was used as the connection function in the model to control the overdispersion effect. Relative risks were used to assess the lag-response relationships of single-day effects.dlnm [Before DLNM analysis, all Baidu search terms were divided into 4 categories: general influenza information, influenza prevention, influenza treatment and medicine, and influenza symptoms. The DLNM was used using R Studio and R software , specifically the dlnm  package.In this research, we built a prediction model for multifeature time series based on the gated recurrent unit and multiple attention mechanisms model . By fullAfter receiving the multifeature inputs, we connected them to the gated recurrent unit layer, which processes the time series and captures the relationship between the step spacing in a time series. Subsequently, we concurrently used 3 different attention mechanism modules, namely, squeeze and excitation attention , channelP<.05), regular correlation (PCC\u22650.4), and strong correlation (PCC\u22650.6-0.7). The R2 and SE were calculated by comparing the predicted and actual data to identify the best model with different correlation levels of Baidu search terms. To identify the influence of different days in advance on the prediction effect, this study distinguished 30 cases where the prediction began 1-30 days in advance. The composite prediction model was constructed using Python .The training interval was from January 1, 2011, to May 31, 2017, the validation interval was from June 1, 2017, to August 31, 2017, and the prediction interval was from September 1, 2017, to July 31, 2018. According to the PCCs, the Baidu search terms were classified into 4 levels, namely, correlation . The terms were classified into 4 categories: basic understanding of influenza, symptoms, treatment and medicine, and prevention . The corThe lag of relevant days varied according to the search term category. Search terms related to the basic understanding of influenza, treatment, and medicine showed no associated lag. Meanwhile, symptoms-related terms showed a lag correlation with influenza activity of 1.4 days in northern China and 3.2 days in southern China. Prevention showed a lag correlation with influenza activity of 5.0 days in northern China and 8.0 days in southern China. The lag correlation trend of Baidu search indexes in the same category was similar between northern and southern China .Upon comparing the effect of different search terms on the prediction, more supplemental search terms were found to occur in northern China, exhibiting a worse prediction effect. Conversely, the best predictions in southern China were demonstrated by supplementing all relevant search terms .R2>0.9 and 0.8 for the influenza-positive trend prediction 14 days and 22 days in advance, while that in southern China was 11 days and 21 days in advance, respectively (We chose the optimization model to test the prediction at different times. Northern China showed an ectively . We alsoPredicting seasonal influenza has proven to be a great challenge based on the traditional surveillance system and simple algorithms. Following the application of big data and updated technology, the World Health Organization proposed a contemporary model. This study explored the application of combining Baidu search data with the traditional surveillance system to predict influenza outbreaks. This will not only provide a reference for southern and northern China but also for a modern surveillance approach.r values \u22650.7 [Baidu search term\u2013based surveillance can be applied to seasonal influenza, COVID-19, and other respiratory infectious diseases. Syndromic surveillance can detect outbreaks of disease or adverse health events earlier than traditional forms of public health surveillance  and avoiues \u22650.7 . In Italues \u22650.7 . TherefoIncreasing the use of big data is not always beneficial. Not all relevant search terms positively affect the accuracy and timeliness of influenza prediction. Inconsistent with previous research, this study demonstrated that increasing the use of Baidu search terms reduces the accuracy and time before influenza prediction. The same conclusion has been confirmed in GFT-related studies, which indicated that GFT data might not provide reliable surveillance for seasonal or pandemic influenza . This maIn light of these findings, it is crucial to recognize the broader public health implications of this study. First, while this study used data solely from the Baidu search engine, it is important to acknowledge that Baidu is primarily used in China and may not represent search behavior in other countries. As a result, the generalizability of these research findings to other regions or search engines may be limited. However, the methodology used in this study could be adapted and extended to incorporate data from other search engines or regions, providing valuable insights for future research. Second, early warning signs must be studied in addition to prediction. Third, we did not test all the methods to make a comparison. Fourth, other factors, such as news reports, may affect search engine search results. Future studies should explore mechanisms to maintain the accuracy of big data prediction. In light of these findings, it is crucial to recognize the broader public health implications of this study. Improving influenza prediction models have far-reaching benefits, including better preparedness and response measures to combat outbreaks. By detecting influenza outbreaks up to 10 days in advance using Baidu search data, we can mobilize health care resources, implement preventive measures, and initiate timely vaccination campaigns. This risk assessment and prediction analysis can also help control emerging respiratory infectious diseases, allowing for prompt interventions to mitigate the scale and impact of epidemics. This approach provides a valuable framework for understanding and managing public health crises, not only in China but globally. By addressing the identified limitations and leveraging the strengths of big data, we can continue to refine predictive models and strengthen global public health preparedness. Early detection and response to infectious disease outbreaks are essential in safeguarding population health, and this study contributes to achieving this critical goal.In conclusion, this study underscores the potential of combining big data and search engine\u2013based surveillance for influenza prediction, offering insights to revolutionize public health preparedness. By harnessing the power of Baidu search data and others and integrating climate variables, we can enhance early detection and response efforts, ultimately protecting communities from the devastating impact of infectious diseases. As we further explore and optimize these surveillance methods, we envision a future where early warning systems enable swift and effective responses to safeguard global public health."}
+{"text": "Reithrodontomys mexicanus, which has a problematic taxonomy history as it is considered a complex species. Here, we evaluate the cryptic diversity of R.\u2009mexicanus using an integrative taxonomy approach in order to detect candidate lineages at the species level. The molecular analysis used one mitochondrial (cytb) and two nuclear (Fgb\u2010I7 and IRBP) genes. Species hypotheses were suggested based on three molecular delimitation methods  and cytb genetic distance values. Skull and environmental space differences between the delimited species were also tested to complement the discrimination of candidate species. Based on the consensus across the delimitation methods and genetic distance values, four species were proposed, which were mostly supported by morphometric and ecological data: R.\u2009mexicanus clade I, R.\u2009mexicanus clade IIA, R.\u2009mexicanus clade IIIA, and R.\u2009mexicanus clade IIIB. In addition, the evolutionary relationships between the species that comprise the R.\u2009mexicanus group were discussed from a phylogenetic approach. Our findings present important taxonomic implications for Reithrodontomys, as the number of known species for this genus increases. Furthermore, we highlight the importance of the use of multiple sources of data in systematic studies to establish robust delimitations between species considered taxonomically complex.Species boundaries are difficult to establish in groups with very similar morphology. As an alternative, it has been suggested to integrate multiple sources of data to clarify taxonomic problems in taxa where cryptic speciation processes have been reported. This is the case of the harvest mouse  Reithrodontomys mexicanus using an integrative taxonomy approach in order to detect candidate lineages at the species level. Our results suggest at least four candidate species supported by molecular, morphometric, and ecological data, which has important implications for the current taxonomy of the genus.We evaluate the cryptic diversity of  Most species have been described using morphological traits Mayden,\u00a0, but an Molecular data have been crucial for studying cryptic species in rodents; they allow both the determination of the number of entities comprising a species complex and the delimitation of those entities  and Interphotoreceptor retinoid\u2010binding protein (IRBP) were obtained from collected specimens and tissue loans from mammal collections  and cytb\u2009+\u2009IRBP  because very few individuals presented genetic information for all loci. The information on the sequences generated in this study and those downloaded from GenBank is included in Appendix\u00a0Sequences were edited using Codon Code Aligner v.8.0.2 (CodonCode Corporation) and aligned against a reference in UGENE v.1.32.0  that best fit each data set were estimated in ModelFinder  using the BI tree and default parameters. The bGMYC coalescent method was implemented in the bGMYC package . This coalescent method is implemented as a package within BEAST2 and uses a modified birth\u2013death\u2010collapsed model for the species tree. The species.tree file generated by STACEY was used as input in the SpeciesDelimitationAnalyzer program  to summarize the posterior tree distribution and calculate the frequency with which each pair of taxa were assigned to the same clade.The cytb gene in MEGAX  had a known genetic identity or (2) were collected within less than 60\u2009km of an individual with a known genetic identity were used in the morphometric analyses. In total, 69 specimens  were selected for morphometric analysis . Digital images of the dorsal and ventral view of the skull of each specimen were taken using an Olympus DP73 Digital Camera and a millimeter rule as a scale bar.We examined 335 adult specimens . Because CVA cannot be performed when the number of variables is greater than the sample size per group  collected within less than 60\u2009km of an individual with a known genetic identity were also included in the ecological analyses Figure\u00a0. To reduto Hall,\u00a0. We remoto Hall,\u00a0. This st2 resolution were downloaded from Wordclim 2.0 : Bio1\u2009=\u2009Annual Mean Temperature, Bio5\u2009=\u2009Max Temperature of Warmest Month, Bio6\u2009=\u2009Min Temperature of Coldest Month, Bio12\u2009=\u2009Annual Precipitation, Bio13\u2009=\u2009Precipitation of Wettest Month, and Bio14\u2009=\u2009Precipitation of Driest Month. These bioclimatic variables were selected based on previous studies that highlighted their importance in ecological studies of small mammals  as a measure of the percentage of vegetation cover. This product was obtained from the MODIS sensor using 24 scenarios corresponding to the year 2020, with an original resolution of 250\u2009m. We rescaled the VCF product to the same resolution as the bioclimatic variables using ArGis 10.8 , with 1000 bootstrap iterations and E\u2009=\u20090.05.We used MaxEnt . Clade II corresponded to specimens distributed in Mexico, Guatemala, and El Salvador and was recovered as the sister group to brevirostris brevirostris from Costa Rica (pP\u2009=\u20091). The remaining individuals of R.\u2009mexicanus were grouped into a clade III with distribution in Colombia and Ecuador and sister to R.\u2009darienensis from Panama (pP\u2009=\u20091).The s Figure\u00a0. The ApoEngstrom\u00a0 were recR.\u2009mexicanus group, the mPTP method identified 15 putative species while 11 were delimited by the bGMYC. The multi\u2010locus method STACEY suggested 10 species (excluding taxa that were not successfully sequenced), although the proposed species were not the same between data sets (cytb\u2009+\u2009Fgb\u2010I7 and cytb\u2009+\u2009IRBP). The three methods supported the recognition of Reithrodontomys darienensis, R. sp. from Volcan Poas, Alajuela, Costa Rica, and R.\u2009garichensis , as species. In addition, Reithrodontomys gracilis from Yucat\u00e1n and Reithrodontomys spectabilis were considered the same species, excluding specimens of R.\u2009gracilis distributed in El Salvador, which were delimited as a distinct species. For R.\u2009brevirostris, each delimitation method proposed different numbers of possible species (from 1 to 3), although bGMYC and K2P genetic distances were congruent in delimiting only one species.The results of the species delimitation methods were not congruent with each other Figure\u00a0. For theR.\u2009mexicanus species complex, the R.\u2009mexicanus clade I was supported at the species level by all three delimitation methods. Within the R.\u2009mexicanus clade II, STACEY (cytb\u2009+\u2009Fgb\u2010I7) demarcated four possible species, while the other methods delimited only two. Following the recognition of two species, one of them is formed of samples from Mexico and Guatemala (R.\u2009mexicanus clade IIA), whereas the other corresponded to individuals from El Salvador, which were considered a distinct species under all the methods (R.\u2009mexicanus clade IIB). Within clade III, the mPTP identified 5 species, whereas bGMYC 2, and STACEY 3 (cytb\u2009+\u2009Fgb\u2010I7). In this clade, the consensus (bGMYC + genetic distances) demarcated two putative species: R.\u2009mexicanus clade IIIA and R.\u2009mexicanus clade IIIB. The cytb genetic distance values between the species delimitation consensus ranged from 4.96 to 18.18, with the highest value generally between R.\u2009mexicanus clade I and the other identified species. The cytb genetic distance values between delimited taxa in the R.\u2009mexicanus species group are shown in Table\u00a0Within the 3.2cytb tree . The most common recent ancestor of the Aporodon clade was at ca. 5.70 mya (95% HPD\u2009=\u20094.53\u20136.84), when the R.\u2009mexicanus clade I diverged from the rest of the species of this subgenus. Deep divergences were mostly well supported (pP\u2009=\u20091), except for the split between the R.\u2009mexicanus and R.\u2009tenuirostris species groups ca. 5.35 mya (95% HPD\u2009=\u20094.31\u20136.48) and the split between R.\u2009mexicanus clade II\u2009+\u2009R.\u2009brevirostris\u2009+\u2009R.\u2009darienensis\u2009+\u2009R.\u2009mexicanus clade III and R.\u2009garichensis\u2009+\u2009R. sp. Volcan Poas . The earliest divergence in the R.\u2009mexicanus species group occurred ca. 4.45 mya (95% HPD\u2009=\u20093.51\u20135.51) when R.\u2009spectabilis and R.\u2009gracilis separated from the rest of the species while the most recent divergences occurred between R.\u2009spectabilis and R.\u2009gracilis from Yucatan . Specimens of the R.\u2009mexicanus clade II did not form a monophyletic group and their relationships with the species with which they were grouped were mostly poorly supported . The IRBP tree showed minor inconsistencies with the cytb tree, and relationships between Aporodon species were, in general, better supported, compared with the Fgb\u2010I7 tree, at least for the BI analysis  or CS . For the dorsal view of the skull, there was no significant correlation between CS and shape . For the ventral view, the CS\u2010shape correlation was significant , with CS explaining only 9.10% of the shape variation hence the allometric effect was considered relatively weak.None of the skull views showed sexual dimorphism in shape  of the delimited species. For the dorsal view, pairwise comparisons showed significant differences among all putative species , a slightly narrower interorbital region (landmark 5), and the braincase with a tendency to narrow toward the caudal region of the occipital bone. Compared with R.\u2009mexicanus clade I, the R.\u2009mexicanus clade IIIB showed a narrowing of the anterior\u2010medial region of the skull  although the braincase is slightly wider toward the ends of the parietal\u2010interparietal bones. Reithrodontomys mexicanus clade IIA and R.\u2009mexicanus clade I differed in most of the landmarks that characterized the skull shape, having the latter a longer rostrum and a braincase that was wider toward the parietal\u2010interparietal region but narrower toward the curvature of the occipital bone.Significant differences were found between the cranial shape  between delimited species .In the ventral shape, only the comparison between R.\u2009mexicanus clade I for the ventral view along the positive CV1 axis . The R.\u2009mexicanus clade IIIA presented on average the highest values of annual precipitation  and precipitation of the driest month  but showed similar average values of precipitation of the wettest month, together with the R.\u2009mexicanus clade IIA . The CVA completely segregated the environmental niches of R.\u2009mexicanus clade IIIA\u2013R.\u2009mexicanus clade IIIB from those of R.\u2009mexicanus clade I\u2013R.\u2009mexicanus clade IIA, but there was still a small region of overlap in their environmental space , highly divergent from each other, which agrees with Arellano et al.\u00a0 from Risaralda (ICN16579) and Antioquia (FMNH78179) constitute a distinct species from the other South American individuals. Poorly supported phylogenetic relationships (by ML and BI methods) between these putative species could be additional evidence of recent speciation processes. However, different sources of genetic information and extensive geographic sampling are needed to obtain a more comprehensive understanding of the evolutionary history of these South American rodents.Arellano et al.\u00a0 analyzedR.\u2009mexicanus clade IIIA with morphological evidence, the ENM delimited its distribution to the northwestern region of the western and central Cordilleras of Colombia, with a habitat characterized mainly by high precipitation values. These individuals could be considered a divergent lineage that is already distinct from the rest of the South American populations in at least three species properties  were validated at the species level by genetic and ecological data, and to a lesser extent with morphometric evidence. Hooper\u00a0. However, the distribution of R.\u2009mexicanus clade IIIA was restricted to a small region of the western and central Cordillera, whereas R.\u2009mexicanus clade IIIB was distributed mainly in the Cordillera Oriental, a region not reported for R.\u2009m.\u2009milleri , distributed only in Ecuador . The congruence between the independent datasets is essential for the delimitation of this clade as a new species since until now, only molecular data had been used to differentiate it.Even though 4.2Aporodon, the R.\u2009mexicanus group currently comprises the species R.\u2009mexicanus, R.\u2009brevirostris, R.\u2009paradoxus, R.\u2009gracilis, R.\u2009spectabilis, R.\u2009darienensis, and R.\u2009garichensis. The overall distribution of this group ranges from Mexico to South America, although most species are concentrated in Central America  than to R.\u2009gracilis. This is consistent with its restricted distribution in eastern Panama  occurred at some point in the Middle Pleistocene , which fall within the intraspecific range values proposed for Reithrodontomys  to demarcate cryptic lineages at the species level has been demonstrated , R.\u2009mexicanus clade IIIA, and R.\u2009mexicanus clade IIIB. In addition, specimens from El Salvador (R.\u2009mexicanus clade IIB) should be reevaluated taxonomically including a better sampling of multiple lines of evidence. For R.\u2009mexicanus sensu stricto, additional analyses are necessary to estimate its phylogenetic relationship with respect to the subspecies R.\u2009mexicanus riparius, which was not included in our analyses but has evident geographic isolation from the other Mexican populations ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); writing \u2013 original draft (lead). Elizabeth Arellano: Conceptualization ; methodology (supporting); resources (lead); supervision (lead); writing \u2013 review and editing (supporting). Francisco X. Gonz\u00e1lez\u2010C\u00f3zatl: Conceptualization ; methodology (supporting); supervision (supporting); writing \u2013 review and editing (supporting). Sandra M. Ospina\u2010Garc\u00e9s: Conceptualization ; methodology (supporting); writing \u2013 review and editing (supporting). Duke S. Rogers: Conceptualization ; methodology (supporting); resources (supporting); writing \u2013 review and editing (supporting).DM\u2010B was supported by a Ph.D. Scholarship Program 2018\u2010000012 01NACF\u201011852LANC from Consejo Nacional de Ciencia y Tecnolog\u00eda (CONACYT), Mexico. Fieldwork season 2019\u20132020 was carried out thanks to the Latin American Field Research Award 2019 from the American Society of Mammologists.The authors declare that they have no competing interests.Appendix S1.Click here for additional data file.Appendix S2.Click here for additional data file.Appendix S3.Click here for additional data file.Appendix S4.Click here for additional data file.Appendix S5.Click here for additional data file.Appendix S6.Click here for additional data file.Appendix S7.Click here for additional data file.Appendix S8.Click here for additional data file."}
+{"text": "Late diagnosis is still a major issue in HIV infection management, leading to important consequences for both patients and community. In this perspective, HIV screening targeted on some clinical conditions (HIV indicator conditions\u2014HIVICs) emerged as a useful strategy, also involving patients not considered at high behavioral risk. We organized an in-hospital HIVICs guided screening campaign named ICEBERG in Milan, Italy, between 2019 and 2021. Among the 520 subjects enrolled, mainly presenting with viral hepatitis or mononucleosis-like syndrome, 20 resulted HIV positive . A significant proportion of them had multiple conditions and advanced immunosuppression, with 40% being AIDS-presenters. As adherence to the screening campaign was modest for non-ID specialists, educational interventions to raise clinicians\u2019 sensitivity are urgently needed. HIV-ICs guided testing was confirmed as a useful tool, but a combined approach with other screening strategies seems to be essential for early HIV diagnosis. Despite enormous progresses in HIV infection management achieved in the last 25 years, UNAIDS 2020 90-90-90 targets  were not universally achieved ,2.3. Most of them had previously sought medical attention on one or more occasion for conditions that should have prompted HIV testing [While in many countries antiretroviral (ARV) treatment availability is still limited, thus hindering the achievement of the second and third UNAIDS 90 targets, in Western Europe, availability of ARV drugs is widespread while the first target seems to be the hardest to reach. In fact, according to epidemiological European reports, up to 1 out of 7 people living with HIV (PLWH), estimated in 120,000 individuals, are not aware of their status . Moreove testing . This de testing ,6,7,8.Therefore, screening strategies and early diagnosis achievement have a major role in the effort to control and eventually stop HIV/AIDS epidemic. Since 2008, HIV indicator condition (HIV-IC) guided screening emerged as an alternative to universal and risk behavior driven testing, that showed suboptimal performances. HIV-ICs are clinical conditions more frequently observed in HIV subjects than in seronegative individuals, and their recognition should elicit HIV testing offer . SullivaDespite some differences in conditions under study, HIV-ICs guided testing is currently recommended by most international and national guidelines, especially in settings with low HIV prevalence ,16,17.Italian epidemiological reports match European data, with almost 60% of newly diagnosed patients presenting with advanced immunosuppression, and more than half of them being AIDS-presenters .Specific informed consent for HIV testing requested by Italian legislation appears as a further obstacle to early diagnosis, sometimes hampering serological screening as routine test and posing HIV on another level than other clinical conditions, contributing to stigma .The primary aim of the study was to verify feasibility and effectiveness of an in-hospital condition-guided HIV screening in terms of number of new HIV cases and immune competence upon diagnosis, evaluating its utility in early HIV diagnosis. A secondary objective was to estimate prevalence of previously undiagnosed HIV within each indicator condition.A HIV-IC guided screening campaign named ICEBERG (HIV sCreening tEst BEyond the taRGet) was organized in ASST Santi Paolo e Carlo, Milan, Italy, from January 2019 to December 2021.Infectious diseases, Dermatology, Hematology, Oncology, Neurology, Gynecology, Gastroenterology wards, and the Intensive Care Unit were involved, since HIV-ICs were expected to be most frequently observed in such units. Each unit provided a dedicated health care professional, in charge of patients\u2019 enrollment, informed consent obtaining, and HIV test prescription. The study was approved by the local Ethical Committee.As in HIV in Europe Guidance, HIV-ICs included AIDS-defining diseases, conditions that may indicate initial immunological deficit , conditions sharing same transmission route with HIV , and those in which an undiagnosed HIV infection could cause severe clinical consequences, such as neoplasm requiring chemotherapy  .Basic demographic variables including sex, age, ethnicity, and nationality besides one or more HIV-ICs, were collected in an anonymous electronic database. For subjects who resulted HIV positive, basal HIVRNA, CD4+ lymphocytes count, and most plausible transmission modality were investigated.According to Italian legislation, informed consent was obtained with opt-in modality. Fourth generation antibody-antigen combined whole blood HIV test was employed as screening assay. In case of reactivity, a second sample was collected to perform confirmatory test and Western Blot.Statistical analysis was performed with Stata , 2021). Distribution of HIV positivity by demographic variables and HIV-ICs was analyzed by Chi square test and Mann\u2013Whitney U test with 95% confidence intervals. Factors associated with HIV positive test were assessed by uni-and multi-variate logistic regression analysis.n = 170, 33%) and non-Caucasian ethnicities  were also included.From January 2019 to December 2021 520 patients presenting with a total of 628 HIV-ICs were enrolled in the study. Most of the population was composed of Caucasian males, mostly Italian, with median age 44 (IQR 32\u201359) years. However, a substantial rate of foreign patients , mononucleosis-like syndrome , and syphilis, both early and latent . Among the total cohort, 96 (19%) subjects were suffering from at least one AIDS-defining disease; in detail: 66 had tuberculosis, 2 disseminated CMV infections, 9 non-Hodgkin\u2019s lymphomas, 5 recurring pneumonia, 3 P. jirovecii pneumonia, 5 esophageal candidiasis, 6 had dementia, and 1 Kaposi\u2019s sarcoma.Most frequently encountered HIV-ICs were viral hepatitis, especially HBV  were diagnosed with a sexually transmitted infection, 148 (35%) suffered from a condition possibly related to mild immunosuppression, 63 (15%) had an AIDS-defining disease, and 6 (1%) performed HIV test before starting anticancer treatment .A total of 95 subjects (18%) presented with multiple conditions, with 82  of them presenting with 2 HIV-ICs, and the remaining 13 patients with three.Subjects presenting with an STI, or multiple conditions were frequently males, while the highest proportion of foreign patients was observed among those with AIDS-defining diseases or multiple conditions .A total of 93 out of the 95 subjects with more than 1 condition had at least 1 STI . Almost Twenty new HIV infections were diagnosed, indicating 3.8% (95%CI 2.4\u20135.9) prevalence. These patients were mainly men, Italian, with median age 39 (32\u201344) years . Most fr3 or AIDS defining disease, and among them 11 had LTCD4 count below 200/mm3, thus defined as \u2018with advanced disease\u2019, so that median LTCD4 count was 49/mm3; immunological markers were apparently worse among patients with multiple conditions or AIDS defining disease.An amount of 15 patients (80%) were late presenters, with LTCD4 count below 350/mmSex, nationality, ethnicity, and age did not emerge as significantly associated with HIV test positivity by Chi square or Mann\u2013Whitney U test, although a trend towards younger median age in HIV positive subjects was observed. Moreover, as expected, patients presenting with multiple HIV-ICs resulted more commonly HIV positive . Both unP. jirovecii pneumonia, Kaposi\u2019s sarcoma, syphilis, and mononucleosis-like syndrome; HIV infection prevalence resulted above 0.1% in thirteen conditions , and HIV prevalence among all patients affected by this condition was around 8%. Despite this clinical presentation is quite non-specific and common among the general population, it might hide both an acute retroviral syndrome and a long-course HIV infection revealing with constitutional symptoms. Similar results were presented in HIDES I and II studies, where HIV prevalence among subjects with mononucleosis-like syndrome was 4\u20135% ,11. BaseIndeed, these symptoms should prompt HIV testing in primary care facilities and/or community pharmacies in which medical staff are likely to see many cases of febrile illnesses, thus potentially increasing the rate of HIV diagnosis in the earliest stages of disease.In a recent study also investigating feasibility of HIV-ICs guided screening in a Sardinian hospital, De Vito et al. found similar results, with 3.7% prevalence of previously unknown HIV infection. Eight out of eleven HIV positive patients had advanced immunosuppression, and five of them had an AIDS-defining condition at diagnosis .Two-thirds of the study period coincided with the COVID19 pandemics. The complete reorganization of healthcare systems negatively affected all screening campaigns and all activities non-SARS-CoV-2 related ,27. AnywIn our cohort, the great majority of HIV tests were prescribed by either infectious disease specialists or by dermatologists, underlying a certain reluctance in test offer by other clinicians, not routinely involved in HIV and STI management. This could be due to a suboptimal knowledge of international recommendations for HIV screening, and of the prevalence of HIV in the disease they are expert of, but also a lack of confidence in facing such topic, commonly considered limited to some specific populations, actually contributing to HIV-related stigma. On the other hand, a British study reported how in 2017 HIV screening was not mentioned in many guidelines regarding HIV-IC management . In thisA possible further deterrent to HIV screening might have been the need for explicit informed consent and pre -test counselling, as required by Italian legislation, being a time-consuming step. This approach adopted in the pre-ART era, sometimes termed HIV-exceptionalism, was overcome in some countries such as the USA with an opt-out strategy, by which patients get tested unless actively declining .Many conditions were extremely rare in our analysis. While some of them may have been rarely observed in fact, some others, such as lymphomas, were presumably much more common than reported and likely triggered HIV test, as part of well-established clinical practice, but were not enrolled in Iceberg study for unknown reasons.Our analysis has some relevant limitations. First, no retrospective phase was designed, so that it is not possible to detect any difference in terms of HIV test coverage with respect to previous standard.Moreover, since the total number of patients with HIV-ICs and patients test refusal were not evaluated, no conclusion can be drawn about test offer nor about the gap between subjects who accepted the screening and those enrolled in the study, and selection bias cannot therefore be excluded. Third, the reasons for previous medical contact in newly diagnosed HIV-infected subjects are unavailable; a detailed analysis of these data may lead to findings in contrast to those we report in the present paper by showing that indicator condition-based testing, if broadly and consistently applied, may be extremely useful in identifying people early in the course of HIV infection.In conclusion, condition-based HIV screening confirmed to be feasible and highlighted a significant prevalence of newly diagnosed infection in healthcare settings, although immunodepression was already advanced in most cases. However, this strategy remains poorly applied by non-ID clinicians, and its implementation is key to identifying the hidden part of HIV infection. A combined approach, including out-of-hospital screening strategies involving asymptomatic subjects, or those with non-specific symptoms  in primary care facilities/community pharmacies together with changes in legislative aspects, such as opt-out modality testing and educational campaigns among non-ID clinicians, seems to be necessary for early diagnosis, achieving the first UNAIDS 95 goal."}
+{"text": "Helicoverpa armigera, otherwise known as the cotton bollworm. Acoustic bat detectors revealed that high activity of Nyctalus spp. and Tadarida teniotis bats were closely synchronized with the arrival of the migratory moths, suggesting this food source is important for both resident and migratory bats to build or maintain energy reserves. Bats of the Nyctalus spp. are likely migrating through the study site using fly-and-forage strategies or stopping over in the area, while resident T. teniotis may be exploiting the abundant food source to build fat stores for hibernation. This study shows that nocturnal migratory insects are abundant in the Pyrenees during autumn and interact during migration, not only with their co-migrant bats but also with resident bat species.High altitude mountain passes in the Pyrenees are known to be important migratory hotspots for autumn migrating insects originating from large swathes of northern Europe. In the Pyrenees, prior research has focused on diurnal migratory insects. In this study, we investigate the nocturnal component of the migratory assemblage and ask if this transient food source is also used by bat species. Three seasons of insect trapping revealed 66 species of four different orders, 90% of which were Noctuid moths, including the destructive pest  This. This2])Pipistrellus nathusii) are thought to feed while actively migrating [Nyctalus lasiopterus) are thought to have tightly comparable migration routes to those of nocturnally migrating passerines, as the bats will feed on their co-migrating birds during this period [T. brasiliensis bats are known to exploit the arrival of migratory moths to provide the nutrients they need to rear their pups [Tadarida species found in Europe (T. teniotis) although sedentary, is thought to rely on migratory insects in order to provide the nutrients needed for their autumn-born pups to develop and to stockpile enough fat reserves for winter hibernation [T. brasiliensis bats in the USA who are thought to feed on approximately four billion migratory moths per night [Bats are known to interact with nocturnal insects and other taxa while on migration in the form of predation. In Europe, insectivorous Nathusius's pipistrelles  nocturnal migratory insects would also use this route in large numbers; (ii) this transient food source would be used by both migrant and resident bats; and (iii) nocturnal weather conditions would influence the abundance of bat and insect migrants.. 2. 2.1Our study took place in the Pyrenean migration hotspot of the Pass of Bujaruelo (2273 m in elevation and 30 m in width). Although autumnal preferred migratory direction is towards the south, the pass is orientated southwest/northeast. Despite this, migration still occurs through the pass due to the channelling effect of the steep-sided Pyrenean valley, forcing the migrants through the pass under certain weather conditions . We docu. 2.2To capture the insects migrating south during the autumns of 2019\u20132021, we used a Robinson moth trap complete with a LepiLED bulb, powered by a 20 000 mAhr battery pack. The moth trap was placed in the same location each season, on the northeast edge of the pass, the light shining east-north-east down the valley towards the Cirque du Gavarnie . Each mo. 2.3Tadarida teniotis bats detected furthest from the Anabat detector [The bat activity data were collected using an Anabat Express Passive Bat Detector fitted with an omnidirectional microphone. This device records at 5\u2013250 kHz in a zero-crossing format (capturing the loudest frequency at any given moment). The detector was placed at the east opening of the pass, approximately 30 m from the moth trap\u2014greater than the 20 m minimum advised to prevent impacts of the moth trap on bat activity [detector . This liBat recordings were imported into Anabat Insight by Titley Scientific (version 1.9.0). All recordings were run through a filtering system, separated by the genus/species specific metrics of their calls. Only four species/groupings were used in the analysis due to data insufficiency. This form of filtering only allows the addition of \u2018species tags' to recordings which may contain any number of species. To combat this, the raw filtered data were extracted to Excel and imported into RStudio and were filtered once again using the same call metrics, on a per pulse basis as opposed to a full recording . This cr. 2.4r-value) and an associated p-value. A GLM was fitted with a log link function and quasi-Poisson family to test the factors affecting the bat numbers. The explanatory variables were the same as the nocturnal insect analysis with the addition of nocturnal insect counts as an explanatory variable.Nocturnal weather conditions including windspeed, wind direction, temperature, cloud cover and total precipitation were obtained from meteoblue weather simulations and recorded between 19.00 and 07.00 . To test. 3. 3.1a). The Noctuidae dominated the nocturnal insects with 90% of the total assemblage (b). The majority (92.7%) of the insects trapped were moths (Lepidoptera), but Limnephilidae caddisflies (Trichoptera) (6.6%), Diptera (0.6%) and Hymenoptera (0.04%) were also recorded . The most numerous species were Helicoverpa armigera , Mythimna vitellina  and Noctua pronuba  (c). For a full species list, see electronic supplementary material, tables S1 and S2.A total of 4187 insects of four orders, 13 families and 66 species (or morpho-species) were collected in the Robinson moth trap over the course of the three seasons from a total of 90 trapping nights, averaging 30 nights per season. There was substantial variation between number of insects trapped per evening with a range of 0\u2013400 and an average of 47 (s.e. \u00b1 7.5) individuals a. The Nosemblage b. The mang, 12%) c. For a . 3.2p < 0.001) (d\u2013f). Temperature positively affected the nocturnal insect count number  while we found a negative effect of precipitation and windspeed on the number of migrating insects . Next, we used AIC model selection to distinguish between various models describing the relationship between insect number and meteorological variables. The best-fit model, carrying 64% of the cumulative model weight, was average nightly temperature. The results of the model selection are displayed within figure 2g; Rayleigh test: 211\u00b0, r = 0.7783, p < 0.001).To investigate the environmental factors important for migration, we analysed the influence of average nightly temperature, total nightly precipitation and average nightly windspeed on the numbers of nocturnally migrating insects. All of these explanatory variables were found to significantly affect the number of nocturnal insects caught migrating through the Pass of Bujaruelo (< 0.001) d\u2013f. Temp. 3.3Nyctalus leisleri/noctule) group, the European free-tailed bat (T. teniotis), the western barbastelle (Barbastella barbastellus) and the common pipistrelle (Pipistrellus pipistrellus) were analysed further. Nyctalus spp. activity was 2.5\u00d7 higher than the next most common group of bats (T. teniotis).To investigate the presence of both resident and migratory bats on moth migration nights through the pass, we collected bat activity data concurrent with moth trapping during the 2019 migration season. A total of 463 511 bat call pulses were recorded with an average of 20 152 per evening and a range of 460\u2013111 337, indicating substantial variation in activity. A total of seven species were identified  and the . 3.4Nyctalus spp. activity levels at the pass were significantly influenced by increased nocturnal insect count  (b) and this remained the case after removal of two outlier peak days . Visualization of peaks of Nyctalus spp. activity and nocturnal insect count reveal clear synchronicity (see peaks on 8 and 19 September in figure 3a). Feeding buzzes of Nyctalus spp. bats were only recorded when insects were present. Of the other groups, only Tadarida activity had significant correlation with nocturnal insect count  (figure 3c), where again higher numbers of insects coincided with increased bat activity levels. Feeding buzzes of T. teniotis were also recorded during these peak dates (figure 3a) but also on days when insects were not caught, suggesting that the bats were feeding on nocturnal insects not attracted to the light from the moth trap. As with the Nyctalus spp. activity, even after the removal of the two outlier data points, Tadarida activity had significant correlation with insect count . Neither the P. pipistrellus nor the Barbastella barbastellus species were significantly correlated with nocturnal insect counts. All bat group activity was found to significantly increase when the wind was blowing from a southwesterly direction . For a full breakdown of the results, see electronic supplementary material, Results: \u2018Influence of meteorological conditions on bats'.The  0.0005) b and thi. 4H. armigera, Mythimna vitllina and Noctua pronuba [Rhyacia simulans), has no previous evidence of migratory behaviour, but it has long been suspected of being a migrant to the UK [We monitored nocturnal migration through the Pyrenees in autumn, focusing on nocturnal insects moving through the Western European flyway and bat activity measured at a pass known to concentrate diurnal insect migrants. During our observation period of three consecutive autumns (September to October) in the years 2019\u20132021, an average of 1396 nocturnal insects were trapped each season, comprising of four orders, 13 families and 66 species. While the majority of the common nocturnal insects caught were well-known migrants such as  pronuba \u201331, the o the UK . Rather Nyctalus leisler/noctula species group, the European free-tailed bat T. teniotis, P. pipistrellus and B. barbastellus. Of the four species groups analysed, only the Nyctalus spp. and P. pipistrellus are known to be migratory [Nyctalus spp. and T. teniotis bats with peaks in the number of nocturnal insects. When removing the high-leverage data points from the analysis, the relationship between these bat species and the number of nocturnal insects remained significant. While the co-occurrence of bats and moths strongly suggests interaction, particularly as peaks of migrating insects coincided with increased numbers of feeding buzzes, we were not able to collect direct evidence, for example from faecal samples. By contrast, neither P. pipistrellus nor B. barbastellus activity synchronized with migratory movements of moths.During 2019, bat activity was also monitored and eight species were recorded, with the four most active being the igratory \u201335. Unfo. 4.1P. pipistrellus activity , no activity \u201341.Nyctalus spp. and T. teniotis bat activities were positively correlated to insect numbers. However, the major activity peak of P. pipistrellus and B. barbastellus occurred during a period when no migratory insects were caught. As a migratory species, P. pipistrellus may be simply migrating through the pass, uninfluenced by the insect migrants but possibly influenced by the lower windspeeds. By contrast, B. barbastellus bats are non-migratory and may be using the pass for a different reason. B. barbastellus bats near exclusively forage just above a forest's canopy [B. barbastellus may be using the pass to traverse between forest habitat or to search for hibernacula [s canopy ,43 and ternacula ,43. The . 4.2Nyctalus spp. bats  in America are thought to rely on the seasonal pulse of southward migrating moths as a form of sustenance while other prey is scarce, to build up reserves prior to the bats' own migration [T. teniotis may more generally be using this influx of migratory insects by using other passes and even feeding when insects are not forced into these passes by strong headwinds. This may be why feeding buzzes are recorded on nights of low moth numbers, where the detectors are still registering bat calls from above and around the pass. This species has previously been shown to use topography, for example through orographic lift, in order to fly at high altitudes, while also reaching maximum sustained self-powered airspeeds of 135 km h\u22121 [ samples . Tadaride autumn , meaningigration ,10,11. I5 km h\u22121 . Therefo. 4.3H. armigera). This species is highly polyphagous as a larva and is a major pest of crops, as are many other of the commonly recorded nocturnal insects in this study [Plutella xylostella). This moth is expected to expand its range northwards and increase its populations due to climate change [The insects caught migrating nocturnally played a range of ecological roles including as pests (79%), pollinators (25%) and in nutrient transfer (100%). The most numerous species comprising 31% of the assemblage was the cotton bollworm  can eat four billion Helicoverpa zea-sized insects  every night [Predation of these pest species by the the bats . An Amerry night . Therefo. 5Our 3-year study of nocturnal insects migrating through a Pyrenean mountain pass has revealed a remarkable diversity of taxa, including many important pest species, following the Western European flyway south in the autumn and provides an important baseline for future comparisons. The relationship between the nocturnal insect migrants and the resident and migratory bat species described here may have important impacts for both the animals and the surrounding ecosystems. Human activities, such as habitat destruction and climate change, light pollution and the presence of wind turbines, have the potential to disrupt these interactions ,62\u201366. A"
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