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+{"text": "We have previously documented significant differences in orthogonal P wave morphology between patients with and without paroxysmal atrial fibrillation (PAF). However, there exists little data concerning normal P wave morphology. This study was aimed at exploring orthogonal P wave morphology and its variations in healthy subjects.120 healthy volunteers were included, evenly distributed in decades from 20\u201380 years of age; 60 men (age 50+/-17) and 60 women (50+/-16). Six-minute long 12-lead ECG registrations were acquired and transformed into orthogonal leads. Using a previously described P wave triggered P wave signal averaging method we were able to compare similarities and differences in P wave morphologies.Orthogonal P wave morphology in healthy individuals was predominately positive in Leads X and Y. In Lead Z, one third had negative morphology and two-thirds a biphasic one with a transition from negative to positive. The latter P wave morphology type was significantly more common after the age of 50 (P < 0.01). P wave duration (PWD) increased with age being slightly longer in subjects older than 50 . Minimal intraindividual variation of P wave morphology was observed.Changes of signal averaged orthogonal P wave morphology , earlier reported in PAF patients, are common in healthy subjects and appear predominantly after the age of 50. Subtle age-related prolongation of PWD is unlikely to be sufficient as a sole explanation of this finding that is thought to represent interatrial conduction disturbances. To serve as future reference, P wave morphology parameters of the healthy subjects are provided. Atrial fibrillation (AF) is the most common sustained arrhythmia of clinical importance. Over the last couple of decades, many important findings have contributed to the enhanced understanding of AF pathophysiology, but much still remains to be clarified. AF is normally characterized as permanent, persistent or paroxysmal (PAF), with the latter being more common in younger persons. Both variants might be asymptomatic but, unfortunately, PAF is none the less still associated with considerably increased mortality and morbidity, thus implying the need for early diagnosis and subsequent prophylactic measures .Using band-pass filtered signal averaging technique, P wave duration (PWD) has been studied in PAF patients with inconsistent findings regarding possible clinical applications -7. The mThe study population was recruited by advertising in local press. The subjects were interviewed and examined by a physician to ensure good health. A 12-lead ECG was performed and blood pressure was measured. Exclusion criteria included history of cardiovascular disease , excessive use of alcohol, or a pathological 12-lead ECG at rest, including non-sinus rhythm. A total of 120 healthy individuals were included. The study was approved by the Ethics Committee of Lund University . Written informed consent was obtained and the study complied with the Declaration of Helsinki.A standard 12-lead ECG was recorded for 6 minutes with the subjects at rest. Data was acquired at a rate of 1 kHz and with a resolution of 0.625 \u03bcV. The data acquisition board used for the recordings was supplied by Siemens-Elema AB, Solna, Sweden.\u00ae , was used for signal processing and analysis. The method has been described in detail earlier ) was defined as the difference, in time, between the P wave onset and end.In Leads X and Y, the maximum amplitude was determined  and also the distance from the onset of the P wave to the position of these maxima . In Lead Z, the maximum and minimum amplitude was determined  along with their locations . In addition to this, the location of the zero crossing in Lead Z was measured when applicable (Z zero pos). The different parameters and a schematic illustration of the method used are shown in Figure All data is presented as mean \u00b1 standard deviation unless stated otherwise. Statistic evaluation was performed using the software StatView 4.5 . The Mann-Whitney U-test and Kruskal-Wallis one-way analysis of variance were used for comparisons between populations of continuous data. For comparisons between groups of proportions the Chi-square test was used. A P-value less than 0.05 was considered statistically significant.The study group was comprised by 60 men (age 50 \u00b1 17 years) and 60 women (50 \u00b1 16), (n.s.). The subjects were evenly distributed in groups of ten men and women for every decade from 20 years of age to 80. All subjects presented data, i.e. 6-minute registrations, available for analysis.The males were significantly taller and heavier than the females . They also had lower heart rate  and higher systolic and diastolic blood pressure . With increasing age, a slight increase of systolic and diastolic blood pressure was also observed but still within normal limits.P wave duration (PWD) was longer in males . PWD also increased significantly with increasing age (P < 0.05). When comparing PWD between subjects aged below and above 50, a slight but significant increase was observed in the older group . Baseline data is presented in Table With the exception of higher P wave amplitude in Lead Y (Y max) for females compared to males, the only significant gender or age-differences regarding amplitudes or positions of P wave maxima or minima were seen in Lead Z. Here females had lower amplitudes of maxima and also earlier location of minima. With older age, the amplitude of the maxima in Lead Z increased while the zero-crossing was located earlier thus making the positive phase of the P wave in Lead Z greater. The numerical data and P-values of PWD as well as the amplitudes and temporal markers of X max, Y max, Z max, Z min and Z zero are shown in Table Of the 120 subjects, 118 had P wave morphology types consistent with those observed in earlier reports; 45 Type 1 , and 73 Type 2 . Two subjects  had P wave morphologies that were classified as atypical, i.e. neither Type 1, 2 or 3.When comparing the distribution of P wave morphology types in the different age groups it was found that the Type 2 morphology was more common with increasing age (P < 0.01). There was also a significant (P < 0.001) increase in Type 2 morphology in subjects older than 50 when comparing equally large groups of subjects younger and older than 50. In the younger group 26 of 60 cases of Type 2 morphology were observed in comparison to 50 of 60 in the older group. The distributions of P wave morphology types in different age groups are illustrated in Figure In 92 % of the cases only one group of P wave morphologies was observed during 6-min long recording. In the remaining cases (8%), additional groups of P waves with a cross-correlation coefficient < 0.90 were obtained. The P waves from different groups appeared to be very similar in shape and of the same morphological type Figure .In the present study, orthogonal ECG registrations from 120 healthy adults of varying age were analysed using PSAECG. Two main P wave morphologies were observed (Type 1 and 2). Type 2 morphology, associated with PAF and other arrhythmia-prone patients, was more common in healthy subjects than earlier perceived  and highIn a report on the anatomy of the human atria Ho and co-workers denoted BB, the muscular fibre band that abridges the anterior inter-atrial groove, the \"superhighway for inter-atrial conduction\" . HoweverFrom an electrophysiological standpoint, BB has been considered the preferential conductive route from RA to LA but other transseptal pathways, close to Koch's triangle and also in the CS area have been recognized . The funPSAECG has previously been used by our group to study orthogonal P wave morphology in patients with PAF, HCM and control patients ,21. ThreIn the reported material, Type 2 morphology is significantly more common with increasing age with a marked increase in prevalence after the age of 50. We did not observe any unexpected changes of the characteristics of the study group that would explain these findings that implicate altered RA-LA activation. For instance, only slight prolongation of mean PWD (\u0394PWD = 7 ms) with age was observed and the longer PWD in men could possibly be explained by their heavier stature. As explained above, interatrial conduction can occur via several pathways. One could speculate that age-related changes in the human heart such as fibrosis that have been reported and suggested to play a role in atrial reentrant mechanisms  could afHowever, other causes of altered P wave morphology are also plausible. These include variations in the atrial pacemaker complex and sinus node morphology but also differences of conduction velocities . FurtherHowever, intraindividual variation of P wave morphology during 6-min long recordings was minimal in the present study. This could indicate that interatrial conduction propagation on an individual basis is stable and that the changes that occur are stable as well. These age-related changes in P wave morphology are therefore unlikely to be caused solely by the age-related decrease in conduction velocity reflected in marginal prolongation of PWD, but could best be explained by changes in the function of interatrial conductive pathways.Even though the selection process was aimed at including only healthy asymptomatic subjects with normal blood pressure, no known diseases and not taking any medication it cannot be ruled out that some subjects with asymptomatic AF may have been included. However, in our opinion possible impact of these single individuals on the overall results would be negligible. Furthermore, influence of atrial dimensions on P wave morphology that has been demonstrated earlier cannot be excluded since echocardiography data for the study group was not available.The presented study describes orthogonal P wave morphology of a material comprised of healthy men and women and provides a reference material for future research. Changes of signal averaged orthogonal P wave morphology , earlier reported in PAF patients, are common in healthy subjects and appear predominantly after the age of 50. Subtle age-related prolongation of PWD is unlikely to be sufficient as a sole explanation of this finding that is thought to represent interatrial conduction disturbances. However, further studies are needed to determine the electrophysiological explanation for these changes and its possible role as a substrate for arrhythmia.The author(s) declare that they have no competing interests.RH participated in the processes of study design, data acquisition and analysis. JC, AH and PP contributed to study design, data acquisition and analysis. CM and BO took part in study design and analysis. All authors participated in the process of drafting the manuscript as well as read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Heart chambers rupture in blunt trauma is uncommon and is associated with a high mortality. The determinant factors, and the incidence of isolated heart chambers rupture remains undetermined. Isolated rupture of the right atrium appendage (RAA) is very rare, with 8 cases reported in the reviewed literature. The thin wall of the RAA has been presumed to render this chamber more prone to rupture in blunt trauma.To report a case of isolated RAA rupture in blunt trauma, and to compare right atrium (RA) and RAA wall thickness in a necropsy study.The thickness of RA and RAA wall of hearts from cadavers of fatal penetrating head trauma victims was measured. Our case of isolated RAA rupture is presented. The main findings of the 8 cases reported in the literature, and the findings of our case, were organized in a table.The comparison of the data showed that wall thickness of the RAA (0.53 \u00b1 0.33 mm) was significantly thinner than that of RA (1.11 \u00b1 0.42 mm) (p < 0.05).In all these 9 cases of isolated RAA rupture, cardiac tamponade occurred, RAA rupture was diagnosed intraoperatively and sutured, and the patients survived. Main mechanisms hypothesized for heart chamber rupture include mechanical compression coincident with phases of cardiac cycle, leading to high hydrostatic pressure inside the chamber. Published series include numerous cases of RA rupture, and only a few cases of RAA rupture.Thus, our data suggests that wall thickness is not a determinant factor for RA or RAA rupture in blunt trauma. Traumatic rupture of the heart occurs in 0.5% blunt trauma (BT)  cases, wobjectives of this study were: 1) To compare the thickness of the RAA and RA walls of hearts of lethal head trauma victims; 2) to present a case report of isolated BT rupture of the RAA, and discuss the role of wall thickness as a possible factor.Among the diverse mechanisms proposed for traumatic rupture of heart chambers ,9,12,13,To evaluate the wall thickness of RAA and RA of trauma victims, twenty-eight hearts that had been removed during autopsy, at the Legal Medicine Institute of the USPSM, from cadavers of penetrating lethal head trauma victims aged from 14 to 84 (mean 31) years, were studied. The hearts were preserved in formaldehyde for anatomic study at the MAPD of the INCOR, CH-USPSM. The study was performed according to the norms of the Ethics Committee of the institution. With the use of a pachymeter, the RAA wall thickness was measured at the apex of this chamber. The RA wall thickness was measured at the antero-superior wall of the RA, at 1 cm from the base of the RAA. The data obtained for the RA and RAA wall thickness (mm) were expressed as mean \u00b1 SEM, and compared using Student's t test. P values < 0.05 were considered significant.The wall thickness obtained for the RA was 1.11 \u00b1 0.42 mm, and for the RAA was 0.53 \u00b1 0.33 mm , and the comparison of these results showed that RAA thickness was significantly smaller than that of RA (p < 0.05).20. Cardiac tamponade was suspected, and confirmed through a subxiphoid pericardial window. A thoracotomy was performed, with pericardiotomy and removal of blood clots from the pericardial cavity. A 0.5 cm bleeding tear in the apex of the RAA was found, and closed with sutures. The thoracotomy incision was closed. The patient was discharged from the hospital on the 20th postoperative day. His injury severity score (ISS) was 50, and his TRISSCAN was 0.31.A 16-year old male, unrestrained passenger was a victim of high speed frontal collision of the car with an electricity pole, and 30 min thereafter was admitted to the emergency room (ER). On admission, he was complaining of thoracic and abdominal pain, and presented free airways, normal chest expansion, BP:70 \u00d7 40 mmHg, HR: 120 bpm, RR: 18 epm, GCS: 15, RTS: 6.4, abdominal tenderness, and a closed right femur fracture. Plain X ray films suggested a widened mediastinum. After 3000 mL of warmed saline infusion, his BP was 60 \u00d7 30 mmHg, and his HR was 120 bpm. A diagnostic peritoneal lavage was positive for blood. As ultrasonography was unavailable, a laparotomy was performed, with splenectomy and cauterization of a superficial liver injury. After infusion of 6000 more mL saline, 8 units PRBC, and 9 units fresh frozen plasma, the patient's conditions worsened, with muffled heart sounds, undetectable peripheral pulses, and CVP of 20 cmHThe main data of this case are presented (see Table The 9 cases (see Table) comprised 5 men and 4 women, aged 15 to 34 (mean: 28) years, all involved in high speed MVC (8 cars and 1 motorcycle). The victims were a motorcyclist (case 1), unrestrained car drivers , or passengers (cases 7 and 9), or unspecified position (case 6). Overall, the main diagnostic findings in these 9 cases included hypotension or persistent unresponsive shock , muffled heart sounds , high CVP values , neck vein distension , thoracic imaging alterations , and traumatic ascitis (case 7). Cardiac tamponade was present in the 9 cases. All the patients underwent a thoracotomy allowing RAA injury identification and repair, and all patients survived. The length of hospital stay ranged from 8 to 20 days.Blunt trauma rupture of heart chambers is more frequent in MVC with great deceleration leading to high energy impact of drivers' thorax on the steering wheel -20. In tAlthough not supported by clinical or experimental data, diverse mechanisms were proposed for heart rupture in BT, including: sudden deceleration , the occAccording to some authors, ventricular versus atrial rupture depends on the phase of the cardiac cycle, atrium rupture being more likely to occur under a forceful compression in late systole, when the atria are most distended with venous blood, and the atrioventricular valves are closed ,24. Giveconclusion, the findings of our study indicate that the RAA wall is thinner than that of the RA. However, given the reported higher incidence of RA rupture than of RAA rupture, our findings do not suggest wall thickness as a predominant factor either for RA or RAA rupture in blunt trauma.In Concerning the present manuscript, the authors declare that there are no personal, or financial, or non-financial competing influence, interests, or conflicts involving them with other people or organizations."}
+{"text": "Dendritic cells (DC) are important for MHC class I processing and presentation of peptide epitopes to memory CD8+ T cells, and could potentially be targeted to activate memory CD8+ T cells to a broad array of HIV-1 epitopes during ART.HIV-1 remains sequestered during antiretroviral therapy (ART) and can resume high-level replication upon cessation of ART or development of drug resistance. Reactivity of memory CD8+ T cells specific for a much broader range and magnitude of Gag and Nef epitopes than do peptides without DC. The DC also reveal novel, MHC class I restricted, Gag and Nef epitopes that are able to induce polyfunctional T cells producing various combinations of IFN gamma, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1 beta and the cytotoxic de-granulation molecule CD107a.We show for the first time that HIV-1 peptide-loaded, CD40L-matured DC from HIV-1 infected persons on ART induce IFN gamma production by CD8+ T cell reactivity in persons on ART that is revealed by DC. This supports the use of DC-based immunotherapy for HIV-1 infection.There is an underlying, broad antigenic spectrum of anti-HIV-1, memory CD8 Our results show that HIV-1 peptide-loaded, mature DC induced IFN\u03b3 production to a much broader range of HIV-1 Gag and Nef epitopes than did peptides without DC. The MHC class I restricted Gag and Nef epitopes included novel ones that could activate polyfunctional T cells producing various combinations of IFN\u03b3 interleukin 2 (IL-2), TNF\u03b1, macrophage inhibitory protein 1\u03b2 (MIP-1\u03b2) and the cytotoxic de-granulation molecule CD107a. This indicates that there is a broader and more robust array of memory CD8+ T cells specific for HIV-1 antigens circulating in persons on ART than has previously been appreciated, and supports use of DC-based immune therapies.Induction of broad and robust T cell reactivity could be particularly important in immunotherapy of HIV-1 infection during antiretroviral therapy (ART) This research was part of the Pittsburgh Multicenter AIDS Cohort Study (MACS), an investigation of the natural history of HIV infection, and was approved by the University of Pittsburgh Institutional Review Board. 7 HIV-1 seropositive homosexual men on ART were randomly selected for study from the Pittsburgh, PA, portion of the MACS . Four HI+ monocytes were positively selected from peripheral blood mononuclear cells (PBMC) using anti-CD14 monoclonal antibody (mAb)-coated magnetic microbeads  to a purity of >96%, cultured for 5 to 6 days in AIM V medium  containing 1000 U/ml of recombinant IL-4  and 1000 U/ml of recombinant granulocyte-monocyte colony stimulating factor (GM-CSF) . Fresh IL-4 and GM-CSF were added every other day. The DC were treated with maturation factor CD40L  for 40 h to induce DC maturation.To obtain immature DC, CD14The number of viable DC was determined by typical morphology in trypan blue dye-stained preparations. The maturation status of the DC was determined by flow cytometry as the percent positive and mean fluorescent intensity of expression of MHC class II (HLA-DR), MHC class I (HLA ABC), CD80, CD86 and CD83. Viable DC displayed a characteristic DC morphology and cell surface marker expression and responded to stimulation with CD40L.+ T Cell Epitope Database A library of HIV-1 peptides (consecutive 15mers overlapping by 11 amino acids) spanning the consensus B HIV-1 proteome was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. These were used as singlets or in pools of consecutive peptides. Known, \u201cA-list\u201d epitopes were identified by the Los Alamos CTL/CD8\u00b0C. Responder cells were autologous PBMC or CD8+ T cells (96\u201398% pure) positively selected from PBMC using anti-CD8 mAb-coated, magnetic microbeads (StemCell). The peptide-loaded DC (stimulators) were washed to remove excess peptide and mixed with responder cells at a responder-to-stimulator [R:S] cell ratio of 10 to 1 and incubated for 18 h with peptide-loaded DC at 37\u00b0C in a 5% CO2 atmosphere. The wells were washed with PBS and treated with biotinylated anti-IFN-\u03b3 mAb  and incubated at 37\u00b0C for 3 hours. Avidin-peroxidase (100 \u00b5l/well) was added after the biotinylated antibody was decanted and the plates were washed four times with PBS-0.05% Tween 20 . Diaminobenzidine solution  was added to each well for 5 minutes at room temperature. The plates were washed and air-dried overnight. A negative control (medium without peptides), and 2 positive controls \u2013 CEF (1 \u00b5g/ml), which is a mixture of human cytomegalovirus, Epstein-Barr virus and influenza A virus 6 cells. The results were considered positive if the number of SFC in the peptide-stimulated cultures was more than 50 and above the mean plus two standard deviations of SFC in cultures with medium alone.An ELISPOT assay modified from AIDS Clinical Trials Group protocol A5181 was used to determine single cell IFN\u03b3 production In some experiments, HLA restriction of the T cell responses was confirmed by ELISPOT assay using a panel of EBV-transformed B cell lines (BLCL) matched with the effector cells at only one MHC class I allele.6/ml in RPMI-10% FCS and rested overnight at 37\u00b0C. The PBMC were then cultured with 2 \u00b5l each of T cell costimulatory mAb specific for CD28 and CD49d , monensin  and brefeldin A  to inhibit extracellular release of the immune mediator CD107a-PECy5 , and peptides or peptide pools (5 \u00b5g/ml). In some experiments, DC loaded with peptide (5 \u00b5g/ml) were used at a 1\u223610 ratio with PBMC but without \u03b1CD28/49d. Negative controls (without peptides) and positive controls  were included in each assay. Cells were incubated for 6 h at 37\u00b0C and then kept at 4\u00b0C for 16 h. The cells were washed, fixed using the Cytofix/Cytoperm kit (BD PharMingen) and stained with mAb CD8-APC Cy7, CD4-APC Cy7, IL-2-APC (BD Biosciences), CD3-PE Cy7, IFN\u03b3-FITC, MIP-1\u03b2-PE (BD PharMingen) and TNF\u03b1-PB (eBiosciences). Following staining, the cells were washed, fixed and analyzed with an LSR II flow cytometer (BD Immunocytometry Systems), with 200,000 to 1,000,000 events collected per sample. T lymphocyte subsets were analyzed by first identifying and gating the whole lymphocyte population according to light scatter properties (FSC and SSC), followed by gating T cell subsets based on the expression of the surface markers CD3 and CD8, as well as the intracellular expression of IL-2, IFN\u03b3, TNF\u03b1, MIP-1\u03b2 and CD107a compared to negative controls. All data were background-subtracted using the non-antigen stimulated control and analyzed by FlowJo  and SPICE (version 4.1.6). The expression of CD107a, IFN\u03b3, IL-2, MIP-1\u03b2, TNF\u03b1 and T cell surface markers was quantified separately and in combination.Frozen-thawed PBMC were suspended to 2\u00d710+ T cell responses to HIV-1 peptides are stronger in the presence than in the absence of DC; (b) CD8+ T responses to HIV-1 peptides exceed PBMC responses both in the presence and in the absence of DC; and (c) relative changes in the level of response to HIV-1 peptides is highly correlated between CD8+ T cells and PBMC both in the presence and the absence of DC. The first two hypotheses were tested using the binomial sign test. In each case, the peptide data was transformed into a binary variable and tested against the null hypothesis (H0\u200a=\u200a0.5) using a one-sided alternative. For the first two hypotheses, we also calculated mean ratios and 95% confidence intervals in order to provide a better sense of the size of the comparative responses. For the third hypothesis, we ranked the size of the response observed to each of the HIV-1 peptides by PBMC and CD8+ T cells both in the presence and the absence of DC. Under both of the latter conditions (presence or absence of DC), we calculated Spearman rank order correlations between the PBMC and CD8+ T cell responses and the statistical significance  of each correlation. We used the Scheffe multiple comparison test and chi-square test for analysis of the polyfunctional T cell results.Statistical analyses were carried out to test three specific hypotheses: (a) PBMC and CD8+ T cell responses + T cells from 3 HIV-1 seronegative, uninfected persons, with or without DC (data not shown). Furthermore, using purified CD8+ T cells as responders, we confirmed that the broad reactivity induced by the peptide-loaded DC was mediated by CD8+ T cells, as there was a correlation between the total response of the purified CD8+ T cells and the PBMC to the HIV-1 peptide pools   for the peptides in the absence of DC, and 1.57  in the presence of DC.We first examined the effects of DC loaded with a library of overlapping 15mer peptides spanning the HIV-1 proteome arranged into 29 pools of 19\u201332 peptides each on T cell reactivity in HIV-1 infected persons on ART. We have previously shown that CD40L-matured DC loaded with pools of \u226432 HIV-1 peptides are optimal for stimulation of CD8P<0.001) . This en+ T cells in the PBMC cultures. These immune responses were of significantly greater magnitude and breadth across the whole HIV-1 proteome (except Vpu) compared to those induced by conventional stimulation with peptides without DC in HIV-1 infected persons on ART.These results indicate that the IFN\u03b3 production in response to DC loaded with HIV-1 15mer peptides was produced mainly by CD8+ T cell counts or viral load in the 7 HIV-1 infected subjects (data not shown).We next focused on HIV-1 specific IFN\u03b3 production in response to CD40L-treated DC that were loaded with single peptides spanning 49 consecutive HIV-1 Nef 15mers overlapping by 11 amino acids in HIV-1 infected persons on ART. As expected, the number of positive responses to Nef varied among these genetically disparate study subjects . Of the Based on MHC class I alleles of the 7 subjects, we next determined the known and potential Nef epitopes associated with the T cell responses. There were 19 known Nef epitopes matched for the MHC class I alleles of the 7 subjects within the 42 Nef 15mers that induced positive responses . Of thesTaken together, these results show that DC from HIV-1 infected subjects on ART can process 15mer peptides for stimulation of responses against a broad range of known and potential Nef epitope in greater magnitude and breadth compared to that stimulated by these Nef peptides without DC.73\u201387 (QVPLRPMTYKAAVDL) peptide. We found that stimulation with peptide-loaded DC, but not with peptide without DC, resulted in positive IFN-\u03b3 responses in PBMC from 3 HIV-1 infected subjects who shared HLA B*2703  stimulated the highest levels of IFN\u03b3 compared to N and C terminal extended and truncated peptides, in a concentration-dependent manner  detected by the ELISPOT assay, i.e., Nef69\u201383, Nef73\u201387 and Nef77\u201391, induced the greatest polyfunctional CD8+ T cell responses when presented without DC (P<0.01 compared to N flanking Nef65\u201379) . With DC, the Nef69\u201383 and Nef73\u20138715mers induced the greatest polyfunctional CD8+ T cell responses (P<0.05 compared to Nef65\u201379 and Nef77\u201391)  (+ T cell response to DC loaded with each of the 5 8-10mer variants (P\u200a=\u200a ns)  , no DC. ef77\u201391) , DC. Amo\u200a=\u200a0.07) , no DC. P\u200a=\u200a ns) , DC. Mos76\u201384, by a conventional ELISPOT in individuals on ART that was unrecognized using direct stimulation of PBMC with peptide alone. Monofunctional and polyfunctional ICS responses supported that these overlapping Nef 15mers contained a dominant CD8+ T cell epitope. However, a range of less definitive, CD8+ T cell activity was noted against Nef76\u201384 as well as to the N and C terminal variants, with and without DC.These results show that DC revealed a new, HLA B*2703 epitope, NefWe next determined HIV-1 specific IFN\u03b3 production in response to CD40L-treated DC that were loaded with single peptides spanning HIV-1 Gag (122 consecutive 15mers overlapping by 11 amino acids) in HIV-1 infected persons on ART. Overall, there were responses to 80 of the 122 peptides by PBMC from the 7 subjects presented with or without DC . SubjectOf the total of 854 possible responses to the 122 Gag peptides among the 7 subjects, there were 114 (13.3%) responses to DC loaded with Gag peptides compared to only 14 (1.6%) responses to peptides without DC (P<0.001). There were 24 (2.8%) common responses to peptides with and without DC. Higher magnitude responses were observed to DC loaded with peptides compared to peptides without DC (P<0.001). DC enhanced the number of Gag peptide-responding T cells by an average of 8.5 fold (P<0.001).+ T cell counts or viral load in the 7 infected subjects (data not shown). Repeat testing of different blood samples from 2 of these subjects resulted in IFN\u03b3 production comparable to the previous responses . IFN\u03b3 production was HIV-1 immune specific, as there was little or no IFN\u03b3 production induced by the Gag peptides with or without DC in 4 HIV-1 negative controls (data not shown).Enhanced T cell responses to Gag were not associated with CD45\u201331, p1773\u2013103, p245\u201327, p2457\u201395, p24117\u2013179 and p24205\u2013227. The breadth of responses varied across subjects  that elicited positive IFN\u03b3 responses with DC in 4 of 7 subjects, and its C terminal overlapping peptide p1721\u201335 (LRPGGKKKYKLKHIV), which was positive in 2 of 7 subjects using DC . Analysis using the HLA Binding Motif Scanner 23\u201331 (PGGKKKYKL) was a potential new epitope for HLA B*5101. ELISPOT results for the predicted epitope showed that the 10mer sequence p1722\u201331 (RPGGKKKYKL) stimulated IFN-\u03b3 production without DC in a concentration-dependent manner      : no DC. o no DC) : DC. Of P\u200a=\u200a ns) : no DC. o no DC) : DC.22\u201331 as a novel, optimal 10mer epitope restricted by HLA B*5101. Polyfunctional CD8+ T cell responses induced by the 15mer peptide-loaded DC supported this peptide as the optimal epitope, but were less discriminatory among the 8-10mer, N and C terminal variants presented with or without DC.Thus, using DC and single cell IFN-\u03b3 production, we identified Gag p17161\u2013175 (FRDYVDRFYKTLRAE), 5/7 to p24165\u2013179 (VDRFYKTLRAEQASQ) and 4/7 to p24169\u2013183 (YKTLRAEQASQEVKN), whereas there were no responses to either the N or C terminal flanking 15mers (161\u2013169 (FRDYVDRFY) was a potential new epitope restricted by HLA A*0101, and p24173\u2013181 (RAEQASQEV) for HLA B*5101.A cluster of positive responses resulted from stimulation with peptides 74\u201376 from the p24 region of Gag, with 4/7 subjects responding to p24g 15mers . 12 of 1161\u2013169 showed that there was little or no IFN-\u03b3 production induced at any peptide concentration without DC in subject 2 , one aa longer than the predicted, optimal HLA B*5101 epitope p24173\u2013181  (+ T cells by p24165\u2013179 (P\u200a=\u200a0.01 compared to p24161\u2013175) and p24169\u2013183 (P\u200a=\u200a0.07 compared to p24161\u2013175)  , no DC, 161\u2013175) , DC.161\u2013169 (FRDYVDRFY) peptide and its 8-10mer peptide variants showed no distinct, polyfunctional CD8+ T cell responses (P\u200a=\u200a ns)  , no DC. P\u200a=\u200a ns) , no DC. P\u200a=\u200a ns) , DC.+ T cell responses were induced by the 5 peptides without DC derived from the putative optimal 9mer p24173\u2013181 peptide, with no distinct response to a particular peptide (P\u200a=\u200a ns)  , No DC. P\u200a=\u200a ns) , DC. In 161\u2013169, and a novel HLA B*5101 10mer epitope p24173\u2013182. N and C terminal variants of these optimal peptides were able to induce appreciable levels of IFN-\u03b3 detected by ELISPOT assay only when using DC as APC. There was no clear immunodominance of these 2 Gag p24 epitopes compared to their 8-10mer variants detected by monofunctional or polyfunctional CD8+ T cell responses when presented with or without DC.Taken together, our data indicate that within this cluster of 3 overlapping Gag p24 15mers, stimulation with peptide-loaded DC revealed a novel HLA A*0101 9mer epitope p24+ T memory responses, particularly against Gag + T cells + T cells to non-HIV-1 viral infections + T cell reactivity in persons on ART. The IFN\u03b3 response induced by peptide loaded DC was mediated by CD8+ T cells, with purified CD8+ T cells exhibiting an enhanced magnitude and breadth of IFN\u03b3 responses relative to PBMC. The 15mer peptides targeted by CD8+ T cells were similar to those targeted by PBMC, but included a broader array of peptides across the proteome of HIV-1. CD8+ T cell responses were noted to DC loaded with 15mer peptides within all 9 HIV-1 proteins except Vpu. Confirming evidence that CD8+ T cells were the predominant responders to HIV-1 peptides was induction of polyfunctional immune mediator reactivity in CD8+ T cells to both Gag and Nef peptides.Control of HIV-1 infection has been related to the magnitude and breadth of HIV-1 CD8Focusing on Nef and Gag specificities, we found that T cells responded to clusters of overlapping 15mer Nef and Gag peptides that contained known, immunodominant epitopes matched to the subjects' MHC class I alleles. Notably, T cell reactivity was induced to 22% of these overlapping 49 Nef peptides by DC compared to only 4% of Nef peptides without DC. Furthermore, the magnitude of the anti-Nef responses was significantly greater for peptides with DC compared to peptides without DC. Similar to Nef, positive T cell responses to Gag 15mers containing known MHC class I epitopes were clustered in well documented, immunodominant regions of the protein that matched the MHC class I alleles of our study subjects. DC revealed T cell responses to 16% of these overlapping 122 Gag peptides compared to only 4% of Gag peptides without DC. The magnitude of the anti-Gag responses was also significantly greater for Gag peptides presented by DC compared to peptides without DC.+ T cell responses to immunodominant epitopes of Nef and Gag has important implications for assessment of T cell immunity in HIV-1 infection. Much of this research relies heavily on use of overlapping 15mer peptides in PBMC without taking into account the role of professional APC + T cell reactivity + T cells were the predominant responders in our study. Our finding implies that the conventional assessment of single cell, IFN-\u03b3 responses to libraries of overlapping HIV-1 peptides without DC is potentially missing a significant number of epitopes in persons on ART.That autologous, mature DC significantly enhance the breadth and magnitude of CD8We noted reactivity to 15mer peptides with no previously reported epitope matched for the study subjects' MHC class I alleles for 52% of the reactive Nef peptides and 55% of the reactive Gag peptides. Most of this reactivity was in response to peptides with DC, i.e., T cells specific for 86% of the reactive Nef peptides and 75% of the reactive Gag peptides were only activated by peptide-loaded DC. Analysis of these peptide sequences by predicted binding to their MHC class I motif 73\u201387 with DC, and the predicted binding motif of HLA B*2703, we defined a novel HLA B*2703 epitope, Nef76\u201384 (LRPMTYKAA). This new HLA B*2703 Nef epitope is found in 34% of circulating HIV-1 subtype B sequences, while the most common peptide is LRPMTYKgA found in 42% of circulating sequences (based on a dataset of 1184 subtype sequences). This is a variable peptide region since 77 additional peptides were identified in the 1184 circulating sequences but 73 of them were found in less than 1% of sequences. Similarly, using DC we found 3 novel epitopes within Gag that would have been missed using peptide stimulation alone. The 10mer HLA B*5101 epitope for p17  is found in 24% of circulating sequences, while RPGGKKKYrL is found in 27% of sequences. The Gag p24161\u2013169 peptide RAEQASQEVK (HLA A*0101 restricted) is the consensus in 49% of circulating HIV-1 sequences, whereas p24173\u2013182 FRDYVDRFY (HLA B*5101 restricted) is extremely conserved, found in 99.5% of HIV-1 sequences.An extensive analysis of a subset of these putative Nef and Gag epitopes indicated that DC indeed revealed novel epitopes for Nef and Gag. Based on single cell IFN-\u03b3 production induced by the 15mer Nef+ T cell reactivity for CD107a, IFN\u03b3, IL-2, MIP-1\u03b2, and TNF\u03b1, and various polyfunctional combinations of these immune mediators. In particular, the novel Gag peptides presented by DC induced greater levels of polyfunctional CD8+ T cells than peptides without DC. However, with or without DC, polyfunctional T cell responses were less discriminatory for optimal epitope specificity than single cell production of IFN-\u03b3 by ELISPOT assay. We have recently noted a similar, limited discrimination by polyfunctional analysis of novel T cell epitopes of human herpesvirus 8 presented by DC We observed that the 15mer overlapping peptides encompassing the 4 novel Nef and Gag epitopes, as well as the 8-10mer epitope variants within these 15mers, induced monofunctional CD8+ T cell responses, the majority of immunodominance is based on the affinity of the peptide for its MHC class I allele on APC, forming a stable number of complexes to activate naive T cells + T cells. However, it is not clear how closely measures of binding of soluble peptides to MHC class I molecules by in vitro affinity assays reflect peptide-MHC binding in DC. Moreover, an increased antigen storage capacity of DC has been linked to their ability to activate T cells by facilitating a continuous supply of MHC class I ligands + T cells There are many factors of mature DC that could relate to their enhancing afferent T cell responses to HIV-1 epitopes. In primary CD8+ T cells by DC is also related to the relative expression of T cell receptor and CD8 molecules on memory T cells Activation of CD8+ T cell responses to HIV-1 epitopes in subjects who have suppressed HIV-1 infection on ART may be misleading. Clearly, the present results indicate that mature DC reveal a broad spectrum of T cell epitopes recognized by CD8+ T cells in persons on ART with suppressed viral load that are not detectable by conventional stimulation of PBMC with peptide alone. These include novel, MHC class I restricted, HIV-1 epitopes that induce monofunctional and polyfunctional T cells producing up to 5 immune mediators that have been linked to control of HIV-1. This suggests that DC could be potent inducers of anti-HIV-1 T cell immunity as an immunotherapy for HIV-1 infected persons on ART.In conclusion, our findings have important implications for T cell immune control of HIV-1 infection. Previously reported, low memory recall, CD8Table S1Characteristics of the HIV-1 infected subjects.(0.21 MB TIF)Click here for additional data file.Table S2MHC class I Nef epitopes detected in 7 HIV-1 infected subjects.(0.03 MB TIF)Click here for additional data file.Table S3MHC class I Gag epitopes detected in 7 HIV-1 infected subjects.(0.03 MB DOCX)Click here for additional data file."}
+{"text": "Cryptomeria japonica being pharmacologically active substances. We investigated whether M1 and M4, end products of steroidal ginseng saponins metabolized in digestive tracts, as well as T-cadinol and calamenene can drive DC maturation from human monocytes in vitro. Human monocytes were cultured with GM-CSF and IL-4 for 6 days under standard conditions, followed by another 2 days in the presence of M1, M4, T-cadinol or calamenene. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR on M1-primed DC, M4-primed DC, T-cadinol-primed DC and calamenene-primed DC were enhanced with a concomitant decrease in endocytic activity. M1-primed DC, M4-primed DC, T-cadinol-primed DC or calamenene-primed DC enhanced the T cell stimulatory capacity in an allo MLR . Na\u00efve T cells co-cultured with allogeneic M1-primed DC, M4-primed DC, T-cadinol-primed DC or calamenene-primed DC turned into typical Th1 cells, which produced large quantities of IFN-\u03b3 and released small amounts of IL-4 depending on IL-12 secretion. In the CTL assay (cytotoxic T-lymphocyte assay), the production of IFN-\u03b3 and 51Cr release on M4-primed DC was more augmented than of immature DC or TNF-\u03b1-primed DC. These results suggest that M1, M4, T-cadinol and calamenene appear to be a good factor to induce DC maturation, or even better in some respect, for the use in clinical DC therapy to induce strong Th1 type immune responses.Dendritic cells (DC) play a pivotal role in the initiation of T-cell-mediated immune responses, making them an attractive cellular adjuvant for use in cancer vaccines. The interaction of T cells with DC is crucial for directing T cell differentiation towards the Th1, Th2 or Th17 type, and several factors determining the direction of the T cell polarization. IL-12 plays a central role in the immune system, not only by augmenting the cytotoxic activity of T cells and NK cells and regulating IFN-\u03b3 production, but also by the capacity of IL-12 to promote the development of Th1 cells. Therefore, it is important to identify factors that might affect the differentiation, maturation and function of DC. Ginseng is a medicinal herb widely used in Asian countries, and many of its pharmacological actions are attributed to the ginsenosides. Moreover, T-cadinol and calamenene are sesquterpenes isolated from the heartwood of  The purity of isolated cells was >95% of CD4+ na\u00efve T cells as determined by flow cytometry using FACSCalibur. Allogeneic CD4+ na\u00efve T cells (5 \u00d7 105 cell/200 \u03bcl) at 1:5 DC/T cell ratio in 96 well U-bottomed tissue culture plates . Some cultures were supplemented with neutralizing Abs to block endogenous cytokines: anti-IL-12 . On day 5, cells were washed extensively and expanded with fresh medium containing 10 U/ml of recombinant human IL-2 (IL-2) . One hundred microliters of culture supernatant was replaced with medium of the same concentration every 3 days. On day 14, cells were washed, counted, and T cells (106/ml) were re-stimulated for 24 h on plates coated with anti-CD3  and anti-CD28 . The cell-free supernatants were collected and frozen at \u221220 \u00b0C until measurement of cytokines using ELISA.Irradiated (30 Gy) DC were co-cultured with na\u00efve T cells (2.5 \u00d7 106/ml) were stimulated with phorbol myristate acetate (PMA) (10 ng/ml) and ionomycin (1 \u03bcg/ml) for 5 h at 37 \u00b0C in a water bath. Brefeldin A (10 \u03bcg/ml) was added during the last 2 h of incubation to prevent cytokine secretion. Cells were collected, fixed with 1% paraformaldehyde, permeabilized with a commercial solution (BD-Pharmingen), and stained with FITC-labelled anti-IFN-\u03b3 (IgG2a) and PE-labelled anti-IL-4 (IgG1) mAbs. The samples were analyzed on a FACS-Calibur with CellQuest software. Ten thousand cells were analyzed per sample.The intracellular cytokine concentrations of the harvested T cells were measured by FACS analysis as previously described . Briefly5/100 \u03bcl) were then added to the upper chambers of the Transwell plates. Transwell cultures were maintained for 5 h in 5% CO2 at 37 \u00b0C, and DC migrated from the upper wells were counted using a Colter Counter. The results were expressed as net migration percent calculated as:/total number of cells loaded in the upper chamber \u00d7 100.Migration of DC induced by macrophage inflammatory protein (MIP)-3\u03b2 was measured using a double chamber system as previously described . Briefly2+ ([Ca2+]i) concentration was carried out as previously described i was monitored with a CAF110 spectrofluorimeter .The determination of intracellular Caescribed . Briefly6 DC using an RNeasy Mini Kit . The cDNA synthesized from total RNA by Moloney murin leukaemia virus reverse transcriptase  was subjected to RT-PCR using 30 cycles at 94 \u00b0C for 0.5 min, 57.5 \u00b0C for 1 min, and 72 \u00b0C for 1 min for CCR7 . The sense and anti-sense oligonucleotide primers for CCR7 were 5\u2032-CGCGTCCTTCTCATCAGCAA-3\u2032 and 5\u2032-GTCCCGACAGGAAGACCACT-3\u2032, respectively. PCR products were identified by electrophoresis on 2% agarose gels that were photographed.Total RNA was extracted from 1\u20132 \u00d7 105) cultured from monocytes of HLA-A24+ donors were loaded with 10 \u03bcM Epstein-Barr virus (EBV)-derived peptide (TYGPVFMCL) for 2 h at 37\u00b0C in 5% CO2. EBV-derived peptide can bind to HLA-A2402 as reported by Lee et al. [6 cells/ml with 10\u03bcM EBV peptide and incubated for 2 h at 37 \u00b0C in 5% CO2. Effector cells were co-cultured with target cells at effector-to-target ratios of 2:1, 10:1 and 20:1 in 96 well round-bottomed culture plates  in duplicate. After overnight incubation at 37 \u00b0C in 5% CO2, IFN-\u03b3-concentration of the supernatant was determined by ELISA.Details of the method used in this assay have been previously described . Brieflye et al. . In 24 w51Cr labelled target cells (1 \u00d7 104) at various effecor-to-target cell ratios for 6 h, followed by supernatant harvesting for measuring radioactivity using as automatic \u03b3 counter. This assay was done in triplicate.Details of the methods used in this assay have been previously described . BrieflyStatistical analysis of the results was performed by ANOVA. Differences were considered statistically significant when p value were less than 0.05.To study the direct effects of M1, M4, T-cadinol and calamenene on the maturation of sentinel DC into effector DC, human monocytes-derived DC were cultured with M1, M4, T-cadinol or calamenene. Human monocytes were cultured with GM-CSF and IL-4 for 6 days under standard conditions, followed by another 2 days in the presence of various concentrations of M1, M4, T-cadinol or calamenene. LPS, CT and TNF-\u03b1 were used as a positive control. The resulting populations of DC were analyzed by flow cytometry. As shown in Because the level of IL-12 production by DC is a major factor driving the development of Th1 cells, we studied the influence of M1, M4, T-cadinol or calamenene on IL-12 production by DC. We measured IL-12p70 production in immature DC and in DC matured for 2 days in the presence of the above factors after stimulation by CD40-L for 24 h. The production of IL-12p70 by M1-primed DC, M4-primed DC, T-cadinol-primed DC and calamenene-primed DC was more augmented than that of LPS-primed DC . In contWe observed that M1-primed DC, M4-primed DC, T-cadinol-primed DC and calamenene-primed DC expressed increased levels of Ag-presenting and the expression levels of CD1a, CD80, CD83, CD86 and HLA-DR as expressed by MFI. Therefore, we next compared the capacity of M1-primed DC, M4-primed DC, T-cadinol-primed DC, calamenene-primed DC, LPS-primed DC, CT-primed DC or TNF-\u03b1-primed DC to stimulate T cells in an allo MLR. M4-primed DC showed higher stimulatory efficiency in an allo MLR than LPS-primed DC , CT-primWe next evaluated the nature of primary allogeneic T cell responses stimulated by M1-primed DC, M4-primed DC, T-cadinol-primed DC and calamenene-primed DC. Allogeneic M1-primed DC, M4-primed DC, T-cadinol-primed DC or calamenene-primed DC co-cultured with na\u00efve T cells at 1:5 DC/T cell ratio secreted sizeable amounts of IFN-\u03b3 , but litin vitro. T-cadinol-primed DC and calamenene-primed DC had a higher migration response to MIP-3\u03b2 than LPS-primed DC (2+ mobilization to MIP-3\u03b2 (2+ mobilization to MIP-3\u03b2 were observed with CT-primed DC and TNF-\u03b1-primed DC (data not shown). On the other hand, migration response and intracellular Ca2+ mobilization to MIP-3\u03b2 in immature DC were low or just detectable  was used as a target cell (negative control)  .in vitro. Here, we demonstrate that culture of immature DC with M1, M4, T-cadinol or calamenene increase cell surface expression of CD1a, CD80, CD83, CD86 and HLA-DR, while decreasing endocytic activity, resulted in cells with a phenotype characterized by efficient Ag-presentation and costimulatory capacity of mature DC. Functionally, M1-primed DC, M4-primed DC, T-cadinol-primed DC or calamenene-primed DC have enhanced primary allogeneic T cell stimulatory activity in an allo MLR.The present study was performed in order to investigate whether M1 and M4, end products of steroidal ginseng saponins metabolized in digestive tracts, as well as T-cadinol and calamenene can change the phenotype and function associated with DC differentiation from human monocytes in vivo anti-metastatic effect by oral administration of ginsenosides may be primarily meditated by their metabolic component M1 and M4. Moreover, Scaglione et al. [The differentiation of monocytes into DC has a critical impact on the immune response. DC interaction with na\u00efve T cells plays a key role in primary immune responses , 3 and te et al.  have inve et al. . The core et al. . M1-primin vitro is very likely due to the lack of IL-12 production by CT-treated DC and may be independent of IL-4. We showed that the production of IL-12 by CT-primed DC that were stimulated CD40-L was low or detectable. Therefore, it suggests that the involvement of these cells in the innate responses to Th1 response-inducing pathogens during the period preceding the initiation of adaptive immunity can create an environment rich in IL-12, which promotes the production of IFN-\u03b3 and the outgrowth of Th1 cells. It has been demonstrated that immune balance controlled by cytokines such as IL-10 and IL-12 plays an important role in immune regulation, including anti-tumor immunity [In contrast, CT-primed DC induced na\u00efve T cells showed a shift towards Th2 effector cell. CT is a powerful mucosal adjuvant that amplifies B cell and T cell responses to mucosally co-administered antigens, stimulating predominant Th2-type responses , 46. Gagimmunity . Our resin vivo. In most clinical trials using DC-based immunotherapy, immature monocytes-derived DC pulsed with tumor antigen peptides were used. However, recent observation suggests that mature DC could be a better anti-tumor adjuvant [One result of DC maturation and activation demonstrate increased motility and a potential to propensity to migrate towards lymphoid organs. During the maturation process, DC down-regulate the expression of inflammatory chemokines and their receptors and up-regulate the synthesis of constitutive chemokines and the chemokine receptor CCR7. We found that M1-primed DC, M4-primed DC, T-cadinol-primed DC and calamenene-primed DC had high migration, high calcium mobilization in response to MIP-3\u03b2 and up-regulated the expression of CCR7, suggesting that M1-primed DC, M4-primed DC, T-cadinol-primed DC and calamenene-primed DC are likely to be mature DC that have the potential to migrate adjuvant . Althougin vivo.Antitumor immunity has classically been measured by the quantity of tumor-antigen-specific CD8+ T cells . Immunotin vitro. Moreover, DC differentiated with M1, M4, T-cadinol or calamenene enhance the differentiation of na\u00efve T cells towards the Th1 type depending on IL-12 secretion. M-4-primed DC produces a stronger CTL response. Although the molecular events leading to the effects of M1, M4, T-cadinol and calamenene on DC function remain to be resolved, DC appear to be potential targets of M1, M4, T-cadinol and calamenene. Further understanding of the mechanisms by which M1, M4, T-cadinol and calamenene modulate DC function may lead to the development of effective immunotherapy for cancer.This is first study on the effect of terpenoids, e.g. ginseng saponins on human monocytes-derived DC giving new insights into the action of terpenoids. M1, M4, T-cadinol and calamenene potentially regulate differentiation of DC from human monocytes in combination with GM-CSF and IL-4"}
+{"text": "Social factors may be of importance causally and act as maintenance factors in patients with anorexia nervosa. Oxytocin is a neuromodulatory hormone involved in social emotional processing associated with attentional processes. This study aimed to examine the impact of oxytocin on attentional processes to social faces representing anger, disgust, and happiness in patients with anorexia nervosa.A double-blind, placebo-controlled within-subject crossover design was used. Intranasal oxytocin or placebo followed by a visual probe detection task with faces depicting anger, disgust, and happiness was administered to 64 female subjects: 31 patients with anorexia nervosa and 33 control students.Attentional bias to the disgust stimuli was observed in both groups under the placebo condition. The attentional bias to disgust was reduced under the oxytocin condition (a moderate effect in the patient group). Avoidance of angry faces was observed in the patient group under the placebo condition and vigilance was observed in the healthy comparison group; both of these information processing responses were moderated by oxytocin producing an increase in vigilance in the patients. Happy/smiling faces did not elicit an attentional response in controls or the patients under either the placebo or oxytocin conditions.Oxytocin attenuated attentional vigilance to disgust in patients with anorexia nervosa and healthy controls. On the other hand, oxytocin changed the response to angry faces from avoidance to vigilance in patients but reduced vigilance to anger in healthy controls. We conclude that patients with anorexia nervosa appear to use different strategies/circuits to emotionally process anger from their healthy counterparts. Anorexia nervosa (AN) is characterized by problems with eating, weight, and shape. Shape and weight concerns often involve social contagion and social evaluation The social processing problems in patients with eating disorders have been synthesized in recent systematic reviews, and impairments in emotional recognition, social hierarchy, and theory of mind have been found Oxytocin is an important neuromodulatory hormone involved in social emotional processing, particularly in association with attentional processes. Oxytocin increases the sensitivity to detect emotional expressions, which is associated with recruitment of attentional resources, as evidenced by pupil dilation It is possible that changes in oxytocin function may account for some of the anomalies in social cognition in patients with AN. A recent review synthesized evidence for abnormal oxytocin functioning in patients with AN The aim of this study was to examine whether oxytocin impacts on social attentional processes in patients with AN. Our first hypothesis, based on previous studies using the dot probe paradigm, is that patients with AN would have vigilance towards threatening cues and away from rewarding stimuli. Our second hypothesis is that oxytocin would attenuates the selective attention towards negative social cues.Sixty-four women  took part in this double-blind, placebo-controlled cross-over study. The first participant entered the study on August 16, 2012, and the last participant was examined on November 30, 2013. The patients with AN were recruited at the Eating Disorders Clinic of Seoul Paik Hospital, Seoul, South Korea. As this study aimed to examine broadly whether oxytocin might be of benefit in the short term for patients with AN, patients were recruited at various stages and phase of illness both from an outpatient clinic (n\u200a=\u200a13) and an inpatient ward (n\u200a=\u200a18). All patients who volunteered to participate were screened during the early phase of treatment. All of them were in an active stage of illness, and none of them were in the weight-recovered stage. The diagnosis of AN was confirmed by the Structured Clinical Interview from the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition The Korean version of the Wechsler Adult Intelligence Scale was used to measure IQ. Healthy participants were tested during the follicular phase of their menstrual cycle. None of the patients were menstruating. Compensation was provided for travelling expenses and time. This study protocol was approved by both the Korean Food and Drug Association Institutional Review Board (12061) and the Institutional Review Board of Seoul Paik Hospital (IIT-2012-096). All participants provided written informed consent prior to participating in the study. Consent was provided by both the patients and their guardians in the case of 16-year-old patients.JW Pharmaceuticals  from oxytocin powder . The solution was prepared by combining 35.2 mg oxytocin (568 U) with 300 mL 0.9% sodium chloride solution, and adjusting the pH to 4.01 with 10\u00d7 diluted acetic acid. The placebo spray (pH 4.01) was formulated with 0.9% sodium chloride solution and acetic acid, but without the peptide. The filtered and sterilized solution was sealed in individual vials (1.5 mL each) and stored frozen. On the day of use, the vials were thawed and kept in a refrigerator (4\u00b0C) until required. A clinician prepared the nasal spray by transferring oxytocin or the placebo from the vial into a nebulizer.The intranasal oxytocin spray was formulated by Oxytocin and the placebo were administered intranasally 4\u20137 days apart 45 min before the neuropsychological tasks. We chose a dose of 40 IU of oxytocin based on a recent review reporting that cumulative evidence from clinical trials shows that short-term use of intranasal oxytocin administered to both male and female humans in dosages up to 40 IU (per dose) results in no detectable subjective adverse effects in a controlled research setting The photographic stimuli used for the dot probe task were obtained from a validated collection of the Korean Facial Expressions of Emotions (KOFEE) Our pool of target stimuli consisted of 60 images of adults showing 15 positive (happy) or 30 negative (15 angry and 15 disgust) expressions, with each adult having a corresponding picture showing a neutral expression (15 non-target stimuli). The target stimuli were paired with matched non-target stimuli.Attentional biases were assessed using a visual probe detection task As the two images disappeared from the screen, one of two probes (\u2018:\u2019 or \u2018..\u2019) appeared where used to be the center of one of the two pictures. The participants were required to press one of two buttons on a keyboard (marked with white stickers) that corresponds to the probe (Q for \u2018:\u2019 and Z for \u2018..\u2019). When a response was made, the probe disappeared and the next trial started. Participants were advised to identify the probe as quickly and accurately as possible. The target stimuli positions and the probe positions were balanced across trials, so each appeared in either location  with equal frequency, following the paradigm of Mogg and Bradley Attention during the visual probe detection task was measured as the time to respond to the probe. The assumption is that the time to respond to the probe is faster if attention was already allocated to the spatial location where the probe appeared. Faster responses to congruent trials (when the probe emerges at the threatening stimulus location) compared to incongruent trials  were interpreted as vigilance to a threat.These tests were comprised of 10 trials in each of six conditions: three target stimuli  and two probe locations (the location of the target stimuli or non-target stimuli), for a total of 60 trials, repeated twice. Test trials were presented in a new random order for each participant. The dot-probe task was presented on E-prime ver. 2 .The Korean version of the clinician rating interview of the EDE The EDE-Q assesses the main behavioral features of an eating disorder over the past 28 days. The questionnaire consists of 36 items on a 7-point forced choice rating scale. It measures weight, shape, eating concerns, and dietary restraint. We used the standardized Korean version of the 12th edition of the EDE-Q, which has high internal consistency and good 2 week reliability.The STAXI is a 44-item self-report questionnaire that is divided into seven scales. It measures the current intensity of anger (State Anger) and disposition toward anger as a personality trait (Trait Anger) and the tendency to express anger when criticized (Angry Reaction). The other scales measure the frequency with which provoked anger is suppressed (Anger-Suppression), the frequency of the expression of anger toward other people or objects (Anger-Expression), and the frequency at which the expression of anger is controlled (Anger-Control). The Korean version of the STAXI was used in this study The AQ is a 50-item, self-administered questionnaire that assesses the degree to which adults with normal intelligence have autism spectrum traits. It has five sub-scales, including social skill, attention switching, attention to detail, communication, and imagination.Depression and anxiety were assessed in each subject using the standardized Korean versions of the Beck Depression Inventory (BDI) The participants were tested in a private room at Seoul Paik Hospital. They were instructed to abstain from alcohol and caffeine on the day of drug administration and food and drink (other than water) for 2 hours before drug administration. Upon arrival, the participants completed baseline measures of physical symptoms, including abdominal, neurological, dermatological, and cardiac symptoms. Subsequently, oxytocin or the placebo was self-administered by inhalation of the spray into each nostril. The order of oxytocin and placebo administration was randomized using Microsoft Excel in this double-blind procedure. The participants completed the self-report measures to assess their psychological state during the 45 minutes after administration of each spray. At the end of the study period, each participant completed follow-up measures for adverse physical symptoms.We followed the standard analysis procedure used for this task Attentional bias scores were calculated for each matched trial type  as PN \u2212 PE, where PN is the mean RT for probes replacing neutral stimuli, and PE is the mean RT for probes replacing emotional target stimuli. Therefore, positive scores suggest increased attention towards the emotional/target stimuli, whereas negative scores indicate attention away from emotional/target stimuli. A score of 0 suggests no bias towards any stimuli.t-test comparisons for each group. Greenhouse\u2013Geisser corrections were applied on the assumption that sphericity was violated. Data are shown as mean and with the effect size (ES) if appropriate . The estimation of ES was based on Cohen's d: described as negligible (\u200a=\u200a0 and <0.15), small (\u22650.15 and <0.40), medium (\u22650.40 and <0.75), large (\u22650.75 and <1.10), very large (\u22651.10 and <1.45) and huge (>1.45) . We defined the drug effect as the difference in the RT score between the administration of the placebo and oxytocin (drug effect \u200a=\u200a RTplacebo \u2212 RToxytocin). Pearson's correlations were used to investigate the relationship between the drug effect on attentional bias and psychological variables. Analyses were performed using SPSS 19.0 , and the two-tailed p-value was 0.05.The attentional responses to shape stimuli were investigated via a series of initial 2 (group: AN and HC) \u00d72 (drug: oxytocin and placebo) \u00d73  repeated-measures analyses of variance (ANOVAs). Post-hoc analyses were conducted through paired 2) and the EDE-Q scales. The AN group also scored significantly higher on the BDI, STAI, and the Social Skill, Attention, and Communication subscales and the total score on the AQ compared to those in the HC group. The EDE interview scores of the AN group were 5.07\u00b16.04 for the restraint subscale, 3.73\u00b14.41 for the eating concern subscale, 4.35\u00b18.33 for the weight concern subscale, and 3.85\u00b13.10 for the shape concern subscale.The demographic and the group characteristics of the participants are shown in F \u200a=\u200a3.568, p\u200a=\u200a0.031, \u03942\u03b7 \u200a=\u200a0.057) was observed in the three-way group (AN vs. HC) \u00d7 drug (oxytocin vs. placebo) \u00d7 emotion  repeated-measures ANOVA.A significant group x drug x emotion interaction effect  \u200a=\u200a4.988, p\u200a=\u200a0.029, \u03942\u03b7 \u200a=\u200a0.078).The two-way group (AN vs. HC) \u00d7 drug (oxytocin vs. placebo) repeated-measures ANOVA identified a group \u00d7 drug interaction effect  \u200a=\u200a0.028, p\u200a=\u200a0.868, \u03942\u03b7 \u200a=\u200a0.000) nor a group \u00d7 drug interaction effect in response to happy stimuli  \u200a=\u200a0.009, p\u200a=\u200a0.925, \u03942\u03b7 \u200a=\u200a0.000).The two-way group (AN vs. HC) \u00d7 drug (oxytocin vs. placebo) repeated-measures ANOVA showed that there was neither a main effect of drug  \u200a=\u200a8.970, p\u200a=\u200a0.004, \u03942\u03b7 \u200a=\u200a0.130) in response to the angry stimuli. Post-hoc tests showed avoidance of angry faces in the AN group and vigilance in the HC group with a moderate to large difference between the AN and HC groups under the placebo condition (t(62) \u200a=\u200a2.510, p\u200a=\u200a0.015, d\u200a=\u200a\u22120.633). Both of these information processing responses were moderated by oxytocin and produced a large increase in vigilance in the AN group (t(30) \u200a=\u200a\u22122.847, p\u200a=\u200a0.008, d\u200a=\u200a0.785) but a tendency to decrease with a small ES in attention in the HC group (t(32) \u200a=\u200a1.218, p\u200a=\u200a0.233, d\u200a=\u200a0.219).The two-way group (AN vs. HC) \u00d7 drug (oxytocin vs. placebo) repeated-measures ANOVA showed that there was a group \u00d7 drug interaction effect  \u200a=\u200a10.120, p\u200a=\u200a0.002, \u03942\u03b7 \u200a=\u200a0.142) with no group \u00d7 drug interaction effect  \u200a=\u200a0.375, p\u200a=\u200a0.542, \u0394\u03b72 \u200a=\u200a0.006) in response to the disgust stimuli. Post-hoc tests showed an attentional bias to the disgust stimuli in both the AN and HC groups under the placebo condition (t(62) \u200a=\u200a3.208, p\u200a=\u200a0.002, d\u200a=\u200a0.597). Attentional bias was reduced under the oxytocin condition in both groups with a small effect in the AN group (t(30) \u200a=\u200a1.602, p\u200a=\u200a0.120, d\u200a=\u200a0.418) and a moderate/large effect in the HC group (t(32) \u200a=\u200a3.138, p\u200a=\u200a0.004, d\u200a=\u200a0.555). The attentional bias scores across conditions in each group are shown in The two-way group (AN vs. HC) \u00d7 drug (oxytocin vs. placebo) repeated-measures ANOVA showed a main effect of drug  i.e., individuals with higher anger expression showed a greater reduction in vigilance with oxytocin. There was no association between STAXI subscales and the oxytocin effect on angry stimuli in patients with AN. No association was observed between the drug effect and aspects of psychopathology typically associated with eating disorders (EDE-Q), depression (BDI), state and trait anxiety (STAI), autism traits (AQ) in either the AN or HC groups. No association between BMI or weight and the oxytocin effect were observed in either the AN group  or in the HC group .The reduced vigilance to angry stimuli in the HC group was positively correlated with STAXI subscales of anger expression in the oxytocin condition  faces produced no attentional bias in patients with AN or the HC group. Our second hypothesis was confirmed, in part, as oxytocin attenuated the selective attention towards disgust in both the patients with AN and the HC group. In contrast, oxytocin converted an avoidant response to anger into vigilance in patients with AN but reduced the vigilant response to anger in the HC group. Oxytocin did not change the lack of attention to happy (smiling) faces in either the AN or HC groups.Our result that smiling faces did not produce an attentional bias in either the AN or HC groups was consistent with other findings in Caucasian patients with AN In the oxytocin condition, the attentional bias was away from disgusted faces in both groups with a larger effect in the HC group. To our knowledge no previous study has evaluated the impact of oxytocin on recognition of the disgust emotion in humans. The results are consistent with another study, which found that oxytocin decreases attention toward negative faces  in healthy people The avoidance response to anger expression in patients with AN and a vigilant response in the HC group under the placebo condition differed from a study that used \u201crejecting\u201d facial expressions in which the opposite pattern was found, i.e., vigilance to the rejecting stimuli in patients with AN These findings raise the possibility that oxytocin treatment may moderate some of these social difficulties and possibly improve the outcome. Resistance to treatment is associated with less activation in social cognition circuits within the brain There are some limitations in this study that need to be considered and which may account for some of the individual variation in this study. First, a third of the patients with AN were recruited from the outpatient clinic and the others were from the inpatient ward. So the patients were at different phases of treatment. The oxytocinergic pathway may differ in the more severely affected patients undergoing refeeding, compared to that of outpatients. However, all of our participants were screened when they initially visited to the clinic and were in the active stage of the illness, regardless of their inpatient or the outpatient status. None of them were weight-recovered. This is a proof of concept study which aimed to broadly examine that oxytocin might be of benefit in the short term across different stages. Further studies should consider controlling the illness stages. Second, the doses of intranasal oxytocin were not adjusted for individual differences in BMI and therefore the underweight patients received a higher doses per weight than healthy controls. However, in our subsidiary analysis, no relationship was observed between the BMI or weight and the oxytocin effect in either the AN or healthy control groups. Further studies examining for a dose relationship may be of interest and reduce the variance. Third, three patients took fluoxetine, which may affect the oxytocin pathway, so may have increased the variance. An additional analysis found no differences in the response of this subgroup to the non medicated patients. Finally, we did not measure other individual factors that are known to impact attention bias. For example, individuals with a short 5-hydroxytryptamine allele have been found to be more vigilant towards threat In conclusion, patients with AN showed an avoidance of angry faces whereas healthy women were vigilant to anger in the placebo condition. This response in the AN group shifted from avoidance to vigilance following oxytocin administration but there was a trend for the healthy women to be less vigilant to angry faces. Patients with AN showed vigilance to faces of disgust similar to the healthy women and this was moderated with oxytocin in both groups reaching a significant effect in healthy women. Neither group had any attentional bias to happy faces under either the oxytocin or control conditions. These findings suggest that further evaluation of oxytocin nasal spray as a treatment to improve emotional processing and social communication particularly in relationship to anger may be of value in patients with AN."}
+{"text": "In response to \u201cMegadose Methylprednisolone (MDMP) for Hemangiomatosis\u201d We are grateful to Dr. \u00d6zsoylu for his valuable comments on our case report concerning the treatment of hemangiomatosis with propranolol . One of Systemic corticosteroids have been the mainstay of hemangioma treatment since the 1960s. Oral or intravenous MP and prednisone are effective for shrinking hemangiomatous lesions in infants. Cushingoid appearance and delayed healing of the cervical ulcerated lesion in our case were attributed to prolonged use of systemic MP. Recent studies have reported successful treatment of hemangiomas with the \u03b2-blocking agent propranolol, even in low birth weight infants ,5. Propr"}
+{"text": "Despite all the knowledge on depression, it is still unclear whether current literature covers all the psychosocial difficulties (PSDs) important for depressed patients. The aim of the present study was to identify the gaps in the recent literature concerning PSDs and their related variables. Psychosocial difficulties were defined according to the World Health Organization International Classification of Functioning, Disability and Health (ICF). A comparative approach between a systematic literature review, a focus group, and individual interviews with depressed patients was used. Literature reported the main psychosocial difficulties almost fully, but not in the same degree of importance as patients' reports. Furthermore, the covered areas were very general and related to symptomatology. Regarding the related variables, literature focused on clinical variables and treatments above all but did not report that many psychosocial difficulties influence other PSDs. This study identified many existing research gaps in recent literature mainly in the area of related variables of PSDs. Future steps in this direction are needed. Moreover, we suggest that clinicians select interventions covering not only symptoms, but also PSDs and their modifiable related variables. Furthermore, identification of interventions for particular psychosocial difficulties and personalisation of therapies according to individuals' PSDs are necessary. Depression is a major public health issue due to its prevalence, high mortality rates , suicideThis evidence suggests that usual management strategies do not address sufficiently relevant areas of depression. One substantial dimension of depression comprises the psychosocial difficulties (PSDs) which people experience. PSDs constitute the impairment on psychological and social daily functioning of individuals, linked with their particular health condition . The broPrevious scientific literature provides different definitions of psychosocial difficulties in depression but has some limitations: the methods either focus only on specific areas or consider the PSDs as a result of depression. Recent studies \u20139, howevOnce this comprehensive definition of psychosocial difficulties has been extracted from the literature, it is very important to incorporate the patient perspective in clinical practice and research. The majority of studies on this topic are quantitative; however, the literature that uses qualitative methodologies\u2014which could provide deeper, richer, and more elaborated data, exploratory analyses on patients' needs and standpoints, and thus more objective results\u2014is sparse. Furthermore, this kind of studies can provide a general view if particular psychosocial difficulties, named by patients as important, are missing in the literature.There are few existing studies using qualitative research to identify a full list of PSDs which depressed people experience. Yet, none of them has applied a comprehensive approach in order to encompass all the psychosocial difficulties from different perspectives. An example is a study by Lasch et al. , which aAn article by Br\u00fctt et al.  based itTo date, no study has analyzed the full set of PSDs and their related variables in depression by including both literature and patient perspective. The aim of the present study is to tackle this research gap and discover whether the recent scientific literature actually reports the PSDs and their related variables that are important for depressed patients. Our objective is to obtain information about whether the latest literature extensively covers the issues that are pointed out as problematic by depressed individuals, or if there are particular areas, which should receive more attention. Moreover, a potential identification of these missing previously-ignored questions in literature will enhance the quality of future research and enable new strategies for treatment and rehabilitation in depression.In order to compare whether the psychosocial difficulties experienced by depressed patients are actually reported by recent literature in depression, we included a step by step methodology.We gathered information from three different studies.First, a systematic review to directly collect information reported in recent literature, consulting the MEDLINE and PsycINFO databases, was conducted. Search terms were adapted to each database combining the MeSH headings of \u201cDepression,\u201d \u201cDepressive Disorder,\u201d and \u201cDepressive Disorder, Major\u201d with \u201cdepress*\u201d (title) in MEDLINE database and (DE=) \u201cMajor Depression,\u201d \u201cRecurrent Depression,\u201d and \u201cDepressive disorder\u201d in PsycINFO. For psychosocial difficulties the following keywords were used: \u201cpsychosocial*,\u201d exp Quality of life/, exp Personal satisfaction/exp Human activities/exp social support/disabilit*, homelessness, environmental factor*, exp Interpersonal relations/, exp Quality of life/, exp personal satisfaction/, exp human activities/, exp paternalism/, prejudice/, psychosocial deprivation/, social values/, exp Social Problems/, Social Adjustment/, social isolation/stereotyping/, exp Social environment/, exp emotions/, exp family/, exp socioeconomic factors/exp life style/exp Disability evaluation/, exp Communication Barriers/, \u201cAdaptation\u201d, exp Psychological/, exp Aggression/, exp Psychological stress/, exp community /, Sexual* or intimacy. Inclusion criteria were articles reporting information on psychosocial difficulties in people with a diagnosis of major or minor depression according to DSM-III-TR, DSM-IV, or DSM-IV-TR \u201316, or aOnce all the included studies were selected, we extracted information about the sample characteristics and then collected the variables, the tools, the psychosocial difficulties and their related variables. Related variables in literature refer to determinants of PSDs since only longitudinal studies were included. Determinants in literature were those variables that were longitudinal predictors for incidence or changing of psychosocial difficulties, so consequently a causal relationship with the PSDs can be hypothesized. Extraction of the information was double checked by two independent reviewers in 20% of the articles (MC and BM) (Kappa = 0.80). Kappa's coefficient was calculated according to Fleiss and Cohen rules [ Hospital Universitario de la Princesa in Madrid were invited to participate. Selection was done by their main mental health care provider (psychiatrist) taking into consideration the maximum variability of sampling in gender, work status, and clinical status , consulting the patients' clinical records. Eight patients were invited to participate and one of them did not consent to participate reporting having no time for this. Participation in this study was not mandatory and only patients with motivation to participate were included in the final sample; therefore a selection bias could have affected our results. Participation was formally agreed after signing a consent informed form. One moderator and one assistant (MC and IL), who had been previously trained, encouraged all the members to participate during the session.On the other hand, the collection of patients' perspectives was performed including information from two studies. The first one consisted of one focus group composed of seven depressed patients. We based our sample size decisions on literature recommendations, \u201cThe ideal size of a focus group for most noncommercial topics is five to eight participants\u201d  and the Four open questions were posed: (1) which psychosocial difficulties are usually experienced due to participant's depression; (2) which ones are more relevant (ranking the five most important ones for each participant); (3) how these difficulties changed overtime; and (4) which events are responsible for the onset or change of these psychosocial difficulties overtime. All the dialogues were digitally recorded and subsequently transcribed. \u201cI have no time to participate,\u201d one had no interest in research studies, and one showed no interest in this particular study. During the initial interview researchers verified the fulfillment of the inclusion criteria. At that point five patients that were derived from the primary care center were excluded because they met diagnostic criteria for other different disorders: bipolar disorders (2), generalized anxiety disorder (1), substance use disorder (1), and complicated grief (n = 1) and thus satisfied one of the exclusion criteria.Finally, in order to gather other difficulties that are not usually reported during focus group sessions because of potential unwillingness of self-disclosure , we inclPatients were singly asked, among other questions, which were the five most disabling psychosocial difficulties that they experienced due to depression and which were the variables that were responsible for the onset or the change of these problems. A causal relationship with the PSDs and extraction of determinants, unlike in the literature, cannot be established, because of the nature of the focus group and the individual interviews; therefore we will refer to these variables of onset or change of PSDs according to the patient perspective as related variables.Santa Hortensia\u201d Primary Care center of Madrid or in the outpatient psychiatric service at the Hospital Universitario de la Princesa in Madrid. In both study sites patients were chosen according to their availability and were invited to participate by their primary care doctors or psychiatrists. All participants signed an informed consent form. Both studies were independently reviewed and approved by the Hospital Ethics Committee for Clinical Research.Trained interviewers  conducted all the individual interviews. Inclusion criteria for individual interviews were patients older than 18 years with a diagnosis of depressive episode or major depression criteria according to ICD-10. All patients were collected in \u201cAfter gathering information from the three different studies, we needed to establish a common language necessary to directly compare outcomes. For that purpose a list of common categories for classifying psychosocial difficulties was agreed on during one research group meeting. Participants were researchers who had been involved in data collection for different health conditions. Researchers involved in the data collection of depression  also participated. All of them were requested to share with the group the PSDs that they had identified in the different studies and the terminology they had used to name them. After each naming, the working group was asked whether they agreed with the terminology proposed. After a brief discussion, stating pros and cons for the proposal, an agreed-on term was decided and documented for each PSD. The same procedure was followed to extract the names of related variables in subsequent working group sessions with the same participants. personal factors we used the definition given by the WHO's International Classification of Functioning, Disability and Health: \u201cPersonal factorsare the particular background of an individual's life and living, and comprise features of the individual that are not part of a health condition or health states. These factors may include gender, race, age, other health conditions, fitness, lifestyle, habits, upbringing, coping styles, social background, education, profession, past and current experience (past life events and concurrent events), overall behaviour pattern and character style, individual psychological assets and other characteristics, all or any of which may play a role in disability at any level. Personal factors are not classified in ICF.\u201d [After obtaining the list of common categories, we associated the different concepts extracted in the studies to the agreed category list. Forin ICF.\u201d . The likin ICF.\u201d . BM and Focusing on the information extracted from the literature, a frequency analysis was performed regarding how many different studies particularly reported the psychosocial difficulties or related variables. In the case of individual interviews, the number of times that the different participants reported the psychosocial difficulties and their related variables was calculated. Finally, for the focus group, digital recordings were analyzed in order to extract the number of times in which psychosocial difficulties and related variables had been a topic during the session . These analyses were performed with the software for qualitative research NVIVO.Tables Comparison between the literature review and the studies reporting patient's opinion showed that literature does cover almost fully the main psychosocial difficulties directly addressed by depressed individuals. There were relatively few PSDs, not covered by the recent scientific literature. Emotional functions [ energy and drive functioning [ cognitive functions [ employment [ relationship with the others [unctions \u201326, enerctioning \u201329, cognunctions \u201332, emplployment \u201335, and e others \u201338 were  Carrying out daily routine was pointed out by ten patients in the individual interviews (making it the fifth most important PSD), but it has been investigated less than three times in the revised literature.However, some main PSDs in the patient reports were not covered enough by the literature.Communication with others was among the most important psychosocial problems, according to the participants in the focus group, but our systematic review did not find it mentioned anywhere in the literature. Moreover, weight maintenance functions and doing housework were highlighted by the patients in the individual interviews, but literature omitted them as significant and important psychosocial difficulties in depression. In addition, there were some main categories, which did not meet the same ranking of significance when the different sources of information were compared. Pain [ sleep [ed. Pain \u201341 and s [ sleep , 43, forRegardless of these few above mentioned PSDs, literature in general addresses substantially the main psychosocial difficulties in depression. Therefore, we conducted an elaborate second level analysis to investigate whether the specific psychosocial difficulties (components within the main PSD categories), reported in the focus group and individual interviews, were also identified in the literature. Once more, the results demonstrated that the literature, with some exceptions, almost fully covers the range of specific psychosocial difficulties, experienced by depressed patients. Loneliness and distress  were among the most important and commonly mentioned difficulties by the patients, whereas these specific PSDs were reported less than three times in the literature. The same is valid for attention and memory (part of cognitive functions), efficiency (employment), and intimate relationships (relationships with others).However, as can be seen in  treatment [ emotional functions as a cause of psychosocial difficulties as well. Specifically, results showed that the literature did not analyse sufficiently cognitive functions, relationships with others, energy and drive, and employment problems as causes of PSDs.Additionally, we investigated whether literature sufficiently reports the most important related variables of change and onset for psychosocial problems in depression that are addressed by patients. All related variables can be seen in reatment \u201346 was aThe current study aimed to analyse whether the recent scientific literature reports those psychosocial difficulties and their related variables that are important for depressed patients. Our findings indicated that the literature does report almost fully the main psychosocial difficulties, experienced by patients with depression, but the degree of importance of each PSD depended on the source of information. The same refers to the specific PSDs, extracted by a second level analysis. Contrary to the main PSDs reported, however, only a few related variables of onset of PSDs were currently reported in literature. In addition, literature was specifically focused on clinical variables as related to the PSDs, whereas it ignored that some PSDs can also become related variables for other PSDs.To our knowledge, this is the first study to throw light on whether the latest scientific literature actually reflects the issues that are indicated as problematic by depressed patients. However, all the subsequent comments on the literature have to be considered with respect to the only 103 existing studies within our period of search that covered the whole spectrum of PSDs in depression and the fact that their average quality rate is not high . In this. Emotional functions stood out as an evident problem for depressed patients, as part of the outcomes of 62 research studies and being mentioned more than 100 times during the focus group and individual interviews. A review by Brockow et al. [ depressive mood and symptoms [ loneliness and distress (emphasized notably by depressed patients but narrowly explored in the literature) can be explained through the literature tendency to focus mainly on those PSDs which belong to the symptomatology spectrum. In addition, according to our results, the literature usually reported major PSD concepts. However, other more specific and smaller categories, also highly affecting the emotional functioning of individuals\u2014like loneliness and distress\u2014were neglected.With regard to the most common psychosocial difficulties, the literature and the patients' outcomes matched almost entirelyw et al. , based osymptoms \u201350 . Literature should therefore consider the positive and negative consequences of treatments on patient's PSDs and not only report the positive impact on them. This type of information would be useful to help clinicians decide among the wide range of interventions available.Regarding the related variables of PSDs, the results of our analysis revealed interesting and alarming gaps in the recent literature. Essentially, only few related variables of onset of PSDs were reported in research. cognitive functions, relationships with others, energy and drive, and employment problems as related variables of PSDs would have to be described as insufficient. These components are fundamental to the understanding of the psychosocial functioning of depressed patients. Hence, if the aim is to reach a general development in this direction, future research needs to focus on these gaps.On the other hand, current scientific literature concentrates on clinical variables and treatments as the only PSDs' related variables, whereas patients additionally highlight the relationships between different PSDs. Specifically, if the patient perspective is considered, literature onOverall, these findings reveal the fact that there is a discrepancy between the patient's and the health science researchers' perspective when analysing not only the biological and psychological factors of depression, but also the socioeconomic and environmental variables. The subject's view of his health and functioning is not usually taken into account when designing research studies in depression. In this sense, future studies may address this wider view in a more accurate way by taking into account additional sources that are primarily focused on the subject and its sociological environment. From a sociological point of view it could be claimed that the mainstream perspective from which most institutions and professionals develop their work is not reflecting the quotidian environmental stressors that interfere with an adaptive and healthy development of everyday life in people with depression. It could be hypothesized that this is a result of the current and most common definition of depression through a list of symptoms facilitated by established diagnostic manuals: the DSM and ICD. They are based on passive categorical labelling , which list a number of prototype behaviours that could occur if certain psychological problems occur. This conceptualization within the medical model does not take into account the life circumstances and biographical context, which the individual interacts with, and thus the psychological sense that provides the key to understanding how this problem has been generated and why it remains is disregarded.The ICF model has been created by WHO as a complementary tool to the diagnostic systems (DSM and ICD) to describe the day-to-day functioning of people. Within the ICF model, psychosocial difficulties are seen as a continuum and as a result of the complex interaction of environmental variables, mental functions, personal variables, activities and participation, and health status. Another option for personalization of medicine and treatment in real clinical settings might be the inclusion of evaluations of daily functional problems experienced by patients. Therefore we suggest customization of healthcare\u2014with therapeutic decisions being tailored to the individual patient. The diagnostic testing has to be adjusted accordingly for selecting appropriate therapies. Eventually, engagement of patients in identifying specific personal problems  could provide more personalized information and change the pattern of diagnosing. For example, ecological momentary assessments of patients via different technologies, eventual self-monitoring, or even more collaborative interactions with therapists and professional carers would enhance research in this direction and give us a new insight. Recent literature based on ecological momentary assessments (EMA) gives us promising results in this line. Different studies , 57 haveEcological momentary assessments have been initially used to identify moment-to-moment patterns and mechanisms of psychopathology , but witMoreover, the patient perspective on PSDs and their related variables has also an impact on the clinical arena. Results of this study highlight the need for change and adjustment of future strategies for treatment and rehabilitation in depression. The process could be more productive, when clinicians select interventions covering PSDs and their modifiable determinants or related variables, and not only symptoms. Solving some activity problems, for example, can help in improving other areas as well. Furthermore, personalization of therapies according to individuals' PSDs will enhance the treatment process. In general, identification of specific interventions for particular psychosocial difficulties is necessary and future research should address this issue. Universitario de la Princesa in general did not allow us to have a more representative sample in terms of age. However, we decided to proceed with the focus group, because, in fact, a more homogeneous sample of participants is often preferable in terms of age, since it might increase the group comfort level when discussing sensitive topics. Therefore we could assess a wide variety of PSDs including sexual problems, relationships with the therapist, and somatic problems. Finally, in order to achieve more reliable results, we conducted individual interviews with 80 patients ranging substantially in terms of age. Thereat, in spite of this limitation, we found a high variability of PSDs and the valuable finding about the discrepancies between literature and patient reports. Further studies reaching saturation points should be done to test if there are other underexplored PSDs.The findings of this study, however, have to be interpreted assuming its limitations. First, only one focus group of seven patients was performed and saturation point\u2014considering the quantity of information regarding the analysed PSDs\u2014was not reached; therefore the focus group can be considered by some as nonrepresentative. As a partial solution we included a heterogeneous group through maximum sampling variation regarding sex, working status, and clinical status. The age range, however, was between 44 and 55 years, which might have caused a potential bias in the identification of PSDs. This is an important limitation, since some psychosocial difficulties, usually experienced in particular ages, like physical pain in older adults, or problems with intimate relationships or loss of life goals in younger adults, might have been omitted by the nonpresence of representatives of these age groups in our focus group. During the selection process, the psychiatrists who were responsible for the recruitment of participants took into consideration the maximum variability of sampling, but the time frame and the profile of patients in the catchment area of the HospitalSecond, a limited period of time was included for the literature search (studies between 2005 and 2010). The search was performed over papers published within the cited period of time because of temporal limitations and because we were interested in the recent literature outcomes rather than general literature. However, we made replication of the original search from 2011 to September 2013 in order to check if any new PSDs will occur in comparison with the initial search and the results did not identify new PSDs. The results of the review have to be read in light of the limitations due to the type of databases consulted. This review study was a component of a larger project that gathered psychosocial factors from several mental health and neurological conditions where only Medline and PsycINFO were included. As a consequence, an amount of studies might have not been identified. As a limitation concerning the reliability of the extraction process we have to indicate that only 20% of articles were independently double checked. Finally, by limiting our search to English literature, we might have omitted relevant papers in other languages.The present study is, to our knowledge, the first to analyse the full set of psychosocial difficulties and their related variables in depression through the literature and the patient perspective. We made an elaborate comparison between both sources of information in order to verify whether recent research literature reported all the PSDs and related variables that are important for depressed patients and to identify the existing gaps in this area. Regarding the research on depression, the results obtained within our study show the existence of many literature gaps and encourage future studies to focus on them more in depth. Concerning the clinical implications, this study emphasizes the need for change and adjustment of future strategies for treatment and rehabilitation in depression. We suggest that clinicians should select interventions that cover PSDs and their modifiable related variables and not only improve symptoms. Furthermore, eventual identification and personalization of therapies according to individuals' PSDs would potentially enhance the rehabilitation process."}
+{"text": "The results were discussed as regards analogous systems where cholesterol (Chol) was the third component. Moreover, the phosphatidylcholine\u2013lauryl gallate (PC\u2013LG) interactions were monitored by the attenuated total reflectance Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry. Besides lipid composition, the addition of LG was found to be a significant factor to modulate the model membrane properties. The LG molecules adjust themselves to the PC monolayer structure. The hydrophobic fragment is dipped into the membrane interior while the hydroxyl groups of phenolic gallate moiety associate with the polar groups of PC mainly through hydrogen bonding inducing the compacting effect. LG is found to be deeply submerged within DOPC, closer to the double bonds, and its insertion practically does not affect the DPPC/DOPC membrane fluidity. This is crucial for getting more profound insight into the role of LG in stabilizing the non-raft domains, mostly exposed to oxidation in which LG can co-localize and serve its antioxidant function.Lauryl gallate (LG) is an antioxidant agent. However, it exhibits poor solubility in water. Its interactions with the membrane result in structure evolution thus affecting the membrane functionality. In this paper the Brewster angle microscope coupled with the Langmuir trough was applied to determine the morphology, phase behaviour, thickness and miscibility of ternary Langmuir monolayers with equal mole fractions of 1,2-dipalmitoyl- Domain formation in the model membranes has been intensively studied since the hypothesis of lipid rafts was put forward  enriched in unsaturated PL susceptible to oxidative attack. Oxidation of unsaturated fatty acids in biological membranes lowers the membrane fluidity, damages its structure and functionality and finally causes undesirable lesions and diseases Yagi . To reduThe aim of this work was to investigate the phase behaviour, morphology and stability of model phospholipid membranes containing cholesterol or antioxidant LG. From the wide spectrum of gallates only LG seemed to optimize high antioxidant activity with sufficient hydrophobicity for the Langmuir monolayer formation. The effect of LG on the binary DPPC/DOPC monolayers was compared to that of cholesterol, which unlike LG is a structural component of biological membranes. The ordering of PL induced by Chol is driven by steric interactions  isotherms . Moreover, the PC\u2013LG interactions in the hydrated multibilayers were monitored by the attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The studies were supplemented with the time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis which identified chemically the lateral distribution of components in the PC/LG monolayers. Formation of intermolecular hydrogen bonds between the galloyl moiety and the head groups of PC was examined based on the signal intensity of characteristic ions.For these purposes, the ternary DPPC/DOPC/Chol (or LG) Langmuir films on the water subphase were investigated using the Brewster angle microscope (BAM) coupled with the Langmuir trough. The kind and magnitude of interactions between molecules in the ternary systems were estimated based on the experimentally determined surface pressure\u2013area per molecule  and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol) and LG of purity >\u200999% were provided by Sigma and used without purification. Chloroform (p.a.) and ethanol   were solvents for compound dissolution. Redistilled water was purified by the Milli-Q system to obtain resistivity of 18.2\u00a0M\u2126cm. Such water was used as a subphase for the Langmuir monolayer formation.PL: 1,2-dipalmitoyl-x\u2009=\u20090.5) and the ternary mixtures of the constant DPPC and DOPC ratio (x\u2009=\u20090.5) with varying Chol or LG mole fraction  were prepared from the respective stock solutions. The droplets of the solution were placed on the subphase surface top by means of a Hamilton microsyringe. After chloroform evaporation, the surface pressure\u2013area per molecule (\u03c0\u2013A) isotherms were recorded during symmetrical monolayer compression with the rate of 10\u00a0mm/min. The solubility of LG in water was estimated for 0.035\u00a0mg/mL, i.e. 1.034\u2009\u00d7\u200910\u22124 M (http://www.hmdb.ca/metabolites/HMDB38720). It was sufficiently low to form the Langmuir monolayers on water in the wide range of compression rates (from 5 to 100\u00a0mm/min) and its monolayers were stable in time even at a high surface pressure. The temperature of 20\u00a0\u00b0C was maintained owing to the water system circulation . The surface pressure was measured by the Wilhelmy plate method. Simultaneously, the morphology of the floating monolayers was monitored by the BAM coupled with the trough. The Langmuir trough and BAM were placed in the laser safety cabinet and isolated from vibrations coming from the surroundings by the active vibration isolation system.The one-component , binary (DPPC/DOPC 1:1) and ternary (DPPC/DOPC/Chol and DPPC/DOPC/LG) Langmuir monolayers were formed on the water subphase using a computer-controlled KSV standard trough . First the compounds were dissolved in chloroform to obtain the total concentration of 1\u00a0mg/mL. Then the binary mixture of DPPC/DOPC  to 10\u22128. As p-polarized light illuminated the pure water surface at the Brewster angle of about 53.2\u00b0, practically no intensity reflection was observed (dark background without contrast). When the monolayer with the refractive index different from that of water (i.e. 1.33) was spread onto the subphase, reflection occurred. The reflected light passed through the objective lens into the analyzer, and finally to the CCD camera . These elements were positioned in the reflected beam path to acquire high quality contrast images of the lateral morphology. Imaging of 720 \u00b5m\u2009\u00d7\u2009400\u00a0\u00b5m areas was performed with the lateral resolution of 2\u00a0\u00b5m. The black glass plate was placed under the water subphase on the bottom of the trough to prevent diffraction of the laser beam and to minimize light scattering.A computer-controlled nanofilm_ultrabam  was used for direct visualization of substructures with a long range orientational order and for evaluation of layer thickness at the air\u2013water interface. The light source of the BAM was a solid-state 50\u00a0mW laser-emitting 658\u00a0nm wavelength light. The laser beam accounts for purely R into the film thickness d  and the mean molecular areas of components in the monolayers (A12), as determined from the \u03c0\u2013A isotherm at 35\u00a0mN/m, first the quantity of compounds needed to cover the support with one statistical monolayer was calculated. Then the concentration and volume of the chloroform solutions were chosen so that after evaporation of the solvent, the 40 statistical bilayers were formed in each case. Such number of bilayers was found to be sufficient for obtaining good quality spectra. Before the solution pouring, the ZnSe crystal plate was cleaned with ultra pure ethanol. After the solvent evaporation the supported layers were transferred to a vacuum (117\u00a0mbar) for 30\u00a0min to remove the possible chloroform residuals, and then placed into the closed chamber with a relative humidity 80% for 30\u00a0min in order to hydrate the dried multibilayers.The multibilayers built of DPPC or DOPC and LG at different mole fractions (0.25 and 0.5) were prepared on the ZnSe crystal by spreading of the chloroform solutions. Knowing the geometric surface area of the ZnSe crystal .Infrared absorption spectra were obtained with a Nicolet 8700A Thermo Scientific spectrometer. The attenuated total reflection (ATR) configuration was used with the Varimax HATR ZnSe crystal plate (45\u00b0 cut). Typically 256 Fourier-transformed and averaged scans were collected for each measurement. Absorption spectra at a resolution of 4\u00a0cmxLG\u2009=\u20090.25) were transferred at 35\u00a0mN/m with the rate of 5\u00a0mm/min from the water subphase onto the mica plates using the Langmuir\u2013Blodgett trough . The samples were dried under vacuum (117\u00a0mbar) overnight. Mass spectrometric measurements were made using a TOF-SIMS 5 device . The primary ion source of Bi3+ was used at 25\u00a0keV. The scanning area of secondary ions was 100\u00a0\u00b5m\u2009\u00d7\u2009100\u00a0\u00b5m with 256\u2009\u00d7\u2009256 pixels. All measurements were performed in the static mode (dose no larger than 1013\u00a0ions/cm2). Two different places of each sample were analysed. No statistical difference was found between them. The post processing data analysis was conducted using the SurfaceLab 6.7 software (ION-TOF). For all samples charge effects were compensated using a flood gun adjusted individually for each sample area. Only positive spectra were recorded and calibrated using the positions of CH3+, C4H7+, C5H9+ peaks. The mass peaks with m/z of 27, 29, 39 and 58 were removed and then the mass spectra were compared after normalization of the intensity, proportionally to the total intensity recorded for each spectrum.The Langmuir monolayers of DPPC, DOPC, LG and mixed PC/LG  mixtures by monitoring differences in the shape of the compression isotherms at the air\u2013water interface. Figure\u00a0s Gaines . Therefos Gaines b.\u03c0\u2013A isotherms which would suggest LG leaving the monolayer for the bulk subphase, or forming a multilayered film. These findings allow to confirm that the LG molecules remain in the mixed monolayer and associate with PC in such a way that changes the polar head configuration yielding smaller areas per molecule.To prove that LG is not \u2018squeezed out\u2019 of the PC monolayer, the isotherm of pure DOPC monolayer was recorded and then taking the same amount of DOPC the mixed DOPC/LG film containing 10\u00a0mol% LG was obtained. The measurements were repeated three times. The same experiment was conducted for the systems with DPPC instead of DOPC. The results are presented in Fig.\u00a0Moreover, as reported previously for the DOPC/LG, POPC/LG and DPPC/LG binary systems in the full range of the LG mole fraction, no LG squeezing out of the PC monolayers was found  there is observed the liquid\u2013liquid phase coexistence (region of coexisting liquid phases) with one phase rich in the unsaturated DOPC and the other in the saturated DPPC and cholesterol which can be seen as dark and bright regions, respectively. Hence the bright regions seem to be equivalent to the liquid-ordered state (Veatch and Keller xChol\u2009=\u20090.5 two liquid phases still coexisting as micron-sized liquid domains are observed and their heterogeneity of size is larger than that at xChol\u2009=\u20090.25. At a high mole fraction (xChol\u2009=\u20090.75) one uniform liquid phase is observed at first as a result of coalescence of domains into larger ones, and finally at ca. 30\u00a0mN/m into one homogeneous phase. Domains coalesce close to a critical point due to a small difference in the dipole density between phases (Keller and McConnell Both DPPC and DOPC form homogeneous monolayers at 35\u00a0mN/m but that of DPPC is tightly packed (condensed) while that of DOPC is loosely packed (expanded) Fig.\u00a0. In the x\u2009=\u20090.25\u201321\u00a0mN/m; at x\u2009=\u20090.5\u201330\u00a0mN/m; at x\u2009=\u20090.75\u201338\u00a0mN/m. At low LG concentration xLG\u2009=\u20090.25 and surface pressure, the area fraction of bright oval domains is small but the average size of the condensed domains increases with the increasing surface pressure up to 35 mN/m. Contrary to DPPC/DOPC, further compression does not lead to the change of domain sizes or shapes but it increases only their number per unity area. Hence the domains can be identified individually. At xLG\u2009=\u20090.5 the domains are much smaller and rather sparsely spread. With the LG mole fraction increase up to x\u2009=\u20090.75 the ternary monolayer is homogeneous at 35\u00a0mN/m  in DPPC/DOPC the difference in x\u2009=\u20090.75 it is as large as 337 mN/m. The drastic increase in the degree of the monolayer condensation, i.e. packing of the lipid molecules, makes it more ordered.The s (Israelachvili a is estimated to be 87\u00a0\u00c52 (http://www.hmdb.ca/metabolites/HMDB38720), the volume of hydrocarbon chain V\u2009=\u2009350.2\u00a0\u00c53, the length of hydrocarbon chain lC\u2009=\u200916.68\u00a0\u00c5, the critical packing parameter s was calculated according to Eq.\u00a0Additionally, the geometric packing (arrangement) of molecules was described in terms of the molecular shape of the constituents. It was predicted based on the critical packing parameter s parameter for LG is 0.24 which is lower than 1/3 suggesting a cone shape of molecule  for which coexistence of condensed DPPC-rich domains and a fluid DOPC-rich matrix can be seen . The thickness values of DPPC/DOPC/LG membranes depend insignificantly on the composition and they are located between those of DPPC/DOPC and LG. The thickness of pure LG monolayer is as low as 0.93\u00a0nm. The difference in thickness of both types of membranes becomes bigger as the mole fraction of the third component increases. As reported earlier Chol exerts a condensing effect on DPPC and DOPC  at different values of the surface pressure were determined directly from the isotherms, and then compared with the values obtained for the ideal miscibility or complete immiscibility of molecules based on Eq.\u00a0A1, A2, A3 denote the mean molecular areas in the one-component monolayers of DPPC, DOPC and Chol (or LG), respectively at a given surface pressure; and x1, x2, x3 are the molar ratios of components 1, 2 and 3 in the mixed monolayer.Miscibility and interactions between molecules in the mixed monolayers were qualitatively analysed in accordance with the additivity rule Gaines . TherefoA123 is the mean molecular area in the ternary films. If Aexc is equal to 0, the dependence of A123 on the composition is linear, and the mixture behaves ideally indicating that the components are completely miscible or immiscible. When Aexc\u2009\u2260\u20090, i.e. any deviation from linearity of the A123\u2009=\u2009f (x) function occurs, this indicates miscibility of the compounds due to the mutual interactions. The non-ideality of these mixed systems was expressed in the excess area (Aexc)-composition plots shown in Fig.\u00a0Then the effect of Chol or LG on the DPPC/DOPC phospholipid films was estimated from the excess area per molecule values according to Eq.\u00a0Aexc are lower  was determined at 35\u00a0mN/m from Eq.\u00a0N denotes the Avogadro\u2019s number, Aexc is the excess area, and \u03c0 is the surface pressure which changes in the range of integration from 0 to 35 mN/m.To explore the magnitude of these interactions and to find out how different amounts of Chol or LG modulate their interactions with the DPPC and DOPC model membranes, the excess Gibbs energy of mixing . Although not corresponding to the more densely packed monolayers, at which xChol\u2009=\u20090.25 (\u2206Gexc\u2009=\u2009\u2212\u20091660.9\u00a0J/mol) (Jurak xLG\u2009=\u20090.5 (\u2206Gexc\u2009=\u2009\u2212\u20091424.2\u00a0J/mol)  Jurak  whereas R is the gas constant and T is the temperature.Furthermore, in order to determine the thermodynamic stability of the mixed systems the total Gibbs energy of mixing was determined Eq.\u00a0:7\\documeGmix are negative regardless of the monolayer composition  of infrared spectra of PC membranes is represented by the bands of antisymmetric and symmetric stretching vibrations of CH2 groups ) of lipid hydrocarbon chains that form a hydrophobic core of the lipid bilayer. These bands are sensitive to the lateral packing of hydrocarbon chains. Spectral parameters (wavenumber of absorption maximum and bandwidth) of the \u03bdas,s(CH2) bands monitor the alterations of the ratio of trans to gauche conformers of lipid CH2 moieties  bands of DOPC/LG to a higher-frequency region and increase in the bandwidths of these two vibrational modes. This indicates that the DOPC/LG bilayers adopt a conformationally disordered gauche-rich fluid state while the DPPC/LG bilayers are enriched in the trans conformer population in the hydrocarbon part of the membrane. The absorption maxima of both \u03bdas,s(CH2) bands for pure LG are between those of DPPC and DOPC, and simultaneously the peaks become broader/tighter and shifted to higher/lower frequencies than those of DPPC or DOPC, respectively. Such observation implies the intermediate ordering of hydrocarbon chains.In Fig.\u00a0+\u2013(CH3)3 stretching vibrations of choline, symmetric and antisymmetric stretching vibrations of PO2\u2212 group, stretching vibrations of the C\u2013O\u2013P\u2013O\u2013C fragment, stretching vibrations of the ester carbonyl group C=O which represents the polar\u2013apolar interface in the PC membrane  and its maximum shifts to the higher-frequency region compared to the pure PC films, and at xLG\u2009=\u20090.5 finally splits into two bands. This is due to contribution of the C=O stretching coming from the C=O ester groups of LG. In the spectrum of pure LG the band ascribed to the stretching vibrations of ester C=O group appears at 1683.28\u00a0cm\u22121. Its shift suggests participation of carbonyl groups in the interaction process. The other characteristics for the LG bands at 1245.81, 1196.76 and 1034.07\u00a0cm\u22121 correspond to the C(=O)\u2013O stretching vibrations of ester groups, in-plane bending vibrations of the phenolic group and O\u2013C\u2013C stretching vibrations of the ester group, respectively  are very sensitive to hydrogen bonds formation  and 1220\u00a0cm\u22121 (in the fully hydrated PC state)  + ion. Other characteristic ions produced by PC head appear at m/z 104.12 ((C5H14NO)+) and m/z 166.07 ((C5H13PO3N)+) +) Fig.\u00a0a intensi)+) Fig.\u00a0b signifi)+) Fig.\u00a0. During .38 Fig.\u00a0b, c. Hig.38 Fig.\u00a0.3+ beam and different packing of LG within the DOPC monolayer.To illustrate how insertion of LG into the DPPC or DOPC monolayer affects the molecular organization, simple models are presented in Fig.\u00a0Interestingly, intensity of molecular ion of DOPC after the addition of LG is 38.3 times higher than that of pure DOPC. This is due to weak binding of LG into DOPC through the hydrogen bond that is favourable for desorption of molecular species. In the presence of LG the yield of DPPC molecular ion increases only eight times indicating that molecules are more strongly bound within the DPPC/LG monolayer due to the additional interactions by hydrogen transfer between the hydroxyl group of LG and the phosphate group of DPPC. As a result, in the molecular desorption region (far away from the impact region)  phase at the experimental temperature. Consequently, because of the presence of C18 chains with one double bond which induces a kink, the DOPC monolayer is less condensed  and \u2206Gexc (1277.8\u00a0J/mol)  Fig.\u00a0 and weak \u2206Gexc 127.8\u00a0J/molLo phase is stronger than for the DOPC-rich liquid-disordered Ld phase , the border of ordered domains blurs which is associated with the fluidizing effect of LG due to disturbing the ordered structure of DPPC-enriched domains. This strongly supports the increased mutual miscibility between DPPC, DOPC and LG in the monolayer state. Hence similarity in the phase behaviour of LG and DPPC/DOPC maintain the membrane fluidity.LG analogously to Chol can modulate the fluidity and ordering of the membrane leaflet, thus influencing its function. LG forms the disordered monolayer at the air\u2013water interface similarly to DOPC Fig.\u00a0. Althougx\u2009=\u20090.25, is lower in the DPPC/DOPC/LG system as the LG area is lower than that of Chol at that surface pressure. However, in this system the thickness and packing of monolayers change only little with the increasing LG due to the similar phase state/condensation degree of the LG and DPPC/DOPC monolayer . Chol is primarily a structural component of biological membranes. LG can interact with the lipid components of membrane and simultaneously being an antioxidant molecule, it is able to protect the unsaturated lipids from oxidation by free radical scavenging. Chol in the DPPC/DOPC monolayers causes their condensation leading to an increase in density of lipid molecules and their ordering, thus improving the film stability. LG practically retains the DPPC/DOPC membrane fluidity and minimizes the unfavourable interactions between DPPC and DOPC also increasing the phospholipid miscibility and stability. As regards the distribution between the coexisting phases, LG preferentially localizes in more fluid areas, which is imposed by antioxidant activity, whereas Chol in more ordered ones. This results from differentiated affinity of both molecules for unsaturated or saturated PL. The favourable interactions contribute to the formation of heterogeneities within the membrane. They seem to reflect the specialization of various regions in the membrane processes."}
+{"text": "Field cancerisation was originally described as a basis for multiple head and neck squamous cell carcinoma (HNSCC) and is a pre-malignant phenomenon that is frequently attributable to oncogenic human papillomavirus (HPV) infection. Our work on \u03b2-HPV-induced cutaneous squamous cell carcinomas identified a novel Lrig1+ hair follicle junctional zone keratinocyte stem cell population as the basis for field cancerisation. Herein, we describe the ability for HPV to infect adult tissue stem cells in order to establish persistent infection and induce their proliferation and displacement resulting in field cancerisation. By review of the HPV literature, we reveal how this mechanism is conserved as the basis of field cancerisation across many tissues. New insights have identified the capacity for HPV early region genes to dysregulate adult tissue stem cell self-renewal pathways ensuring that the expanded population preserve its stem cell characteristics beyond the stem cell niche. HPV-infected cells acquire additional transforming mutations that can give rise to intraepithelial neoplasia (IEN), from environmental factors such as sunlight or tobacco induced mutations in skin and oral cavity, respectively. With establishment of IEN, HPV viral replication is sacrificed with loss of the episome, and the tissue is predisposed to multiple cancer stem cell-driven carcinomas. Human papillomavirus (HPV) infection is associated with oropharyngeal and anogenital cancers in both men and women. Approximately 90% of all cervical cancers are attributed to high-risk alpha-genus HPV (\u03b1-HPV) infections, also \u223c60% of squamous cell carcinomas (SCC) of the vulva, vagina, anus and penis are due to infection of \u03b1-HPV . HPV infSequential genetic and epigenetic changes occur over several years and provide the transformational basis for both intraepithelial neoplasia (IEN) and ensuing epithelial cancers (carcinoma). The proportion of transformed cells within IEN can be graded and used to define the risk of invasive disease . Progres1), hence the American Association for Cancer Research Task Force on the treatment and prevention of IEN recognizes the importance of early treatment to prevent invasive disease    . Howeverigure 1) . Within igure 2) . TherefoFigure 2). Clonal selection and expansion may result in a single clone in continuous epithelia  or multiple clones in discontinuous epithelia (breast and lung)  . Within nd lung) . Hence, Figure 2)  . Howeverigure 2) . As a coigure 2) . Hence, Human papillomavirus (HPV) binds epithelial cell heparan sulfate proteoglycans and cell specific receptors to gain entry by both clathrin-dependent and -independent endocytosis . InfectiMost HPV infections are spontaneously cleared. For example, the risk of \u03b1-HPV female genital infection over a lifetime is up to 80% , but witIn cervical lesions caused by the \u03b1-HPVs, the viral oncogenes E6 and E7 increase proliferation of suprabasal epithelial cells. Along with E1 and E2, viral replication requires E6 and E7 for entry into S-phase. Upon leaving the basal layer, keratinocytes enter into a program of terminal differentiation in order to produce a protective barrier. However, in HPV infection, suprabasal cells continue to proliferate and are prevented from entering terminal differentiation . OncogenFluorescent labeling studies in mice using lineage tracing have concluded that stem cell division is prominently (\u223c90%) asymmetric; in which there is renewal of the stem cell and a daughter cell that is committed to terminal differentiation . StochasAdult tissue stem cell expansion, as proposed for the mechanism of HPV-induced field cancerisation, is dependent on symmetrical division of existing stem cells. As discussed, HPV viral oncogenes will drive proliferation of infected adult tissue stem cells by targeting p53 or pRb. Importantly, the binding of E7 to pRb releases repression of both sex determining region Y-box 2 (Sox2) and octamer-binding transcription factor 4 (Oct4) . Similarin vitro of increased colony forming efficiency  or cutaneous warts. Similarly, mucosal HPV lesions include condyloma or leukoplakia within the genitalia and oral mucosa . In addiThe transition from benign to premalignant lesion has been characterized by TP53 immunostaining, resulting from mutation acquisition, and manifesting as a small micro-clonal expansion comprising of 60\u20133000 cells presenting clinically as an actinic (solar) keratosis or leukoplakia . In the Figure 2)  . For exaigure 2)  Similarligure 2) . Hence, igure 2) .Field cancerisation emerging from HPV induced amplification of adult tissue stem cells results from additional environmental induced mutations. The area of IEN can be large, in the oral cavity it can be over 7 cm in diameter and is predisposed to multiple primary HNSCC and therefore poor prognosis . IntriguMany cancers exhibit hierarchical growth with evidence of differentiation consistent with the cancer stem cell model . Whereinin vitro and in vivo assays (Head and neck squamous cell carcinoma (HNSCC) identification and characterisation of cancer stem cells has been supported by o assays . Similaro assays . The enso assays . Overallo assays . High nuo assays . Hence, This review has focused on HPV infection, notably oncogenic genotypes from both the alpha and beta genus. Within the tropic tissue that was breached to allow viral entry, persistent infection requires that resident adult tissue stem cells are infected. HPV-infected adult tissue stem cells, similar to other HPV-infected cells are forced to proliferate, leading to their expansion as adult tissue stem cells beyond their native niche. This expansion renders them susceptible to environmental carcinogens. In the case of skin, \u03b2-HPV genotypes induce hair follicle junctional zone keratinocyte stem cells to proliferate and expand into the overlying epidermis, whereupon they are susceptible to UV-induced mutations. Transformational mutations result in field cancerisation, with additional driver mutations, causing clonal selection as IEN progresses from mild to severe. Additional mutations then can give rise to multiple cancers. Hence, HPV-induced stem cell expansion predisposes to and, through viral oncogene expression, induces the generation of cancer stem cells, which in turn can define the fate of tumor and patient prognosis. Hence, we propose that the ability of oncogenic HPV infection to manipulate adult tissue stem cells underpin its ability to drive cancer growth through promotion of cancer stem cells.CO, SL, and GP conceived the idea. CO, SL, CB, MG, and GP drafted the manuscript with inputs from all authors. All authors have made final approval for the final version to be submitted.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": "Stem cells and cellular plasticity are likely important components of tissue response to infection. There is emerging evidence that stem cells harbor receptors for common pathogen motifs and that they are receptive to local inflammatory signals in ways suggesting that they are critical responders that determine the balance between health and disease. In the field of papillomaviruses stem cells have been speculated to play roles during the viral life cycle, particularly during maintenance, and virus-promoted carcinogenesis but little has been conclusively determined. I summarize here evidence that gives clues to the potential role of stem cells and cellular plasticity in the lifecycle papillomavirus and linked carcinogenesis. I also discuss outstanding questions which need to be resolved. Human papillomavirus (HPV) infection occurs via micro wounds which allow the virus access to the basal layer of stratified epithelia. This target site of infection has been the subject of intense scrutiny, leading to significant progress regarding the molecular mechanisms governing the attachment and eventual entry of the virus into initially infected cells ,2. HowevPapillomaviruses can productively infect both mucosal and cutaneous stratified epithelia. Infection and the ensuing life cycle of the virus are intimately linked to the differentiation program of the tissue ,9 thus oStudies using HPV virus like particles (VLPs), or infectious virions for attachment and viral entry argue against the exclusive targeting of a small subpopulation such as the tissue stem cells. Rather, the virus associates with the exposed basement membrane at a site of wounding, and furin cleavage leads to conformational changes on the capsid which allow it to attach to nearby basal cells via a receptor which recognizes L1 capsid protein . The higIf one takes for granted the more likely scenario that both stem and committed cells can be the targets of infection, then a vital question concerns the fate of infection in a stem as opposed to a committed cell. A popular hypothesis has been that long-term maintenance of the viral DNA can take place within infected tissue stem cells ,5. This Epidermodysplasia verruciformis (EV) patients, transplant recipients etc.) [Hair follicle stem cells have also been proposed as the reservoirs for human cutaneous HPV infections. DNA of \u03b2-papillomaviruses is frequently isolated from plucked eyebrow hairs ,18 suggets etc.) .The inability to define the concrete characteristics of the human cervical stem cells has complicated studies aiming to understand the maintenance of the human mucosotropic viruses. It is thought that the transformation zone which is characterized by a transition from columnar to squamous epithelium is the site of the so-called reserve cells which may act as the cervical stem cells. Some markers have been proposed for reserve cells , p63, Keratin 7 (K7), etc.) ,22 but tEscherichia coli [Infected tissue stem cells are of interest due to their potential links to carcinogenesis. However, more recently stem cells have also been implicated in the tissue response to infection. There is an emerging understanding that tissue stem cells have evolved to respond directly both to commensal and pathogenic microbes as evidenced by the expression of pattern recognition receptors (PRRs) in tissue stem cells ,24,25. Ihia coli  mobilizeWhile the effects of infection on tissue stem cell dynamics are less well understood in cutaneous and mucosal epithelia compared to the gut, studies investigating the expression of viral gene products on skin stem cell populations suggest that important changes occur. Compelling evidence regarding the changes in stem cell dynamics during papillomavirus infection comes from studies using transgenic animals for both mucosotropic ,29 and cInitial focus has been on uncovering how the behavior of stem cells may change upon papillomavirus infection. There is however also evidence which supports an alternate, not mutually exclusive scenario: that during infection it is possible that reprogramming events can contribute to the emergence of stem-cell like cells.The increased stem cell mobilization seen in transgenic animals is also concurrent with the expression of stem cell markers such as Keratin15 (K15) outside the typical stem cell niche ,29. The In light of technologies describing cellular reprogramming developed after Yamanaka\u2019s seminal discoveries in 2006 , the re-Mycobacterium leprae [M. leprae infection leads to such events are incompletely understood, initial evidence suggests that they involve in part the innate immune response and inflammation which precede such reprogramming events [One of the most intriguing and compelling reported examples of reprogramming in vivo implicates the intracellular m leprae . The bacm leprae . While Sg events .Inflammation is naturally of relevance to the HPV lifecycle as well, particularly if one takes into account the mode of infection. Both infection-associated and sterile inflammation are understood to contribute to a regenerative response and inflammation is emerging as an evolutionarily conserved mechanism of tissue regeneration . RegenerOther than leading intracellular lifestyles HPVs do not have many commonalities with mycobacteria at a first glance. However, upon closer inspection of the available evidence there is good reason to suspect that the virus may be contributing to similar events. The viral oncogenes have prominent targets which are implicated in stem cell biology and may contribute to epigenetic reprogramming via their inactivation.INK4A/ARF locus, some of which are implicated in papillomavirus pathogenesis are also important during reprogramming [Work aimed at illuminating the critical steps during the process of reprogramming cells to pluripotency, clearly implicated prominent tumor suppressors in stem cell biology. p53, retinoblastoma protein (pRb) and other key players in tumor suppression have been shown to control checkpoints during cellular reprogramming ,52,53,54Hox genes. Critically this expression has been linked to the high p16 expression characteristic of HPV positive cancers and may represent a way of targeting HPV-positive cancer cells since they are dependent on its continued expression [In addition to targeting tumor suppressors which have been linked to cellular reprogramming there is also evidence to suggest that the viral oncogenes may contribute to cellular reprogramming in ways which are independent of their ability to target p53 or pRb. High-risk HPVs  have recently been shown to upregulate Kruppel-like factor 4 (Klf4) and contribute to its hypoSUMOylation . This uppression . The E7 pression . High-ripression ,66. Of cThere is a dearth of studies which would allow definitive conclusions about the upregulation of stem cell related proteins in the viral life cycle. However, there is an accumulating body of evidence suggesting they may be linked to disease. The upregulation of genes related to stem cells may be a way in which the virus contributes to the formation and maintenance of cancer stem cell populations . RecentlINK4A as a useful biomarker it remains to be seen whether the use of these are of added prognostic benefit. Nevertheless, the expression of these proteins in cancer biopsies and cervical cancer cell lines may provide insights into potential roles in the carcinogenic process. Critically, the links of early gene products, particularly the viral oncogenes of human papillomaviruses to the upregulation of such markers lends credibility to this notion.The expression of stem cell related markers is now increasingly reported in HPV-related cancers. Proteins such as Sox2, Nanog, and Oct4 are now thought to serve not only as biomarkers tumor stage and therapy response in cancer but also may be directly implicated in the process of carcinogenesis. In HPV-associated cancers stem cell markers Oct4, Sox2  , and LriA source of skepticism for studies supporting a changing cellular state during infection, derives from the refractoriness of keratinocytes to cellular reprogramming and perceived lack of plasticity. It is clear that no condition has been described thus far where a viral gene product or set of products have been shown to independently lead to an isolatable stem cell. However, it is critical to bear in mind that infection occurs in the context of a wound. It is an aspect of infection which has been poorly accounted for particularly since many experimental models used in papillomavirus research do not take it into consideration. In virion-infected monolayer keratinocytes, organotypic cultures of stably transfected keratinocytes or transgenic animals expressing viral gene products, the wound environment is not routinely modelled. However, during real-life infection, in addition to the viral invasion which triggers an inflammatory response via the presence of pathogen associated molecular patterns (PAMPs) there are also damage associated molecular patterns (DAMPs), and reactive oxygen species (ROS) released as a result of the wounding. Thus, both infection-associated, as well as sterile inflammation should be kept in mind as additional important factors which likely remove constraints towards increased cellular plasticity .The role of stem cells and cellular stemness has been proposed to be important in the lifecycle and disease promotion by HPVs. Viral genomes have been detected in anatomical locations which are consistent with those of stem cells yet conclusive evidence as to whether infected stem cells are the sites of viral maintenance of all HPVs or the cancer initiating cells of HPV-related cancers is to this day tenuous. Early gene products, and particularly the viral oncogenes have been shown to modify stem cell dynamics and cellular stemness, however the extent to which this is critical for the viral lifecycle or for ensuing disease remains elusive. As HPV-related cancers account for about 5% of human cancers  and pot. Critica"}
+{"text": "Fcgr2b knockout or antagonistic Fc\u03b3RIIb antibody inhibited A\u03b21-42-induced tau hyperphosphorylation and rescued memory impairments in AD mouse models. Fc\u03b3RIIb phosphorylation at Tyr273 was found in AD brains, in neuronal cells exposed to A\u03b21-42, and recruited SHIP2 to form a protein complex. Consequently, treatment with A\u03b21-42 increased PtdInsP2 levels from PtdInsP3 to mediate tau hyperphosphorylation. Further, we found that targeting SHIP2 expression by lentiviral siRNA in 3xTg-AD mice or pharmacological inhibition of SHIP2 potently rescued tau hyperphosphorylation and memory impairments. Thus, we concluded that the Fc\u03b3RIIb-SHIP2 axis links A\u03b2 neurotoxicity to tau pathology by dysregulating PtdInsP2 metabolism, providing insight into therapeutic potential against AD.Amyloid-\u03b2 (A\u03b2)-containing extracellular plaques and hyperphosphorylated tau-loaded intracellular neurofibrillary tangles are neuropathological hallmarks of Alzheimer's disease (AD). Although A\u03b2 exerts neuropathogenic activity through tau, the mechanistic link between A\u03b2 and tau pathology remains unknown. Here, we showed that the Fc\u03b3RIIb-SHIP2 axis is critical in A\u03b2DOI:http://dx.doi.org/10.7554/eLife.18691.001 In Alzheimer\u2019s disease, damage to neurons in the brain gradually causes memory loss and difficulties with thinking. The main hallmarks of this damage are seen in the accumulation of proteins in and around neurons. First, a protein called amyloid beta forms aggregates outside the cell. This appears to lead to the build up of an abnormal form of a protein called tau inside the cell. These abnormal tau proteins have excessive numbers of phosphate groups attached to them, and so are known as \u201chyperphosphorylated\u201d. The molecular mechanism underlying amyloid beta\u2019s role in the hyperphosphorylation of tau proteins was not known.Amyloid beta binds to many different receptor proteins \u2013 including one called Fc gamma receptor IIb \u2013 on the surface of neurons. Kam, Park et al. investigated whether interactions between amyloid beta and Fc gamma receptor IIb might regulate the phosphorylation of tau within neurons. Adding amyloid beta to mouse neurons caused tau proteins to become hyperphosphorylated. However, removing Fc gamma receptor IIb from the neurons, or stopping it from binding to amyloid beta, abolished this effect.When amyloid beta was bound to Fc gamma receptor IIb, the receptor became phosphorylated. This in turn triggered a series of further phosphorylation events, culminating in an increase in the level of a molecule that relays signals from cell receptors \u2013 called SHIP2 \u2013 inside the neurons. This molecule increases tau phosphorylation when added to neurons. Reducing the activity or amount of SHIP2 in mice that present the symptoms of Alzheimer\u2019s disease reduced the hyperphosphorylation of the tau protein in their neurons and restored their memory to normal levels.Kam, Park et al. also looked at samples taken from the brains of human Alzheimer's disease patients. Unlike samples taken from people without Alzheimer\u2019s disease, neurons in these samples contain both phosphorylated Fc gamma receptor IIb and hyperphosphorylated tau proteins.By uncovering the molecules that link amyloid beta with tau hyperphosphorylation, Kam, Park et al.\u2019s results suggest new targets for therapies to treat the symptoms of Alzheimer\u2019s disease. More research is now needed to investigate whether this could lead to the design of new drugs.DOI:http://dx.doi.org/10.7554/eLife.18691.002 Alzheimer's disease (AD) is characterized by the progressive loss of memory and the neuronal degeneration . The patAPP transgenic mice prevented A\u03b2 pathologies, including learning and memory impairment P2, and PtdInsP2, are known to play a major role in signal transduction upon cellular stimulation P2 have been relatively well characterized in cell survival, proliferation, and synaptic function via their binding proteins P2, PtdInsP2 and PtdInsP3 are formed when cells respond to signals P2 , such as PtdInsPmulation . Among tproteins , but the signals . SH2 domsP2 . Increasmeric A\u03b2 , and rece models . HoweverUntil now, A\u03b2 was reported to bind to many receptors, including alpha7 nicotinic acetylcholine receptors (\u03b17 nAChR), NMDA receptor, receptors for advanced glycation end- products (RAGE), A\u03b2-binding alcohol dehydrogenase (ABAD), the Ephrin-type B2 receptor (EphB2), cellular prion protein (PrPc), and paired immunoglobulin-like receptor B (PirB) . Althoug1-42 to mediate A\u03b2 neurotoxicity, synaptic dysfunction, and memory impairment in AD pathogenesis  to increase PtdInsP2 levels for tau hyperphosphorylation. Further, Fcgr2b or Inppl1 deficiency in 3xTg-AD mice or pharmacological inhibition of either protein abrogates all of these observations, highlighting the importance of the Fc\u03b3RIIb-SHIP2 axis in the A\u03b2-induced tau pathology.Recently, we showed that Fc gamma receptor IIb (Fc\u03b3RIIb) is also expressed in neurons and directly interacts with A\u03b2ogenesis . Here, w1-42 and a mediator of memory impairment in hAPP-J20 mice expressing only familial mutant APP , Thr231/Ser235 (AT180), and Ser202 (CP13), but these phosphorylations were abrogated in Fcgr2b knockout (KO) neurons (1-42 in the neuron-specific enolase (NSE)-positive cortical neurons, but not in the Fcgr2b KO neurons . Each group of mice was tested for spatial working memory in a Y-maze task. Although the total number of arm entries was not significantly different between groups, Fcgr2b deficiency improved the spatial working memory of 3xTg-AD mice  region of wild-type (WT) mice  mice , the micWT) mice . Interesbehavior , object behavior , and pasbehavior . On the behavior . From weted mice . In contted mice . Thus, w1-42  in the cytosolic region of Fc\u03b3RIIb in SH-SY5Y cells  or cytoplasmic region (\u0394cyto), or substituting the tyrosine residue with phenylalanine (Y273F), and examined their effects on A\u03b222 cells . Moreove22 cells , blockediated it , indicat neurons . On the ns , but not in normal and mild cognitive impairment (MCI) patients (stage III) . In addi1-42  is known to bind to the phosphorylated ITIM region of Fc\u03b3RIIb and inhibit downstream responses triggered by immune receptors . Given t1-42 , suggest1-42. Treatment of SH-SY5Y cells with A\u03b21-42 enhanced subcellular localization of SHIP2 to the plasma membrane and thus colocalization of SHIP2 with Fc\u03b3RIIb P2 P2 with a protein lipid overlay (PLO) assay using the purified His-tagged pleckstrin homology (PH) domain of tandem PH domain-containing protein-1 (TAPP1) as a probe PsP2 , we exam a probe . From tho A\u03b21-42 . In contwn cells . Converscells P3 was decreased by 17% P2 and PtdInsP2 levels. On the other hand, the levels of PtdInsP2 were lowered by A\u03b21-42 in WT neurons, consistent with a previous report P2 and PtdInsP3 levels in neurons via Fc\u03b3RIIb.When we directly measured the levels of phosphoinositides with an ELISA assay, we observed that the levels of PtdInsPd by 17% . Howevers report , and als neurons . We furtmembrane . In contwn cells . As repown cells , hydrogeficiency . These o2 by A\u03b21-42 can influence tau phosphorylation, we directly delivered phosphoinositide into living neurons using a carrier  in primary cortical neurons in a dose-dependent manner P2, PtdInsP2, and PtdInsP3, failed to do so. Consistent with the activation of GSK3\u03b2 by A\u03b21-42, PtdInsP2 treatment also reduced the inhibitory phosphorylation of GSK3\u03b2 at Ser9 in neurons , as well P carrier . Comparet manner . Interes neurons . Moreove as well . We furtectively . We confl1 siRNA . Combinel1 siRNA , these r1-42 in control cells, knockdown of SHIP2 expression in SH-SY5Y cells abrogated tau phosphorylation by A\u03b21-42 . We confirmed that infection with lenti-siInppl1 reduced SHIP2 levels in both HT22 cells and primary cortical neurons  in primary cortical neurons . These results indicate that SHIP2 is critical to memory impairment and tau hyperphosphorylation in 3xTg-AD mice.Then, we examined the role of SHIP2 (INPPL1) in memory impairment in vivo using  neurons . Consist neurons . When wenjection . When we-AD mice . The ame1-42 in mice, we directly delivered AS1949490 into mouse brains because of its poor bioavailability   . Taken t1-42, with Fc\u03b3RIIb accounts for such neuropathogenic defects of AD as an initiation step, the selective interaction of oligomeric A\u03b21-42 with Fc\u03b3RIIb may hint at why A\u03b21-42, but not A\u03b21-40, is important in tau pathology . Thus, it is likely that Lyn phosphorylates the ITIM of Fc\u03b3RIIb in neuronal cells in response to A\u03b21-42. Moreover, we also observed this phosphorylation of Fc\u03b3RIIb in AD brains (stage V and VI) in which tau was highly phosphorylated. Then, how is Fc\u03b3RIIb different from other A\u03b2 receptors? It is reasonable to propose that different mechanisms or even the same mechanism exerts multiple effects at different stages of disease progression (Unlike other Fc\u03b3Rs that have an immunoreceptor tyrosine-based activating motif (ITAM), Fc\u03b3RIIb has a unique ITIM in its cytoplasmic tail and thus acts as an inhibitory receptor in B cells . Excitingression . For insgression  and ABADgression . In casegression . Furthergression . Thus, wgression .1-42 to tau by binding to the phosphorylated Fc\u03b3RIIb. Many studies on SHIP2 have focused on its inhibitory effect on insulin signaling P2 is scarce under normal conditions and increases through signaling P3 are known to overlap to some degree in their functions, they apparently have distinct roles in neurodegeneration. Recently, it was shown that PtdInsP2, but not PtdInsP3 and PtdInsP2, potentiates glutamate-induced cell death in neurons P2 and leads to neurodegeneration in brains  also dephosphorylates PtdInsP3 at position 3 and generates PtdInsP2 P2 deficiency P2, and leads to AD pathogenesis.Dysregulation of phosphoinositide metabolism is increasingly recognized as important in various diseases, such as cancer, diabetes, and myopathy . The phoignaling . While P neurons . In addin brains . We alsosP2 . The redsP2 . More reficiency . Becausels in AD , as we a1-42 induces Fc\u03b3RIIb phosphorylation to recruit SHIP2, leading to disruption of phosphoinositide metabolism for tau hyperphosphorylation and memory impairment in neurons and AD model mice. Together with the A\u03b2-lowering strategy, our results provide new ways for AD therapeutics to rescue A\u03b2 and tau pathology: i) selective inhibition of the interaction between Fc\u03b3RIIb and A\u03b21-42, ii) inhibition of a kinase  which phosphorylates Fc\u03b3RIIb upon A\u03b21-42 stimulation, and iii) inhibition of SHIP2 which disrupts phosphoinositide metabolism.In conclusion, the Fc\u03b3RIIb-SHIP2 signaling axis provides the missing link between A\u03b2 and tau pathologies. Notably, A\u03b2Fcgr2b KO C57BL/6 .WT (C57BL/6),  C57BL/6 , 3xTg-ADg for 20 min at 4\u00b0C, aliquoted and stored at \u201380\u00b0C until use. The supernatants were subjected to SDS-PAGE.Hippocampal tissues from AD (Braak V-VI) , MCI (Braak III)  and non-AD patients  were kindly provided by the Harvard Brain Tissue Resource Center . Hippocampal tissues of AD patients were homogenized in ice-cold Tris-buffered saline (TBS) buffer . The homogenates were clarified by centrifugation at 14,000 Inppl1 (5\u2032-GAA GGG AGG GCA CGT TAA TTT-3\u2032) (Sigma-Aldrich) was used for injection and pLKO.1-Neo-CMV-tGFP non-target virus was used as a control. The lentivirus  was stereotaxically injected bilaterally into the dentate gyrus (2 \u03bcl per hemisphere at 0.4 \u03bcl/min) with the following coordinates: anteroposterior\u00a0=\u00a02.1 mm from bregma, mediolateral =\u00a0\u00b1\u00a01.8 mm, dorsoventral\u00a0=\u00a02.0 mm. After the injection, the cannula was maintained for an additional 5 min for a complete absorption of the virus. Behavior tests were performed 20 and 30 days after the injection. For western blot analysis, brains were removed 25 days after viral injection, hippocampal region was dissected and its protein samples were prepared as described above. Intracerebroventricular injection of PBS or A\u03b21-42  was performed as described previously  and xylazine (10 mg/kg). Lentivirus expressing shRNA against mouse eviously . In the Behavior tests for double transgenic or injected mice were performed as described previously . All app1-42 oligomers were prepared from lyophilized monomers . The hydroxyfluroisopropanol (HFIP)-treated A\u03b21-42 peptide was dissolved in dimethylsulfoxide (DMSO) and then diluted in phosphate-buffered saline (PBS). The stock solution was incubated at 4\u00b0C for 24 hr and stored at \u201380\u00b0C until use. Before use, the solution was centrifuged at 12,000 g for 10 min and the supernatant was used as an oligomeric A\u03b2 (ADDLs). The oligomeric status of A\u03b21-42 was evaluated by western blot analysis and atomic force microscopy  kit  following the manufacturer\u2019s instructions. Briefly, the mice were anesthetized and the brains were microdissected. The hippocampus was carefully isolated and homogenized in 10 volumes of ice-cold guanidine buffer  and then mixed for 3 hr at room temperature. The brain homogenates were further diluted 1:10 with cold reaction buffer  and then centrifuged at 16,000 g for 20 min at 4\u00b0C. The diluents were mixed 1:1 with standard dilution buffer in the assay kit.A\u03b2 levels in the 3xTg-AD and 3xTg-AD/2. Primary neurons were transfected using Lipofectamine 2000 reagent (Invitrogen), whereas other cells were transfected using Polyfect reagent  according to the manufacturer\u2019s instructions. If required, cells were treated with SB-415286, roscovitine (Sigma-Aldrich) or AS1949490  as indicated.Primary cortical and hippocampal neurons were cultured from embryonic day 17 (E17) mice. The neurons were plated on poly-L-lysine -coated glass coverslips and maintained in neurobasal medium containing 2% B-27 supplement (Invitrogen) and 0.5 mM L-glutamine (Invitrogen). Half of the medium was exchanged every 3 days. Primary neurons were not authenticated, and were not tested for mycoplasma. SH-SY5Y, HEK293T and CHO cells , which were authenticated by ATCC and were negative for mycoplasma, were cultured in DMEM  supplemented with 10% fetal bovine serum (FBS) (HyClone), penicillin and streptomycin (Invitrogen). Cells were grown at 37\u00b0C under an atmosphere of 5% COFCGR2B and human INPPL1 shRNAs were synthesized, annealed and cloned into the pSUPER-neo vector. Human tau (0N4R) cDNAs were subcloned into pcDNA3-HA and pEGFP-C1 vector as described previously (All primers used in this study are listed in eviously . His-tagFCGR2B shRNAs or pINPPL1 shRNAs for 36 hr and then cultivated in the selection medium containing 1 mg/ml G418 (Invitrogen) for at least two weeks. A single cell was further cultivated to form stable cell colony and the expression of each cell lines was analyzed by western blotting and reverse transcriptase (RT) PCR.SH-SY5Y cell were transfected with pcDNA3-HA, pFCGR2B-HA, pSuper-neo, p3VO4, 1 mM NaF, 1 \u03bcg/ml each of aprotinin, leupeptin and pepstatin A). The lysates were centrifuged at 14,000 g for 10 min at 4\u00b0C and the supernatant was separated by SDS-PAGE and blotted onto PVDF membrane. The blots were blocked for 1 hr at room temperature and incubated with following antibodies: anti-phospho-Fc\u03b3RIIb, anti-Fc\u03b3RIIb , anti-A\u03b2 , anti-phospho-tau , anti-NSE , anti-phospho-GSK3\u03b2, anti-p35/25 , anti-GSK3\u03b2 , anti-SHIP2, anti-mFc\u03b3RIIb, anti-GFP, anti-His , anti-\u03b1-tubulin and anti-\u03b2-actin (Sigma-Aldrich), Nu-1 , PHF1, CP13 and TG5 , and 12E8 . Membranes were rinsed three times with TBS-T , further incubated for 1 hr with peroxidase-conjugated secondary antibodies and visualized using ECL detection system.Cells were lyzed in lysis buffer  (Sigma-Aldrich) for 10 min, rinsed three times with PBS and then permeabilized with 0.1% Triton X-100 in PBS. After blocking with 5% BSA in PBS, neurons were incubated overnight at 4\u00b0C with the following antibodies: AT180 (1:200), AT8 (1:200), anti-NSE (1:200), anti-Fc\u03b3RIIb (1:500) and anti-SHIP2 (1:250). After rinsing three times with PBS, cells were incubated with FITC- or TRITC-conjugated secondary antibodies (Jackson Laboratory Inc.) at room temperature for 1 hr. The coverslips were placed with mounting solution (Sigma-Aldrich) and observed on a confocal laser scanning microscope .1-42-treated SH-SY5Y cell extracts were incubated with anti-Fc\u03b3RIIb or anti-SHIP2 antibodies in IP buffer  for 12 hr at 4\u00b0C, and then pulled-down by protein G-Sepharose beads . For co-immunoprecipitation assay, HEK293T cells which transiently overexpressed His-tagged SHIP2 with either GFP-tagged Fc\u03b3RIIb or Fc\u03b3RIIb Y273F mutant were lyzed and incubated with anti-GFP or anti-His antibodies for 12 hr at 4\u00b0C, and then pulled-down by protein G-Sepharose beads. After a short centrifugation, the beads were washed three times with IP buffer and subjected to western blotting.For endogenous immunoprecipitation (IP) assay, A\u03b21-42 were harvested with the buffer  and then mechanically disrupted using a 26-gauge needle with passing it 20 times. Cell lysates were centrifuged at 1000 g for 10 min at 4\u00b0C to remove the nuclei or unbroken cells. The supernatant was collected into new tube and again centrifuged at 100,000 g for 1 hr at 4\u00b0C with Beckman SW41 rotor. The pellet was resuspended in lysis buffer and used as a crude membrane fraction, whereas the supernatant used as a cytosolic fraction. The separated fractions were confirmed with western blot analysis using anti-Fc\u03b3RIIb and anti-\u03b1-tubulin antibodies for the membrane and cytosolic markers, respectively.SH-SY5Y cells treated with A\u03b2The lipid extraction from primary cortical neurons or SH-SY5Y cells was performed as described . After sE.coli BL21 cells were transformed with the plasmids and cultured to reach an OD600 of 0.6, before induction with 1 mM IPTG. After incubation for 18 hr at 16\u00b0C, cells were harvested and lyzed by sonication. His-fused TAPP1-PH and GRP1-PH were purified from cell lysates using Ni-NTA chelating agarose CL-6B . PLO assays were performed as described previously with minor modification P2 and PtdInsP3 diC16  were reconstituted in a 1:2:0.8 solution of chloroform, methanol and water, and used as positive controls. The lipid extracts from the cells were serially diluted and spotted on PVDF membranes which were pre-wetted in methanol, washed in TBS-T buffer and then air-dried. The membranes were dried completely and blocked with 3% BSA in TBS-T buffer for 1 hr. The blots were incubated overnight at 4\u00b0C with gentle rocking in the fresh blocking buffer containing purified 10 \u03bcM His-TAPP1-PH or His-GRP1-PH. The membranes were washed 5 times over 50 min in TBS-T buffer and incubated with anti-His antibody for 1 hr at room temperature. The membranes were further incubated for 1 hr with peroxidase-conjugated secondary antibodies and bound proteins were visualized using ECL detection system. PtdInsP2, PtdInsP2 or PtdInsP3 levels were quantified by ELISA kit (Echelon) following the manufacturer\u2019s instructions.For the purification of recombinant proteins, the PH domain of TAPP1 and GRP1 was inserted into pET-28a vector. fication . LyophilThe synthetic phosphoinositides diC16 were incubated with histone carriers (Echelon) with a 0.5\u20133:1 molar ratio for 15 min with a vigorous vortexing. The histone-phosphoinositides complex was diluted 1:10 with neurobasal media and added to primary cortical neurons. The phosphoinositides without carriers and the only carriers without phosphoinositides were used as negative controls.t-test. Differences among multiple means were assessed by one-way ANOVA, followed by Tukey\u2019s post-hoc test. Error bars represent s.d. or s.e.m. as indicated.Statistical analyses were performed with GraphPad Prism software. Differences between two means were assessed by paired or unpaired  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.Thank you for submitting your article \"Fc\u03b3RIIb-SHIP2 axis links A\u03b2 to tau pathology by disrupting phosphoinositide metabolism in Alzheimer's disease model\" for consideration by The work describes how Fc\u03b3RIIb deficiency protects neurons against A\u03b2-induced toxicity. Especially the identification of SHIP2 linking A\u03b2 to Tau pathology is novel and potentially of high interest. Overall the referees were in support of publication, but before reaching consensus a few critical questions should be addressed. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Essential revisions:eLife is not misled in the sense that this paper resolves the controversies in the field. Therefore, critical literature should be included in the Introduction and Discussion, and some balancing of the conclusions in the current manuscript versus other work should be provided. Why do the authors think that their receptor is the major driver of pathology in AD and why are other receptors not considered? Does this need any qualifiers?1) Overall the paper should take more into account other publications in both Introduction and Discussion sections. There are in the main time at least ten other receptors for A\u03b2 oligomers described. Also some critical perspective on what is meant with A\u03b2 oligomers is indicated. For instance, the 7PA2 cell line also produces 'zeta' peptides and the relation between these in vitro generated mixtures of A\u03b2 peptides and the in vivo situation is far from clear. It is important that the broader non-specialized reader of 2) There are two major gaps in the proposed cascade and this should be mentioned at least in the Discussion to clarify this for the reader. First there are no mechanistic data in this work to link A\u03b2 to Fc\u03b3RIIb phosphorylation. The authors mention lyn kinase briefly  but don't show any data. The second gap is a mechanistic link between increased PtdIns and tau pathology. GSK-3-\u03b2 kinase that catalyzes tau hyperphosphorylation, uses ATP as a donor of phosphate groups, so phosphoinositides are not a substrate. Therefore a possible link could be PtdIns-mediated regulation of GSK-3\u03b2.3) Two referees have concerns with the idea that the action of Fc\u03b3RIIb is cell autonomous. First there are issues with the expression of Fc\u03b3RIIb in neurons: Figure.1\u2014\u2014figure supplement 1 A shows in fact that a large part of Fc\u03b3RIIb mRNA is in glia and not in neurons. Additional evidence to proof that Fc\u03b3RIIb is expressed in neurons is needed, it could be that the weak signal in the experiments in neurons is background. The authors could consider in situ hybridisation to check their claim. In addition, Fc\u03b3RIIb is highly expressed in microglial cells and may play a role in microglial activation. Therefore, the impact of Fc\u03b3RIIb KO on tau pathology in 3X TG mice can be also mediated by reduced microglial activation (cell autonomous effects vs non-autonomous effects). Did the authors check microglial activation and cytokine levels in these mice (WT 3XTG vs. KO 3Xtg)? Moreover, have the authors looked at whether Fc\u03b3RIIb plays a role in sensing A\u03b2 in microglial cells?4) The authors should provide in the next version the pictures of 5) In the last paragraph of the subsection \u201cFc\u03b3RIIb is essential for tau hyperphosphorylation and memory deficit in 3xTg-AD mice\u201d, it is said that 9months old 3xTgAD mouse are \"plaque-free\". What happens in the cortex of these 3xTg mice where A\u03b2 plaques should be present, is tau phosphorylation more pronounced there? And is that protected by Fc\u03b3RIIb deficiency? Does the age of mice affect tau hyperphoshorylation, i.e. is tau pathology still absent in 3xTg Fc\u03b3RIIb KO mouse even at a very advanced age when there are loads of A\u03b2?6) In Figure.3\u2014figure supplement.2A the authors analyze the effects of the receptor mutations on A\u03b2-induced cell death. \"Cell death was determined after 24h under fluorescence microscope\". What method/dye/criterion was used? How many spots were analyzed per sample per condition to produce statistics on panel B? [\u2026] Essential revisions:1) Overall the paper should take more into account other publications in both Introduction and Discussion sections. There are in the main time at least ten other receptors for A\u03b2 oligomers described. Also some critical perspective on what is meant with A\u03b2 oligomers is indicated. For instance, the 7PA2 cell line also produces 'zeta' peptides and the relation between these in vitro generated mixtures of A\u03b2 peptides and the in vivo situation is far from clear. It is important that the broader non specialized reader of eLife is not misled in the sense that this paper resolves the controversies in the field. Therefore, critical literature should be included in the Introduction and Discussion, and some balancing of the conclusions in the current manuscript versus other work should be provided. Why do the authors think that their receptor is the major driver of pathology in AD and why are other receptors not considered? Does this need any qualifiers?Until now, A\u03b2 was reported to bind to many receptors, including alpha7 nicotinic acetylcholine receptors (\u03b17 nAChR), NMDA receptor, receptors for advanced glycation end-products (RAGE), A\u03b2-binding alcohol dehydrogenase (ABAD), the Ephrin-type B2 receptor (EphB2), cellular prion protein (PrPc) and paired immunoglobulin-like receptor B (PirB) . Although these receptors were shown to be responsible for A\u03b2 neurotoxicity, especially memory impairment in AD mice, their role as neuronal receptors in A\u03b2-induced tau pathologies was limitedly shown in \u03b17 nAChR and NMDA receptor . Of particular note, while \u03b17 nAChR was reported to mediate A\u03b2-induced tau phosphorylation, the finding was based on in vitroand ex vivosystem . It thus remains to be confirmed in detail using animal models of AD. Furthermore, evidence showing a correlation of the proposed molecular mechanism with pathologic evidence was not much provided. In particular, the CAMKK2-AMPK at down-stream of NMDA receptor was recently proposed to mediate the synaptotoxic effects of A\u03b2 oligomers through tau phosphorylation and this event is very likely caused by NMDA receptor-induced increase of intracellular calcium, not by direct interaction of NMDA receptor with A\u03b2 . Therefore, a neuronal receptor that is important in A\u03b2- induced tau pathology has not been identified and needs to be elucidated for controlling overall AD pathology. We added this information in \u2018Introduction\u2019 (fourth paragraph).Then, how is Fc\u03b3RIIb different from other A\u03b2 receptors? It is reasonable to propose that different mechanisms or even the same mechanism exerts multiple effects at different stages of disease progression . For instance, RAGE is now believed to mainly function to transport A\u03b2 in the blood brain barrier  and ABAD acts for mitochondrial toxicity as an intracellular binding partner of A\u03b2 . In case of PrPc, it's debatable whether it is involved in A\u03b2-induced memory impairments and thus needs to be further characterized . Further, compared to those receptors, our observations that the phosphorylation of Fc\u03b3RIIb at tyrosine 273 is found in the brain of AD patients and is required for both oligomeric A\u03b2 neurotoxicity and tau hyperphosphorylation can make it distinct from other A\u03b2- binding receptors. In the case of PirB that shares structural similarity with Fc\u03b3RIIb and also acts as an A\u03b2 receptor for synaptic plasticity, the phosphorylation of PirB is not associated with A\u03b2 signaling . Thus, we believe that Fc\u03b3RIIb facilitates tau phosphorylation and neuronal loss in AD brains, consistent with the proposed role of tau in AD pathogenesis, such as severe memory impairment and neuronal loss . Following the reviewer\u2019s suggestion, we added the specificity of Fc\u03b3RIIb in \u2018Discussion\u2019 (fourth paragraph).n oligomers, and the proposals on which species of A\u03b2 oligomers are responsible for the pathogenesis are a little in debate . We have here used 3 different sources of A\u03b2 oligomers; synthetic A\u03b2 oligomers, naturally secreted A\u03b2 oligomers (7PA2 cells), and A\u03b2 of 3xTg-AD model mice. Although synthetic A\u03b2 oligomers are well-characterized and have been used widely for neurotoxicity, the acting concentration of synthetic A\u03b2 (\u03bcM range) is relatively higher than that in AD brains. Compared to synthetic A\u03b2 oligomers, conditioned medium from 7PA2 cells mainly contains not only A\u03b2 dimers and trimers but also the different pools of A\u03b2 oligomers, including zeta peptide, and show more potent neurotoxic properties (nM range) . Moreover, the oligomers generated in 3xTg-AD mouse brain may be more complicated and needs to be identified. Nonetheless, we propose here that Fc\u03b3RIIb plays a crucial role in tau phosphorylation and neurotoxicity in vitroand in vivoin response to these species of A\u03b2, probably a certain common species among the different sources of A\u03b2 oligomers. We added above information in \u2018Discussion\u2019 (second paragraph).In general, A\u03b2 oligomers play a key role in AD pathogenesis, while soluble A\u03b2 oligomers are still heterogeneous, including low or high 2) There are two major gaps in the proposed cascade and this should be mentioned at least in the Discussion to clarify this for the reader. First there are no mechanistic data in this work to link A\u03b2 to Fc\u03b3RIIb phosphorylation. The authors mention lyn kinase briefly  but don't show any data. The second gap is a mechanistic link between increased PtdIns and tau pathology. GSK-3-\u03b2 kinase that catalyzes tau hyperphosphorylation, uses ATP as a donor of phosphate groups, so phosphoinositides are not a substrate. Therefore a possible link could be PtdIns-mediated regulation of GSK-3\u03b2.1-42 treatment in primary cortical neurons and SH-SY5Y neuronal cells . Thus, we initially hypothesized that and tested whether Fc\u03b3RIIb was phosphorylated by Lyn after treatment with A\u03b2. We found that Lyn was activated (phosphorylation at Tyr 397) by A\u03b2al cells . In addial cells . To furtal cells  and A\u03b2-ial cells . Moreoveal cells . Taken tThe second gap; the previous study showed that GSK3\u03b2, a well-known tau kinase, is stimulated by ER stress . We previously demonstrated that ER stress is elicited by the interaction between A\u03b21-42 and Fc\u03b3RIIb, and is responsible for A\u03b2 neurotoxicity . Thus, we decided to examine the gap among SHIP2, ER stress, and GSK3\u03b2. To determine the relationship between PtdInsP2 (a SHIP2 product) and ER stress, we first analyzed the alteration of GRP78, a typical marker of unfolded protein response (UPR), following the delivery of various phosphoinositides into primary cortical neurons. Among the phosphoinositides, we found that PtdInsP2 only increased the level of GRP78. We also found that PtdInsP2 abolished the inhibitory phosphorylation of GSK3\u03b2 at Ser9 (GSK3\u03b2 activation) and increased tau hyperphosphorylation  Two referees have concerns with the idea that the action of Fc\u03b3RIIb is cell autonomous. First there are issues with the expression of Fc\u03b3RIIb in neurons: To evaluate neuronal expression of Fc\u03b3RIIb, we have really paid great attention and by performing many experiments.First, as already shown in our previous manuscript , Fc\u03b3RIIb mRNA (RT-PCR) and proteins  were found in the neurons of mouse and AD patient brains . MoreoveFcgr2b2 mRNA that is selectively increased by oligomeric A\u03b21-42 treatment in primary cortical neurons   mice of Fc\u03b3RIIb is the best. However, as you known well, generation of neuron-specific knockout (nsKO) mice of Fc\u03b3RIIb and of Fc\u03b3RIIb nsKO/3x AD double transgenic mice will take too long time (3 more years). Thus, as an alternative approach, we generated a dominant negative mutant of Fc\u03b3RIIb  lacking its cytoplasmic region (a.a. 232-300) and thus unable to transmit the signal into cells, and confirmed its protective activity in A\u03b2-induced neuronal cell death after its transient expression in neuronal cells (data not shown). We then generated a transgenic mouse expressing Fc\u03b3RIIb- \u0394Cyto-HA under ., 1996) . We conf., 1996) . To addr., 1996) . The beh., 1996) . These rhttp://www.brain-map.org/). Although fcgR2b expression is quite low, fcgr2b-positive ISH signals were detected in the hippocampus of P56 male mouse  results provided from Allen Brain Atlas  Cahoy et al. [J. Neurosci. (2008)] created a transcriptome database of the expression levels of >20,000 genes by gene profiling from astrocytes, neurons and oligodendrocytes. Based on their list, Fc\u03b3RIIb is significantly expressed in postnatal day 16 (P16) neurons and P17 forebrain. (2) Suemitsu et al. [Neuroscience (2010)] detected Fc\u03b3RIIb protein in parvalbumin neurons, whereas Fc\u03b3RIII and Fc\u03b3RI proteins were detected in microglial cells.In summary, we confirmed that, though neuronal expression is very low compared to non-neuronal cells, Fc\u03b3RIIb is evidently expressed in neuronal cells as well. Again, please note that its expression is increased at least several folds in primary neurons by A\u03b2 treatment and in AD brains compared to age-matched non- AD brains. Moreover, presence and distinct function of a neuron-enriched Fc\u03b3RIIb2 provides more compelling evidence for the pathogenic role of Fc\u03b3RIIb in AD.Fcgr2b KO in the cortex .While we have mainly focused on the role of neuronal Fc\u03b3RIIb in tau pathology of AD model, we were also attempted to examine the role of Fc\u03b3RIIb in the microglial activation in 3x Tg-AD mice. From immunohistochemical analysis, we identified that, compared to that of age-matched control mice, expression of Iba1, a microglia marker, was significantly reduced by e cortex  and slige cortex  of 3x Tg-AD mice . Though 4) The authors should provide in the next version the pictures of We are sorry for missing the pictures. We added the figures and separated those into 5) In the last paragraph of the subsection \u201cFcJRIIb is essential for tau hyperphosphorylation and memory deficit in 3xTg-AD mice\u201d, it is said that 9months old 3xTgAD mouse are \"plaque-free\". What happens in the cortex of these 3xTg mice where A\u03b2 plaques should be present, is tau phosphorylation more pronounced there? And is that protected by Fc\u03b3RIIb deficiency? Does the age of mice affect tau hyperphoshorylation, i.e. is tau pathology still absent in 3xTg Fc\u03b3RIIb KO mouse even at a very advanced age when there are loads of A\u03b2?Fcgr2b KO in the same age of 3x Tg-AD mice . Since we mostly analyzed tau phosphorylation at the age of 8-9 months, we performed additional experiments to identify the effect of Fc\u03b3RIIb KO in more aged mice following the reviewer\u2019s suggestion. While transgenic expression of human tau (detected by HT7 antibody) in 3x Tg-AD mice was not altered during aging, tau phosphorylation in 20 month-old 3x Tg-AD mice was heavily detected and occurred more than that of 6 month-old 3x Tg-AD mice . Compare-AD mice . These r-AD mice  (subsect6) In Apoptotic cell death was determined by morphology-based death assay as described in the previous literatures . After transfection with the DNAs, GFP-positive cells showing condensed and fragmented nuclei after staining with Hoechst 33258 were counted under a fluorescence microscope. We counted at least 900 cells in 3~4 random spots per sample per condition. We added this information into the figure legend of the revised manuscript  legend."}
+{"text": "Optimizing economic welfare in environmental governance has been criticized for delivering short-term gains at the expense of long-term environmental degradation. Different from economic optimization, the concepts of sustainability and the more recent safe operating space have been used to derive policies in environmental governance. However, a formal comparison between these three policy paradigms is still missing, leaving policy makers uncertain which paradigm to apply. Here, we develop a better understanding of their interrelationships, using a stylized model of human-environment tipping elements. We find that no paradigm guarantees fulfilling requirements imposed by another paradigm and derive simple heuristics for the conditions under which these trade-offs occur. We show that the absence of such a master paradigm is of special relevance for governing real-world tipping systems such as climate, fisheries, and farming, which may reside in a parameter regime where economic optimization is neither sustainable nor safe. Economic optimization in environmental governance was criticized for delivering short-term gains at the expense of long-term environmental degradation. Here, the authors use a stylized model of human-environment tipping elements to show no paradigm guarantees fulfilling another paradigm. Yet, it remains challenging to translate these aims into concrete policy implementations, accounting for non-linearities, such as tipping elements3, regime shifts5, and multi-stabilities6, as well as multiple kinds of uncertainties9, and extreme events10.The Sustainable Development Goals11 and how these findings relate to the three real-world systems of climate, fisheries and farming.To support the decision making processes in these contexts, we ask the question how the three prominent decision making paradigms of economic welfare optimization, sustainability and safe operating space compare with each other. Specifically, we investigate the parameter regimes for synergies and trade-offs when applying these paradigms to the management of tipping elements13. Most often, the present value of macroeconomic social welfare, i.e., the sum of discounted future benefits minus costs, is the target to be optimized. Such optimization approaches have been criticized regarding the discount rates used, delivering short term gains at the expense of long-term environmental degradation15. Further criticism targets the lack of a systems perspective required to understand the structural landscape of model dynamics, as well as the assumptions made due to imperfect information10. This critique is partly dealt with in optimization variants, such as robust16 or viable19 control, which are dealing with multiple types of uncertainty20. Naturally, other or multiple objectives21 and criteria23 with possible constraints24 can be optimized as well. In this work, we use the term solely in the narrow economic sense of maximizing the present value as defined in Eq.\u00a0Optimization approaches have emerged as the primary guiding principle to derive a policy strategy for environmental governance25 the policy paradigm of sustainability has emerged in the scientific and political discourse27. The economics of sustainability has brought up many definitions of sustainability alone31. In these analyses sustainability is usually imposed as a constraint within an economic welfare optimization paradigm. Trade-offs to economic welfare optimization are well known32. However, these classic social welfare optimization approaches are challenged through the increasing recognition of non-linearities, such as tipping points, regime shifts, uncertainties and the risk of catastrophic outcomes9. Taking up these challenges, e.g., non-convexities33 and climate tipping elements35 have been studied within an economic framework. Here, we derive our formal definition of sustainability from the Brundtland report26. Its design is deliberately simple and targeted to the mathematical framework we use (see below). We do not intend our definition to be applicable to a general model of a welfare economy27.In recognition of increasing environmental and social threats36 or safe operating spaces38 as a policy paradigm to derive concrete actions from39. These concepts originate from resilience thinking40 and a precautionary principle41 to deal with potential dangerous tipping elements in the environmental governance system. Trade-offs but also synergies with optimization thinking have been therefore discussed42. Also formal analyses studying relations between resilience as a system property and sustainability were conducted44.Recent advances in sustainability science have brought forth tolerable windowsHowever, the reciprocal relationships between these three paradigms of economic optimization, sustainability and safe operating space is still insufficiently explored. Such an understanding is important in order to judge, for example, when economic optimization is, or is not, an appropriate policy goal. Also, guidance is required when a sustainability paradigm may conflict with a safe operating space paradigm and vice versa.Here, we report progress towards a better understanding of the mutual relationships between these three paradigms of economic optimization, sustainability and safe operating space by applying them to a stylized model of a human-environment tipping element. We do so because of the increasing importance of tipping points and regime shifts in environmental governance. Our model is deliberately stylized, thereby applicable across multiple cases and scales, to gain a deeper understanding more complex models might miss. The formal definitions of the three paradigms are designed to fit our mathematical framework (see below). Since we do not focus on intragenerational justice in this article, one agent suffices as a decision making subject, in contrast to a multiagent setting. We find that there exists no master paradigm between the three examined, i.e., a policy can be any combination of optimal or not, sustainable or not and safe or not. This is of special relevance to the climate system which may reside at the edge in the parameter regime where economic welfare optimization becomes neither sustainable nor safe. This suggests the use of more advanced paradigms to support decision making in climate policy.46, in which an agent makes decisions about how to interact with its environment , but risks triggering a collapse of the environment to the degraded system state with non-zero probability \u03b4 and no immediate reward at all. From there, only the low pressure action opens the option to recover to the prosperous state with non-zero probability \u03c1.We use the mathematical framework of Markov Decision Processesent Fig.\u00a0. Our pard r Fig.\u00a0. At the For example, the high pressure action could correspond to emitting a business-as-usual amount of carbon to the atmosphere yielding a reward of high, short-term economic output as long as the system has not tipped. The low pressure action resembles emitting a reduced amount of carbon, assuming a lower short-term economic output for the guarantee to not trigger climate tipping elements into a disastrous state.\u03c0 is a function that specifies what action a to apply at a system state s. The agent receives reward rt at time step t. The value v\u03c0(s) of a state s under a given policy \u03c0 is given by the expected value of the normalized accumulated discounted rewards r with discount factor 0\u2009\u2264\u2009\u03b3\u2009\u2264\u20091 when starting in state S0\u2009=\u2009s and following policy \u03c0:A policy \u03b3\u2009=\u20091 corresponds to no discounting , whereas \u03b3\u2009=\u20090 corresponds to completely myopic, fully discounting agents.Note that the discount factor actually denotes the farsightedness of the agent. Thus, \u03c0r(p)\u2009=\u2009h, \u03c0r(d)\u2009=\u2009l), applying the high pressure action at the prosperous state and the low pressure one at the degraded state and the cautious policy (\u03c0c(p)\u2009=\u2009l, \u03c0c(d)\u2009=\u2009l), applying the low pressure action at the prosperous, as well as the degraded state.We classify policies according to whether they are economic welfare optimal or not, sustainable or not, and safe or not. For the sake of simplicity we focus on two deterministic policies, distinguishing whether the agent should apply the low or the high pressure action at the prosperous state Fig.\u00a0: the ris\u03c0 is defined as optimal (in the economic welfare sense) if its value v\u03c0(s) (Eq.\u00a046.A policy \u03c0(s) Eq.\u00a0 for ever26 a sustainable policy should fulfill two requirements: First, meet the needs of the present. We translate this formally into the agent evaluating the present state s as acceptable , if its value  has much similarity with the notion of weak sustainability48.Note that this reduction of sustainability to the one-dimensional value 37 is typically defined as a subset of the whole state space 49. In practice, the position of these potential tipping thresholds is always uncertain and the boundaries are placed at the lower end of the uncertainty zone. In that way the definition of the safe operating space states constitutes a normative judgment about the risk the decision maker is willing to tolerate. In the subsequent analyses we take the extreme position of no risk tolerance and identify the SOS with only the (more favorable) prosperous state, independent of the collapse probability \u03b4.The Safe Operating Space (SOS)\u03c0 as safe if every state the agents eventually visits under policy \u03c0 lies within the SOS.We define a policy In contrast to acceptable and unacceptable states, safe states are independent of the policy used.\u03b4, \u03c1, \u03b3, rl/rh, rmin/rh: the probability of a collapse from the prosperous to the degraded state under the high pressure action \u03b4, the probability of recovery from the degraded to the prosperous state under the low pressure action \u03c1, the agent\u2019s discount factor \u03b3, the high reward receivable from the high pressure action when staying at the prosperous state rh, the low reward receivable by taking the low pressure action at the prosperous state rl, and the normatively chosen minimum acceptable reward rmin a state value must have to be perceived as acceptable under a certain policy. Since all three rewards come in arbitrary units, the policy classification only depends on their ratios.In summary, our stylized model of a human-environment tipping element depends on the five parameters \u03b4, \u03c1, \u03b3, rl/rh, rmin/rh) .Further, we find a decreasing critical collapse probability with increasing farsightedness rl\u2009\u2265\u2009rmin, then the cautious policy is sustainable for all parameter combinations \u03b4, \u03c1, \u03b3, rl/rh . This is because in this parameter region the risky policy is acceptable also at the degraded state (Methods).Provided the low pressure reward exceeds the normative minimum acceptable value threshold, \u03b4, \u03c1, \u03b3, rl/rh, rmin/rh . This yields a classification of policies into eight different categories Fig.\u00a0.Fig. 3Parmin is too large. The cautious policy does not return enough value to be sustainable  and the risky policy at the degraded state produces too little future reward to be sustainable, due to the low chance of recovery and lack of farsightedness.In particular, optimal policies are not necessarily sustainable  which additionally accounts for social indicators51, such as the number of people living in extreme poverty. Thus, SAJOS policies can be interpreted as the overlap of safe with sustainable policies. Within our model, we can give a definite criterion for when this form of SAJOS exists: as long as the reward from a low pressure action rl exceeds the normative threshold value rmin (rl\u2009>\u2009rmin), the cautious policy is both safe and sustainable . Additionally, we added a parameter sensitivity analysis by visualizing the likelihood of ending up in a certain parameter regime by color gradients between regimes , the climate system is likely to reside in a parameter sweet spot (gray area), where applying an optimization, sustainability or SOS paradigm results in the cautious policy as the advisable way of governing the climate system. However, if the collapse probability per time step is smaller  the situation is different. Here, an SOS and a sustainable paradigm would still yield the cautious policy . At the collapsed and degraded state the conditions for the fishers are not acceptable. Therefore they have to leave the system and cannot wait for the fish\u2019s recovery. But further investigation is needed to reduce the uncertainty with respect to the other parameters.For fishery systems, both transition probabilities certainly depend on a variety of factors, e.g., fisher\u2019s technical and cultural traits or the dominant fish species in the system, as well as external factors such as climate change influencing habitat condition63. Nevertheless, land degradation by farming has been identified as a tipping element by Kinzig and others64, where the authors discuss the case of the western Australian wheatbelt with a typical collapse timescale of about 100 years. Soil recovery is estimated to take place within 20 to 1000 years65, which is roughly consistent to Kitzing et al., where the duration to reach equilibrium again is estimated with up to 300 years. We therefore assume a typical recovery timescale of about 300 years. In contrast to climate and fisheries, the transition probabilities we associated with the process of land degradation by farming suggest, that here an optimality paradigm is very likely to yield the risky policy which is neither sustainable nor safe despite considerate parameter uncertainty \u2009=\u2009h, \u03c0r(d)\u2009=\u2009l, (2) \u03c0c(p)\u2009=\u2009l, \u03c0c(d)\u2009=\u2009l, (3) \u03c03(p)\u2009=\u2009h, \u03c03(d)\u2009=\u2009h, (4) \u03c04(p)\u2009=\u2009l, \u03c04(d)\u2009=\u2009h. We concentrate on deterministic policies only to simplify the calculation without loss of generality, because if an optimal policy exits there exits also a deterministic optimal policy46. We further focus here only on the first two policies, named the risky and the cautious policy, since the remaining two apply a high pressure action at the degraded state. This will trap the agent at this position for eternity without receiving any reward. The math on these policies is left to the interested reader.There are four deterministic policies in our Markov decision process model: (1) \u03b4, \u03c1, \u03b3, rl, rh). From Eq.\u00a0\u03b3\u2009<\u20091 one can derive the recursive relationship between state values, known as the Bellman Equation69:p) being the probability to enter state s\u2032 given the agent has started in state s and applied action \u03c0(s).In the following we derive the analytical expressions of the state values of these policies as functions of the parameters \u2009=\u2009h, \u03c0r(d)\u2009=\u2009l) we solve the system of equationsTo obtain the explicit state values for the risky policy (\u03c0c(p)\u2009=\u2009l, \u03c0c(d)\u2009=\u2009l) we solve the system of equationsTo obtain the explicit state values for the cautious policy  by multiplying the stationary state of the effective Markov chain with the reward vector For P\u03c0 of the effective Markov chain readThe components of the transition matrix \u03c3\u03c0 is the normalized eigenvector of the transition matrix with eigenvalue one. Hence,The stationary state \u03b3\u2009=\u20091 the value v\u03c0 can be obtained by simply inserting \u03b3\u2009=\u20091 into Eqs.\u00a0Performing this calculation for risky and cautious policy explicitly yields consistent results with the calculation for 0\u2009\u2264\u2009\u03b3\u2009<\u20091 from above. For To derive the analytical expression of the hypersurface in parameter space that separates the regions where either the risky or the cautious policy is optimal we set \u03c0 we apply the definition from Eq.\u00a0To obtain the hypersurface that separates state s being acceptable from being not acceptable under policy For the risky policy at the degraded state we set For the cautious policy at the prosperous state we set For the cautious policy at the degraded state we set To get from acceptability to sustainability for the risky policy one has to logically combine Eqs.\u00a0The division of the parameter space according the safe operating space paradigm is obvious from its definition. Only the cautious policy is a safe policy since it will eventually end up and remain in the prosperous, safe operating space state. The risky policy switches recurrently between the prosperous and the degraded which makes it, by definition, not safe.p be the probability per time step that a system state will transition into another state. The average number of time steps the system will be in that state is given by \u2329N\u232a\u2009=\u2009(1\u2009\u2212\u2009p)/p. Inverting yields p\u2009=\u20091/(\u2329N\u232a\u2009+\u20091). We map a model time step to a year. Thus, a collapse time scale of e.g., 50 years corresponds to a collapse probability of \u03b4\u2009\u2248\u20090.02. Supplementary Tab.\u00a0Let by \u2329N\u232a\u2009=\u2009\u2009\u2212\u2009p/p. Ihttps://github.com/wbarfuss/Paradigms.Python code for the reproduction of the reported results plus interactive versions of the figures can be downloaded from Data sharing not applicable to this article as no datasets were stored on disk during the production of the figures (see Code availability).Supplementary InformationPeer review file"}
+{"text": "We have read with great interest the article by P. Faverio et al. published in this journal . We thanWhile the authors have tested the methodology of NIV interruption well, the number of days at each stage of weaning, NIV during afternoon and night or nocturnal NIV only, was at physician discretion. This is an exciting aspect and confusing as well because, eventually, the criteria may be influenced by wide individual variability, which may make it impossible to extrapolate the results. Good numbers (39%) of the patients in the chronic domiciliary NIV group were not able to be weaned by the new method and continued to be in the domiciliary NIV settings. As there are no clear criteria for such patients, it will be worth knowing the settings, whether these patients were finally stabilized, and if yes, \u201chow.\u201d These aspects will help readers in better application or exclusion of the new interruption-based weaning in this subgroup of patients in the future. Pneumothorax due to severe bullous emphysema is considered by the authors as a risk factor for not weaning. While we agree that such patients are prone to not weaning, pneumothorax it is more related to the selected positive inspiratory and end-expiratory pressure levels selected . HoweverWe again thank the authors for another interesting observation of no recurrence of AHRF during hospitalization after weaning by the new method. This is interesting and raises speculation whether this can be considered as a protective aspect of the NIV. A prospective randomized study will give us more evidence on these aspects, especially which subgroup of patients will be benefited by this new technique."}
+{"text": "Cth\u2212/\u2212) pregnant mice display any features of preeclampsia. Cth\u2212/\u2212 females developed normally but showed mild hypertension (~10 mmHg systolic blood pressure elevation) in late pregnancy and mild proteinuria throughout development/pregnancy. Cth\u2212/\u2212 dams had normal numbers of pups and exhibited normal maternal behavior except slightly lower breastfeeding activity. However, half of them could not raise their pups owing to defective lactation; they could produce/store the first milk in their mammary glands but not often provide milk to their pups after the first ejection. The serum oxytocin levels and oxytocin receptor expression in the mammary glands were comparable between wild-type and Cth\u2212/\u2212 dams, but the contraction responses of mammary gland myoepithelial cells to oxytocin were significantly lower in Cth\u2212/\u2212 dams. The contraction responses to oxytocin were lower in uteruses isolated from Cth\u2212/\u2212 mice. Our results suggest that elevated homocysteine or other unknown factors in preeclampsia-like Cth\u2212/\u2212 dams interfere with oxytocin that regulates milk ejection reflex.Elevated plasma homocysteine levels are considered as a risk factor for cardiovascular diseases as well as preeclampsia\u2014a pregnancy disorder characterized by hypertension and proteinuria. We previously generated mice lacking cystathionine \u03b3-lyase (Cth) as cystathioninuria models and found them to be with cystathioninemia/homocysteinemia. We investigated whether Cth-deficient ( Elevated plasma levels of homocysteine, a sulfur-containing amino acid intermediate in methionine metabolism, are widely known as an independent risk factor for cardiovascular diseases (CVDs) such as myocardial infarction, stroke, and venous thromboembolism ,3,4,5,6.Cbs\u2212/\u2212 survivors  were found to be infertile (our unpublished observation), pregnancy complications could not be investigated. In our previous study, we generated mice lacking cystathionine \u03b3-lyase (Cth), which is another essential transsulfuration enzyme downstream of Cbs, as a model for cystathioninuria (MIM 219500)  219500) . Our hom 219500) N-acetylcysteine administration [2S) levels and increased the blood pressure/irregular branching of the fetal vasculature of the placenta, both of which were reversed by the application of GYY4137, a slow-releasing H2S donor [Cth\u2212/\u2212 dams. In this study, we report preeclampsia-like hypertension/proteinuria during late pregnancy as well as partial lactation (milk ejection) failure due to impaired oxytocin responses in Cth\u2212/\u2212 mice. Our results shed light on the novel, clinically relevant roles of Cth and homocysteine in the systemic circulation.Previous studies have also shown that the Cth expression was upregulated in the livers of lactating mouse  or rat  dams, anstration . Moreovestration  while Ct2S donor . TherefoCth+/\u2212 and homozygous Cth\u2212/\u2212 female mice grew in a similar manner as wild-type (WT: Cth+/+) females during juvenile development, gestation, and lactation periods  than those of the respective WT females (left). Systolic blood pressures during lactation periods and diastolic blood pressures in all periods  were indistinguishable between the Cth genotypes . Urinary protein levels  were significantly higher (p < 0.05) in virgin Cth\u2212/\u2212 mice than those in the respective WT mice, which lasted to G15.5 , the females C left. Sto G15.5 A. Urinar+/\u2212 mice A. Serum Cth+/\u2212 pups born to Cth\u2212/\u2212 dams  were significantly higher (p < 0.01) than that in Cth+/\u2212 pups born to WT dams   in nearly half of the litters of Cth\u2212/\u2212 dams while not all the Cth+/\u2212 littermates in any of the litters of WT dams died (data not shown). In the remaining half of the litters born to Cth\u2212/\u2212 dams, all the Cth+/\u2212 pups survived to adulthood even though they showed slightly retarded growth in early development  in \u2212 males) A. Comparf hunger B. Althoual hours F. In conCth\u2212/\u2212 dams was equivalent to that of the mammary glands in WT dams . Histological analyses localized Cth expression in the myoepithelial cells of WT mouse mammary glands, but this finding was absent or unclear in those of Cth\u2212/\u2212 mouse mammary glands , its levels were indistinguishable between WT and Cth\u2212/\u2212 dams , WT uteruses markedly responded to oxytocin (0.1 unit/mL) with increased frequency (\u00d74.50) and higher peak values  (Cth\u2212/\u2212 uteruses were much milder (\u00d71.95) in contraction frequency (Cth\u2212/\u2212 mice show homocysteinemia [Cth\u2212/\u2212 uterus. Although the application of high concentrations of methionine (2 mM) failed to affect both the contraction frequency and magnitude, homocystine (200 \u00b5M) showed a trend toward lower contraction magnitudes in both spontaneous and oxytocin-induced conditions  without altering the contraction frequency (Cth was expressed in the uterus A and oxyificant) A\u2013C. In crequency A\u2013C. As Cteinemia , we nextrequency A\u2013C.Cth\u2212/\u2212 female mice, their responses to vasopressin, another peptide hormone that resembles oxytocin (only two amino acid substitutions in a nine amino acid peptide) and that acts on the Oxtr-ortholog receptor, vasopressin receptor 2 (V2r) [Cth\u2212/\u2212 female mice .If the systemic oxytocin signaling is impaired in  2 (V2r) , could a 2 (V2r) ; howeverCth\u2212/\u2212 dams. To evaluate another oxytocin-mediated signaling in the CNS [Cth\u2212/\u2212 male mice. When a male mouse is exposed to an \u201cunfamiliar\u201d female in his home cage, he spends much of his time in the anogenital inspection of the novel individual during the brief social encounter. Upon the repetitive exposures to the \u201cfamiliar\u201d female, the time spent for investigation usually declines with a full recovery following the introduction of a new female [Cth\u2212/\u2212 male mice displayed declines in the investigation time upon the repetitive exposures to the \u201cfamiliar\u201d females , and the recoveries of the time upon new females in a similar fashion  that can be regulated by oxytocin within the central nervous system (CNS) as well as substantially impaired peripheral oxytocin-mediated responses, milk ejection and uterine contraction, in  the CNS , social w female ,37,38. WCth\u2212/\u2212 virgin adult female mice exhibited serum amino acid profiles that were different from those of the respective WT mice as we previously reported in 2-week-old female/male-mixed pups  Cth\u2212/\u2212 mice display (hyper)homocysteinemia/cystathioninemia [Cth\u2212/\u2212 mice require dietary cyst(e)ine as an essential amino acid (unlike WT mice) [Cth\u2212/\u2212 mice show reduced endogenous H2S levels [Cth\u2212/\u2212 mice display increased vulnerability to paraquat [Cth\u2212/\u2212 mice appeared normal under normal condition and were fertile [Cth is a transsulfuration enzyme essential for homocysteine clearance, cysteine biosynthesis, endogenous production of the gasotransmitter Honinemia ; (2) CthWT mice) ; (3) CthS levels ,40,41, aparaquat , dietaryparaquat , acetamiparaquat ,44, cardparaquat , unilateparaquat ,46, and paraquat . However fertile . Patient fertile . TherefoCth\u2212/\u2212 dams showed hypertension during pregnancy and mild proteinuria  during development/pregnancy  and in the initial nursing  [Cth\u2212/\u2212 and heterozygous Cbs+/\u2212  dams, as previously reported in Cbs+/\u2212 dams by Sonne et al. [Cth\u2212/\u2212 dams  . We did e et al. . In addi\u2212/\u2212 dams  that couResponses or behaviors displayed by the female that specifically support the development and growth of her pups constitute a set of responses termed as maternal behavior. These include (1) parturitional responses such as neonate stimulation, amniotic fluid consumption, and placentophagia; (2) offspring-directed responses such as retrieval, licking/grooming, nursing/crouching (warming), and nest building; and (3) offspring-related responses, including maternal aggression, increased food consumption, and reduced anxiety to enhance exploratory activities . Among tq-coupled Oxtr and thereby activates phospholipase C and intracellular calcium signaling, which triggers various downstream signaling pathways. Previous studies using Oxt\u2212/\u2212 and mice lacking Oxtr (Oxtr\u2212/\u2212) revealed that both Oxt\u2212/\u2212 and Oxtr\u2212/\u2212 females have normal fertility, pregnancy, and parturition, but they were unable to eject milk; therefore, all pups born to these mice die within 24 h of birth due to the deficiency of milk [Cth\u2212/\u2212 dams could survive  reduced uterine contraction magnitudes  although both levels were much higher than those in their respective WT mice  [Cth\u2212/\u2212 dams influenced on (high) tHcy in their whey  oxytocin signaling less active in ctively) . This coeir whey  but not eir whey , and mayIn conclusion, this study revealed preeclampsia-like features and partial lactation failure in cystathioninuria model mice. Cth-deficient cystathioninuria patients are currently buried because they are considered free of apparent clinical manifestations and, unlike Cbs-deficient homocysteinemia patients, they are not detected in the current newborn screening that detects hypermethioninemia . As lactCth+/\u2212 mice were generated and backcrossed for 10 generations to C57BL/6J inbred strain  [Cth+/\u2212 males and females were bred to obtain Cth\u2212/\u2212 mice. Mice were housed in an air-conditioned room , kept in a 12-h dark/light cycle, and allowed free access to a CE-2 standard dry rodent diet (CLEA Japan) and water. Before the surgeries, mice were anaesthetized with isoflurane. All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals, 8th Edition published by the US National Research Council and were approved by the Animal Care Committees of Showa Pharmaceutical University ., Japan) . The N10Blood pressure was measured using a BP-98A tail-cuff manometer  ,28. BrieBlood and urine were collected from isoflurane-anaesthetized mice through cardiac and urinary bladder punctures, respectively. Serum (or urinary) levels of total protein, creatinine, urea nitrogen, AST, and ALT were measured using commercial colorimetric assay kits from Fujifilm-Wako . For the blood oxytocin measurements, blood was collected from the retro-orbital plexus. Serum was prepared and its protein components were denatured by 0.05% trifluoroacetic acid and removed by the MonoSpin C18 column  with acetonitrile elution. After acetonitrile evaporation, oxytocin contents were measured using the Oxytocin ELISA Kit  according to the manufacturer\u2019s instruction.For examining the nesting activity score, postpartum mice were monitored using an infrared camera, and pictures were taken every 10 min before the birth and until 6 h after birth, or during 6\u201312 h after birth. When the dam crouched over more than half of her pups in one place, the dam was judged to have a nest. When the dam had a nest in 0\u20139, 10\u201318, 19\u201327, and 28\u201336 shots among a total of 36 shots, the nesting activity scores were evaluated as 0, 1, 2, and 3, respectively. For the retrieving activity, the latency to retrieve 1\u20134 pups after 5 pups were placed in the diagonal position of a rectangle cage (225 mm \u00d7 338 mm \u00d7 140 mm high) was examined. When more than half of the pups headed to their dam under her arms or belly in 0\u20139, 10\u201318, 19\u201327, and 28\u201336 shots among the total 36 shots, the breastfeeding activity scores were evaluated as 0, 1, 2, and 3, respectively. Namely, the breastfeeding activity score becomes higher when the dam has likely more chance to breastfeed their pups. The actual confirmation of breastfeeding was not executed not to stimulate lactating dams.Mammary glands were isolated from lactating dams after isoflurane anesthetization. For whole-mount Carmine alum staining , isolateFor Cth immunostaining, after the deparaffinization with xylene/ethanol, the samples (on the slide glasses) were immersed in Immunosaver  at 98 \u00b0C for 45 min for antigen activation and blocked with 10% normal donkey serum, 1% casein, and 0.1% Tween 20 in phosphate buffered saline (PBS). The samples were incubated with anti-CTH rabbit polyclonal antibody ,59 (1:10w/v) Triton X-100, 0.1% (w/v) phenylmethylsulfonyl fluoride, and Complete EDTA-free Protease Inhibitor Cocktail ) using a MicroSmash-100R (MS-100R) homogenizing system  and zirconia beads . Homogenates were centrifuged at 10,000\u00d7 g for 10 min at 4 \u00b0C, and the supernatants were recentrifuged in the same way. After heating  with the sample buffer, samples (20 \u00b5g/lane) were resolved on 10% pre-cast PAGE gels, and then transferred to the Immobilon PVDF membrane (Millipore) and subjected to western blotting. Cth was detected with anti-CTH mouse monoclonal antibody ; 1:5000 in Can Get Signal immunostain Solution 1 (Toyobo)) and mouse IgG\u03ba BP-HRP -Tween 20). Oxr was detected with anti-Oxr rabbit monoclonal antibody ; 1:5000 dilution in Can Get Signal immunostain Solution 1) and anti-rabbit IgG, HRP-linked whole Ab donkey antibody . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected with anti-GAPDH rabbit monoclonal antibody ; 1:10,000 in TBS-Tween 20) as a loading control. Chemiluminescence detection was performed using Chemi-Lumi One Ultra  and the ATTO WSE-6100 LuminoGraph I imager .Each tissue aliquot (~100 mg) was homogenized in a lysis buffer (50 mM Tris-HCl (pH7.4), 150 mM NaCl, 1 mM EDTA, 1% ) at 37 \u00b0C. After 5 min of holding, methionine , homocystine , or vehicle (AEF) was applied to the chamber. After 30 min of incubation, oxytocin  was applied. After 5 min of recoding the contraction frequency, the chamber was washed once with AEF and filled with AEF containing 25 mM KCl to obtain the maximal contraction. The contraction (/relaxation) frequency per min and average peak heights (magnitudes) relative to those of 25 mM KCl were recorded.Adult female mice (8\u201310-week-old) were subcutaneously injected with 10 \u00b5g of 17\u03b2-estradiol (in 100 \u00b5L of 50% DMSO/50% PBS) on their backs and, after 2 days, uteruses on both sides were dissected out from anaesthetized mice. The uterus (~1.5 cm long) was mounted between two steel hooks in an isolated tissue chamber of the conventional Magnus apparatus containing artificial extracellular fluids  was placed in the home cage of each male mice (n = 5 each) for a 1-min confrontation. Then the stimulus female was removed to an individual cage for 10 min. We repeated this sequence on the same male and female for four trials with 10-min intervals. In the 5th dishabituation trial, we introduced a new unfamiliar female. Behavior was video-recorded and the duration (s) spent for anogenital inspection was counted during each 1-min confrontation.Social memory/recognition test was performed as described previously . Both 8\u2013g for 10 min at 4 \u00b0C to remove the fat contents, and then at 150,000\u00d7 g for 10 min at 4 \u00b0C to collect the whey samples. The serum and whey samples were analyzed for their amino acid concentrations using amino acid/thiol-derivatization reagents as previously described in detail [Lactating dams (whose pups were removed before 2 h) were subcutaneously injected with oxytocin (4 units) and breast milk was collected by nipple massaging. The breast milk was centrifuged at 2300\u00d7 n detail .n: sample numbers). Statistical comparison was performed using the two-tailed unpaired Student\u2019s t-test, Mann\u2013Whitney U test (only in p values less than 0.05 denoted a significant difference.Data were expressed as mean \u00b1 SD ( only in E,G, or K"}
+{"text": "We investigate the effectiveness of network attack strategies when the attacker has only imperfect information about the network. While most existing network attack strategies assume complete knowledge about the network, in reality it is difficult to obtain the complete structure of a large-scale complex network. This paper considers two scenarios in which the available network information is imperfect. In one scenario, the network contains link errors  due to measurement errors, and in the other scenario the target network is so large that only part of the network structure is available from network sampling. Through extensive simulations, we show that particularly in a network with highly skewed degree distribution, network attack strategies are robust against link errors. Even if the network contains 30% false links and missing links, the strategies are just as effective as when the complete network is available. We also show that the attack strategies are far less effective when the network is obtained from random sampling, whereas the detrimental effects of network sampling on network attack strategies are small when using biased sampling strategies such as breadth-first search, depth-first search, and sample edge counts. Moreover, the effectiveness of network attack strategies is examined in the context of network immunization, and the implications of the results are discussed. The network attack problem has received much attention, and several network attack strategies have been proposed \u20134. The nWhile most existing network attack strategies assume complete knowledge about the network , 2, 4, iMelchionna et al.  performeWhile the effects of link errors on network attack strategies have been studied, the effects of network sampling, which is a typical cause of errors introduced in the networks, on network attack strategies have not been studied before. The networks of interest nowadays are huge, and network sampling is unavoidable to analyze such large-scale networks \u201319. HoweIn this paper, we aim to reveal the effectiveness of network attack strategies when the attacker has only imperfect information about the network, extending the work by Melchionna et al. . We consThe main contributions and findings in this paper can be summarized as follows.We evaluate the effectiveness of CI, a state-of-the-art network attack strategy, under imperfect information. Our results show that CI is effective when the complete knowledge on the network is available whereas CI is sensitive against network uncertainty. When the level of network uncertainty is large, the effectiveness of CI is comparable with that of the conventional Degree strategy.We investigate how the network sampling affects the effectiveness of network attack strategies. We show that the attack strategies are far less effective when the network is obtained from random sampling, whereas the detrimental effects of network sampling on network attack strategies are small when using biased sampling strategies such as breadth-first search, depth-first search, and sample edge counts.We evaluate the effectiveness of network attack strategies in the context of network immunization , 24. OurG =  when a proportion p of all nodes was removed based on the attack strategy. For this study, we assumed that perfect information about network G was unavailable and the attackers only know network G\u2032 = , a network with unknown errors. We used the attack strategies to determine which nodes of network G\u2032 to attack. We then investigated the connectivity of the true network G with the same nodes removed.We investigated the connectivity of the undirected network G. One is a network that expresses connections between autonomous systems (AS) , and the other is a network that expresses connections between peers, namely a peer-to-peer (P2P) network [http://snap.stanford.edu/data/p2p-Gnutella04.html). We also used two types of synthetic network for network G. For generating synthetic networks, we used the Barab\u00e1si\u2013Albert (BA) model [m = 2 for generating networks with similar average degree with the AS network. For the GRG model, the radius for connecting nodes was 0.015 to generate connected networks while making the average degree be not far from other networks. BA graphs and GRG graphs were generated with R igraph package (https://igraph.org/). The characteristics of each network are shown in We used two types of real network for network  network , 26 (avaA) model  and the G\u2032 from network G: a network with link errors and a sampled network. We created the network with link errors using the same procedure as in Melchionna et al. [E|\u03b4 (0 \u2264 \u03b4 \u2264 1) randomly selected links from network G and added |E|\u03b1 (0 \u2264 \u03b1 \u2264 1) links to randomly selected node pairs within the network to create incomplete network G\u2032. Here, \u03b1 and \u03b4 are the parameters that controlled the magnitude of the incompleteness. To create the sampled network, we followed the procedure outlined by Tsugawa and Kimura [ps of nodes from network G to create incomplete network G\u2032. Sampling node v enabled us to obtain the associated links. In incomplete network G\u2032 = , V\u2032 is the set of sampled nodes, and E\u2032 is the set of links between the set of nodes in V\u2032.We created two incomplete networks a et al. . Specifid Kimura . Specifid Kimura , and  a strategy based on degree centrality (Degree) [G and then evaluated its connectivity by using the size of its GC as the evaluation index.Using incomplete network (Degree) , (ii) a (Degree) , and (ii(Degree) . We remoG\u2032 to determine which nodes to attack. We immunized the nodes to be attacked in network G and simulated the virus diffusion based on the susceptible\u2013infected\u2013removed (SIR) model [t = 0, we randomly selected one non-immunized node in network G and changed its state to infected. The remainder of the non-immunized nodes are susceptible (but not infected). At any given time t, infected nodes v cause adjacent susceptible nodes u to become infected at infection rate \u03b2. In addition, infected nodes transition to the removed state at recovery rate \u03b3. To serve as an index to quantify the extent of virus spread, we determined the number of non-infected nodes (susceptible and immunized nodes) at the time when the aforementioned stochastic processes converge and node states stop changing. The stochastic processes were realized using the random number generator in the Python NumPy module (https://www.numpy.org/).Furthermore, in this study we simulated the spread of viruses to evaluate the effectiveness of attack strategies when applied to the immunization problem. We used incomplete network R) model . The SIRG\u2032 for each parameter used to determine the magnitude of incompleteness. For each incomplete network G\u2032, we determined the nodes to attack, and calculated the size of the GC, and the average of the GCs were obtained. In addition, we conducted 1,000 independent virus spread simulation runs for each incomplete network G\u2032 to determine the average number of non-infected nodes. We used l = 1 and l = 2 as parameters for the CI attack strategy. In addition, we used an infection rate of \u03b2 = 0.1 and a recovery rate of \u03b3 = 0.01 in the virus spread simulations.For the ensuing experiments, we created 100 incomplete networks p of nodes removed from the AS, P2P, BA, and GRG networks and the proportion of the largest connected components when the nodes are removed. These figures compare different magnitudes of incompleteness.First, we investigated how link errors affected the effectiveness of each attack strategy. Figs In the AS network, the magnitude of incompleteness has hardly any effect on the size of the largest connected components under each of the attack strategies . In contl = 2) is more effective than other strategies when the complete network is available. By contrast, when the magnitude of incompleteness is large, the attack strategies deliver similar effects. Even in the GRG network, the difference between CI and other strategies is small. These results suggest that CI, which is a state-of-the-art attack strategy, experiences a decline in effectiveness similar to that with other strategies when the magnitude of incompleteness is large.Next, we compared the attack strategies under the same magnitudes of incompleteness Figs , 8 and 9G and node ranking obtained from incomplete network G\u2032. To investigate how the node rankings based on the attack strategies are changed by link errors, we investigated the Spearman\u2019s rank correlation between the node ranking obtained from the ground-truth network Next, we investigated how node sampling affects the effectiveness of the attack strategies. Figs Our results show that random sampling has an extremely strong effect on the Degree strategy in each network. However, the effects of other sampling methods are much smaller than those of random sampling. Next, we compared different attack strategies under the same sampling methods and sample sizes Figs  and 18. G and node ranking obtained from sampled network G\u2032 to investigate how the node rankings based on the attack strategies are changed by node sampling. For calculating the rank correlation, we considered that non-sampled nodes have the lowest rank. We again investigated the Spearman\u2019s rank correlation between the node ranking obtained from the ground-truth network Finally, we investigated the effectiveness of the attack strategies when applied to the network immunization problem. The size of the GC in the experiments thus far expresses the extent of the damage from virus spread in the worst case. For this section, we conducted virus spread simulations based on the SIR model with immunized nodes, and we evaluated the average extent of the damage from virus spread. Figs l = 2) in the GRG network, we confirmed that the effects of link errors are not so large.These results show that in most networks, the effectiveness of each attack strategy at controlling virus spread is essentially the same, for networks with link errors as well as complete networks. Except for the results of CI Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S3 Fig(EPS)Click here for additional data file.S4 Fig(EPS)Click here for additional data file.S5 Fig(EPS)Click here for additional data file.S6 Fig(EPS)Click here for additional data file.S7 Fig(EPS)Click here for additional data file.S8 Fig(EPS)Click here for additional data file.S9 Fig(EPS)Click here for additional data file.S10 Fig(EPS)Click here for additional data file.S11 Fig(EPS)Click here for additional data file.S12 Fig(EPS)Click here for additional data file."}
+{"text": "The fabricated passive probe is tested under a 3-coil inductive link to evaluate power transfer efficiency (PTE) and power delivered to a load (PDL) for feasibility assessment. The minimum PTE/PDL at 137 MHz were 0.76%/240 \u03bcW and 0.6%/191 \u03bcW in the air and lamb head medium, respectively, with coil separation of 2.8 cm and 9 k\u03a9 receiver (Rx) loading. Six hermetically sealed probes went through wireless hermeticity testing, using a 2-coil inductive link under accelerated lifetime testing condition of 85 \u00b0C, 1 atm, and 100%RH. The mean-time-to-failure (MTTF) of the probes at 37 \u00b0C is extrapolated to be 28.7 years, which is over their lifetime.A new class of wireless neural interfaces is under development in the form of tens to hundreds of mm-sized untethered implants, distributed across the target brain region(s). Unlike traditional interfaces that are tethered to a centralized control unit and suffer from micromotions that may damage the surrounding neural tissue, the new free-floating wireless implantable neural recording (FF-WINeR) probes will be stand-alone, directly communicating with an external interrogator. Towards development of the FF-WINeR, in this paper we describe the micromachining, microassembly, and hermetic packaging of 1-mm As neural interfaces strive to more effectively interact with the brain, neural recording and modulation of the future will need the ability to simultaneously interface with multiple sites distributed across large areas of the brain ,2. TheseA major barrier on the path to clinical viability of the intracortical BMIs is their failure in neural signal acquisition over extended periods in the order of the patients\u2019 lifetime . MultiplTo replace the existing centralized, and thus large and anchored, neural interfaces, a new class of stand-alone BMIs are under development based on the idea that sufficiently small untethered implants with smooth surface and rounded corners, which are free-floating with the brain, can reduce these effects, improve biocompatibility, and extend the electrodes effective lifetime ,22. TensL1), the resonator coil underneath dura (L3), and the receiver coil around the ASIC (L4) [L4, will be used as an antenna to transmit raw neural data across a short distance to a headstage on top of the head. The headstage relays the neural data to a common server for post-processing through a microcontroller unit (MCU) with built-in Bluetooth Low Energy (BLE). The battery-powered headstage also includes an efficient power amplifier (PA) that supplies power to tens to hundreds of FF-WINeRs, distributed over wide cortical areas of interest, through the 3-coil inductive link.SIC (L4) . The samThe fabrication and assembly of the FF-WINeR probe are key aspects of the design that must be addressed at the early stages of development because they directly affect the design decisions made for the other components, such as area and power available for the active circuitry. Exploring the assembly and packaging of the mm-sized passive floating probe, which are similar to the final probes in every aspect, except for the ASIC, is necessary to discover the recording failure mechanisms due to hermetic sealing, manual handling, surgical procedure, or the adverse effect of foreign body reaction and scaring in the brain tissue. Even though there have been many studies concerning brain tissue response to insertion and presence of implantable neural interfacing devices, these were primarily constrained to traditional tethered electrodes ,25,26,27In this paper, we focus on microfabrication, assembly, wireless powering, and wireless hermeticity testing of the mm-sized FF-WINeR probes. Hermetically sealed passive probes can be utilized to explore their longevity and robustness in-vitro for future experiments toward the actual FF-WINeR system implementation and in-vivo testing. 2 IMDs via 2-coil inductive link with 1 cm transmitter-to-receiver (Tx-Rx) coil separation is in the order of 1% [Q) resonator , which offers considerably wider coverage and higher PTE [A comparative study shows that inductive power transfer has the highest power density among several power sources for mm-sized implantable medical devices (IMDs) . To enerer of 1% ,32. The gher PTE .The minimum required power delivered to a load (PDL), based on power consumption of a recent state-of-art neural recording ASIC is ~80 \u03bcW per channel: 2.6 \u03bcW for analog front-end (AFE) , 2.4 \u03bcW Vs and Rs represent the PA and its output resistance, respectively. The PA drives the Tx coil (L1) that is coupled to the resonator coil (L3) with coupling coefficient k13, which is in turn encompassing and coupled to one or more Rx coils (L4) with coupling coefficient k34. R1, R3, and R4 represent parasitic resistances of L1, L3, and L4, respectively, and RL represents the load resistance. Using tuning capacitors, C1, C3, and C4, all coils are tuned at \u03c90, the frequency of the sinusoidal carrier signal that is generated by VS at the PA output.L1 to the resonator coil, L3, can be determined as [Q1 = \u03c90L1/R1, Q3 = \u03c90L3/R3, and Q4L = Q4QL/(Q4 + QL), in which Q4 = \u03c90L4/R4 and QL = RL/\u03c90L4. Q1,Q3, and Q4 are the quality factors of the coils and QL is the load quality factor. PTE between L3 and the Rx coil(s), L4, can be approximated as [The PTE from mined as , (1)\u03b713=total = \u03b713 \u00d7 \u03b734, L3-L4 link should be optimized first, followed by L1-L3 link. More specifically, we optimize Q4L with pre-determined geometry of L4, which is defined by the size of ASIC in this application. k34 is highly dependent on the geometries of L3 and L4, and less so on their number of turns since both their mutual- and self-inductances of L3 and L4 increase with their number of turns [L1-L3 and L3-L4, represented by d13 and d34, respectively, are the other key parameters affecting PTE and PDL, which nominal values  are selected based on the human head and brain anatomy, as shown in To maximize \u03b7rms. Under these constraints, the paramters in the Rx coil that can be optimized are the coil pitch, wire diameter, and number of turns. With the choice of an insulated bonding-wire , wire diameter is fixed at 25 \u03bcm. The coil pitch is somewhat difficult to control when the coil is wound using a manual a wire-bonding machine. Finally, the optimal number of turns is chosen to maximize Q4L using the High Frequency Structural Simulator .The size of Rx coil is often limited by constraints of the application ,36. For L4 is shown in Q4L vs. number of turns of L4 at 135 MHz, which varies from 11.5 to 17.2, indicating that the optimal number of turns is 7.The HFSS simulation model for 34. Here we have considered the outer resonator diamer, Do3 = 3.6 cm, to provide enough room for about 100 FF-WINeRs inside. The outer diameter of the Tx coil, Do1, is derived considering the distance between the Tx and resonator coils, d13 = 2.8 cm [There is a trade-off between the number of FF-WINeRs that fit inside each resonator, set by the diameter of the resonator and \u03b7= 2.8 cm , (3)Do1=L1 and L3 (d13), and L4 horizontal misalignment from the center of L3 (x34), as shown in The 3-coil inductive link is implemented based on the above design considerations to explore the achievable PTE and PDL versus the coil distance between L1 to L4 in the presence of L3 on the same plane with L4 using a ZVB4 vector network analyzer . The twisted red-white feeding line to L4 in L4, and can diminish the measurement accuracy. Therefore, we have adopted a calibration method, known as de-embedding, which is described in [x34 and d13 in the air is depicted in x34 = 0 mm, the min and max S21 are \u221224.45 and \u221221.2 dB when d13 = 42 mm and 28 mm, respectively. At d13 = 28 mm, the nominal Tx-resonator coils\u2019 distance that maximizes S21, the min and max of S21 are \u221221.2 dB and \u221215.1 dB when x34 = 0 mm and 12 mm, respectively. Similarly, the minimum S21 is measured \u221222.26 dB in a lamb head medium, as shown in x34 = 0 mm and d13 = 28 mm. Based on the measured S21 and S11, the PTE can be derived as [Z0 is the characteristic impedance, 50 \u03a9. The achieved minimum PTE (with no rotation) in the air and lamb head medium using Equation (4) are 0.76% and 0.6%, respectively. Since the source power of the vector network analyzer is fixed at 15 dBm, the achieved minimum PDL in the air and neural tissue medium can be calculated as 240 and 191 \u03bcW, respectively. This level of PDL is sufficient to operate the state-of-art neural recording ASICs with small number of channels [The S-parameters are measured from ribed in . The de-rived as , (4)PTE=channels .L4 to be perfectly aligned with L3. This is expected to reduce the achievable minimum PTE and PDL of the 3-coil inductive link due to angular misalignment between L3 and L4. We measured the PTE and calculated PDL of the proposed link while rotating L4 from 0\u00b0 to 90\u00b0 in the lamb head tissue medium. The measurement was repeated 6 times for better accuracy because of difficulty in setting the angular rotation of L4 in the lamb head. L4 angular misalignment at x34 = 0 mm and d13 = 28 mm. With 15\u00b0 L4 angular misalignment, PTE and PDL decease to 0.54% and 171 \u03bcW, respectively, with ~10% measurement error. They drop further down to 0.2% and 60 \u03bcW, respectively, with 45\u00b0 misalignment. Therefore, for state-of-art operating power of 80 \u03bcW in [The brain surface is curved and filled with gyri and sulci, which make it unlikely for the small mm-sized 80 \u03bcW in ,33, the L4 is only 191 \u00b5W, its temperature elevation is negligible. On the other hand, circuit simulation using the fabricated coil parameters in L3 under abovementioned operating conditions. Considering the model and measurements presented in [2 surface area, is will result in ~1.5 \u00b0C. However, the surface area of L3 is 15 times larger than the implant presented in [For chronic implantation, the thermal elevation in the brain tissue surrounding the proposed 3-coil inductive link should be considered, even though a detailed modeling and measurement of thermal effects are out of the scope of this manuscript. Since PDL to ented in , if thisented in , and it Fabrication of the FF-WINeR probes requires a novel process flow due to the extremely small size of the device. The process includes micromachining, microassembly, and hermetic packaging. Fabricated probes should undergo a hermeticity testing to estimate their lifespan under harsh operating conditions inside the body.2 dice on a blank Si wafer, based on previously developed processes described in [For passive Si die fabrication, ultraviolet (UV) photolithography is used to define an array of 1 mmribed in ,41,42,43The procedure to microassemble the FF-WINeR probes is illustrated in L4 is one of the key steps in passive probe fabrication. One way is to utilize a fully-automated wire-bonder which bonding tip is programmed to move along a pre-defined trajectory around the core [C4, is a tuning capacitor to resonate with L4 at the operating frequency and the other is the load capacitor, CL, low-pass filtering the output of a voltage doubler for AC-to-DC conversion.Implementing the mm-sized the core . For proTo hermetically seal the FF-WINeR against the harsh environment in the body and maintain biocompatibility, a 5-\u03bcm thick parylene-C coating is applied on the assembled passive probe by vapor deposition . Even though parylene-C is biocompatible, a layer of Sylgard 184 PDMS coating  is added over parylene-C to extend the probe life-span and create a smooth and soft surface. The uncured PDMS mixture has many trapped air bubbles, which should be removed in a dessicator connected to a vacuum pump until the air bubbles are removed. The passive probe is then dipped in bubble-free PDMS, and hung upside down from a hook with the electrode tip inserted in a piece of styrofoam for at least 24 h to cure at the room temperature. Since biological fluids contain NaCl, KCl, phosphates, carbonates, enzymes, and other proteins, they create a harsh environment for the probe, while the surface of the probe can create a harmful environment for the surrounding cells . In suchiL, attached to a network analyzer with the device-under-test (DUT), which is the FF-WINeR Rx L4C4 tank coupled by the coupling coefficient, k14, in this case, placed in proximity. The basic principle is measuring the changes in reflected impedance of L4 and its tuning capacitance, C4, plus its parasitic capacitance, \u0394C4p DUT due to water ingress in the package. iR and R4 are the parasitic resistance of iL and L4, respectively. The DUT reflected impedance onto the interrogator coil side is depicted in iL and L4. Moisture in the package can either nullify C4 by shorting the SMD capacitor or increase C4p by increasing dielectric constant. The relative dielectric of water is 80.1, 29.9 times larger than that of PDMS, which is 2.68 [L4 since the permeability of water is very close that of vacuum. The electrical characteristics of iL and L4 in the current FF-WINeR prototype are summarized in Our hermeticity testing method was inspired by the wireless capacitive sensing presented in . Figure  is 2.68 . Water lL1, the frequency at which it peaks, fZpeak can be estimated by solving Equation (6) for \u03c9 [i and \u03c94 are the resonant frequencies of iCiL and L4C4, respectively. Variations in fZ11peak, based on solving Equation (6) in Mathematica , are plotted in C4p, shifting fZ11peak by less than in a few 100 kHz, which is very small compared to the original fZ11peak, 384 MHz. On the other hand, the short circuited C4 can cause considerable frequency shift from 384 MHz down to 379.8 MHz. To estimate C4p when C4 is shorted, the FF-WINeR coil impedance, Z44, was measured when it was soaked in water. C4p can be estimated ~1 pF when L4 = 92 nH.If we calculate Z11, the input impedance seen through 6) for \u03c9 , (6)1\u2212 in years and describes as [hour is the failure rate per hour that can be defined as, F and T are the number of failures and test hours per device. The MTTF in this case is estimated to be 28.7 years. The small amount of water penetration into the hermetic package may be considered as a hermetic sealing failure. It should be noted that since the passive FF-WINeR has two layers of polymer coating, PDMS over parylene, water ingress into the PDMS layer does not necessarily lead to device failure despite causing a small change in the fZ11peak. The 5 \u03bcm parylene-C coating layer is considerably more resistant to water than the PDMS coating, and maintains functionality of the active circuit even though the L4C4 resonance frequency may shift as a result. This shift can be remedied, however, using the auto resonance tuning (ART) method, described in [In a widely used model for accelerated lifetime testing, the acceleration factor (AF) is defined based on the Arrhenius equation as , (7)AF=MTTFribed in .We have presented the structure, microfabrication, and assembly steps of a mm-sized implant toward a distributed free-floating wireless implantable neural recording (FF-WINeR) system, which can be used for safer and less invasive neural interfacing with potentially less damage to the neural tissue. The silicon chip that houses the ASIC is micromachined after standard CMOS processing to also acts as a substrate and provide mechanical support for the microwire electrodes and the bonding-wire coil, which is wound around the ASIC to offer higher Q compared to on-chip coils. Wireless power transmission to the FF-WINeR is designed based on 3-coil inductive link, using a high-Q planar implantable resonator in the same plane as the FF-WINeR probes and encompassing them. Preliminary experiments show that the required PTE and PDL for the desired functions in the ASIC are achievable with Tx coil separation of 2.8 cm. A simple method is developed for wireless hermeticity testing of the dual polymer coated FF-WINeR probes based on accelerated lifetime testing technology. We are now developing the ultra-low power and area efficient active circuitry for this probe and further improving the hermeticity testing methodology to achieve more accurate estimates of the FF-WINeR probe lifetime. Future steps also include in-vivo experiments on rat animal model."}
+{"text": "Treatment of Class II malocclusion accompanied with a skeletal discrepancy is challenging. The approach of correction depends on several factors such as the status and pattern of growth, severity of the malocclusion, and patient cooperation. This case report describes a successful management of a 12-year-old young adolescent boy that was presented with a Class II division 1 malocclusion with an underlying skeletal discrepancy in horizontal and vertical dimensions. Growth modification was achieved by means of bite opening and unlocking the mandible together with Class II elastics and mechanics. Treatment was highly effective and efficient by achieving all treatment goals within a period of 18 months. Class II dental malocclusion considered as the second most prevalent malocclusion after Class I, with a prevalence range of 13%-24% . As descTreatment approach and modality for Class II division 1 with an underlying skeletal problem depend on several factors that should be considered by the clinicians such as chronological age, growth potential, skeletal maturity, severity of the condition, and patient motivation and cooperation , 11. TheSeveral studies showed that over the long-term treatment results achieved with Class II elastics are similar or comparable to those achieved with functional appliances, given that treatment started at an appropriate timing \u201317. BothIn the current case report, we discuss the treatment of a Class II division 1 malocclusion accompanied with a severe Class II skeletal discrepancy and hypodivergent growth pattern in a growing adolescent.12 years and 8 months young boy referred to the orthodontic specialty clinic with a chief complaint of \u201cprotruded front teeth\u201d. Medical and dental history indicated no significant findings. Extraoral clinical examination revealed a symmetrical euryprosopic face type, convex soft tissue profile, retruded mandible, deep mentolabial sulcus, decreased lower facial third, and on smiling lower lip was trapped by the upper central incisors .st and 2nd molars. Mild anterior crowding with rotated canines . Patient had poor oral hygiene, plaque accumulation, and mild gingival inflammation. All permanent teeth are erupted while the 3rd molars are still developing  impinging overbite, deep curve of Spee (COS) of 3\u2009mm, and unilateral posterior cross-bite on the upper left 1Cephalometric analysis revealed a severe skeletal Class II relationship (ANB = 7.8\u00b0) with horizontal growth pattern tendency , and anterior facial height is markedly reduced. Cervical vertebral maturation (CVM) indicates cervical stage 1 (CS1). Lower incisors are proclined (L1 \u2212 MP = 99.3\u00b0), and both lips are anterior to the E-line by 1\u2009mm .The following were the list of treatment objectives: (1) improve the sagittal and vertical skeletal relationships; (2) enhance and guide mandibular growth; (3) resolve increased overjet; (4) establish Class I canine and molar relationship; (5) resolve the impinging overbite, deep COS, and traumatic occlusion; (6) resolve the left posterior cross bite; (7) resolve crowding and rotations; and (8) improve facial profile and smile esthetics.Given the patient's age and problem list, a nonsurgical and nonextraction treatment plan based on growth modification to enhance mandibular growth was developed. The primary plan presented to the patient and his family was to start with growth modification using a removable Class II functional appliance or cervical pull headgear with anterior bite plane followed by leveling and aligning with a fixed edgewise appliance. The option was not considered by the family due to increased cost from the use of additional device. In addition, the treatment duration could be prolonged especially if it was divided into two phases. Therefore, an alternative plan addressing those concerns was presented which involved bonding a fixed edgewise appliance, anterior bite turbo, and simultaneous growth modification using intermaxillary elastics taking advantage of the patient's early adolescence and prepubertal stage of development.The patient demonstrated a poor oral hygiene and few carious lesions, so before starting orthodontic treatment, oral hygiene instructions were given, and patient was referred for periodontal and restorative clearance.\u2033 \u00d7 0.028\u2033 MBT was bonded in both arches. Leveling and aligning lasted for 6 months, which were achieved by the following NiTi wire sequence  then followed by stainless steel (SS) working wires . SS arch wire expansion and criss-cross elastic from palatal of upper left first molar to buccal of lower left molar were used to correct the unilateral posterior cross bite on left molars.Once cleared, a full-fixed straight wire appliance of slot size 0.022In addition, to address the vertical and sagittal skeletal problems, anterior bite turbos palatal to upper central incisors were cemented ; gradualAfter 10 months in treatment, the antro-posterior canines and molars relationships were corrected. Final detailing and finishing included arch wire adjustments and power chain use for some remaining spaces closure. The fixed appliance was debonded after 18 months of active treatment. Clear vacuum retainers were delivered, and after 1 month, they were replaced by Hawley retainers with anterior bite plate on the upper appliance.The overall outcome of treatment was successful, and the patient's chief complaint was addressed. Treatment objectives were achieved in an efficient treatment duration (18 months and 14 visits). The skeletal and dental relationships were significantly improved. Facial profile showed great improvement as facial proportions were restored, mandible was advanced, convexity was reduced, and restored normal mentolabial sulcus. Moreover, smile esthetics were improved, and lip position was corrected with no entrapment . Intraory-axis 64.7\u00b0) to (y-axis 68.1\u00b0). Maxillary incisors were retroclined, and the deep bite was corrected by relative intrusion of a combination of molar extrusion and mandibular incisors intrusion and proclination. In addition, mandibular molars moved mesially while maxillary molars slightly distalized  to posttreatment , facial convexity decreased from 12.9\u00b0 to 6.9\u00b0, mandibular plane angle increased from  to , total face height (Na-Gn) increased from 105.2\u2009mm to 116.9\u2009mm, lower face height (ANS-Gn) increased from 59.7\u2009mm to 65.5\u2009mm, and the hypodivergent growth pattern improved from ( Figures .At 1-year follow-up, the occlusion was stable with no significant relapse, and the patient demonstrated poor oral hygiene with mild gingival inflammation . LateralThis case report showed a successful management of a 12-year-old adolescent boy presented with a Class II division 1 malocclusion accompanied with a severe skeletal Class II discrepancy. Several key factors contributed to the success of this treatment mainly including early intervention at prepubertal stage, pattern of skeletal growth, patient's cooperation, and mechanics applied.The time of intervention in this case was of great impact in correcting the skeletal discrepancy. The treatment started when the patient was in the prepubertal stage of growth. The patient was at early stage of CVM CS1, , secondaIn the current case, upon opening the patient's bite and releasing the locked mandible from the impinging overbite, a great improvement in sagittal skeletal relation was achieved. Angle indicated that in Class II malocclusion, the lower dentition is locked in distal occlusion, and thereby, the mandible will be locked as well . SeveralIt was well noticed that most of the sagittal discrepancy was improved after removing the restriction from the impinging deep bite and unlocking the mandible, while the use of Class II intermaxillary elastics was for a short period still effective to add to the final sagittal correction. Class II elastics carried out further guidance and enhancement of mandibular growth toward normal skeletal relation, added considerable restriction to the growth of the maxilla, and improved the vertical skeletal relationship . DespiteThe vertical skeletal relationship improved significantly from the correction of the deep bite by use of reverse COS wires, Class II elastics, and bite turbos. Those mechanics helped to extrude the molars, intrude and procline the incisors, and facilitate further mandibular growth. As mentioned earlier, with Class II elastics, there is a clockwise rotation of the occlusal plane and mandible with a final outcome of increase in lower anterior face height , 20, 22.The skeletal growth pattern of the patient was a favorable adjunct in the management of his case and helped in accommodating the current treatment plan. The vertically hypodivergent mandibular plane angle, the forward rotation of the mandible, and the underdeveloped anterior face height facilitated in toleration of the applied extrusive mechanics and the resulted backward rotation of the mandible. On the opposite, cases of vertical growth pattern with high mandibular plane angle are challenging and difficult to treat, and applying such extrusive mechanics should be avoided as they would deteriorate the final results and will not work in favor of the patients while more control of the vertical dimension should be considered .Among the challenges that were faced in this case was to maximize the orthopedic changes using Class II elastics while minimizing elastics possible side effects. Proclination of lower incisors was significantly increased  from ClaClass II elastics considered a viable orthodontic option for correcting Class II skeletal discrepancy in growing patients, given the careful case selection based on timing of growth spurt, pattern of skeletal development, severity of the malocclusion, and patient's cooperation and motivation.Moreover, orthodontists should consider the benefits and potential side effects of Class II elastics use, plan that in the treatment, and be able to handle and direct them to the patient's favor once executed in treatment."}
+{"text": "Penile Mondor\u2019s disease is a rare condition characterized by thrombophlebitis of superficial penile dorsal veins. We report a case of a 26-year-old male who developed a lump on the dorsal surface of the penis following intensive masturbation after prolonged sexual abstinence. Physical examination revealed a firm, cord-like swelling with beaded texture with no localized regional lymphadenopathy. The patient was successfully managed with non-steroidal inflammatory drugs and was symptom-free on a follow-up. Mondor\u2019s disease (MD) is a self-limiting and benign condition, recognized clinically by the presence of palpable cord-like induration on the body surface. The first case was reported in 1939 as a result of generalized thoracoabdominal phlebitis and as an isolated superficial penile veins thrombosis known as Penile Mondor\u2019s disease (PMD) in 1958\u00a0. Other sA 26-year-old male with no significant past medical history and sexual abstinence of more than six months presented in the outpatient department with a cord-like swelling on the dorsal surface of the body of the penis for one day. He first noticed a small lump on the dorsal surface at the distal shaft that suddenly progressed to swelling, extending distally and wrapping like a cord around the shaft of the penis just proximal to glans. The patient had mild pain at rest and moderate discomfort on erection. He reported masturbating three times a day in the last seven days. He denied any recent systemic symptoms such as fever, urinary frequency/urgency, dysuria, hematuria, urethral discharge, local trauma, or any history of current or past sexually transmitted diseases. Physical examination revealed a firm cord-like swelling with beaded texture on the dorsum of the distal penis, extending and wrapping around the shaft just proximal to glans Figure . No locaMD is a rare clinical entity with a reported incidence of 1.39%. It is described as sclerosis of superficial thrombophlebitis of the veins of the anterior thoracoabdominal wall. PMD or Mondor\u2019s cord has been historically reported as affecting the superficial veins on the dorsal surface of the penis. Table PMD is usually a clinical diagnosis. History and physical examination are reliable diagnostic tools that can reveal a palpable, firm cord-like lesion on the dorsal aspect of the penis. Doppler ultrasound can be utilized as next for differential diagnosis or if the physical examination is unclear. We used clinical judgment to diagnose our patient.\u00a0The presence of thrombosed segment and uncompressible vein with no signal flow on ultrasound plus high resistance, low-velocity blood flow in cavernous arteries on Doppler are supporting features for disease and helps to differentiate it from sclerosing lymphangitis . MRI useThe condition is usually self-limiting resolving within four to six weeks. Treatment is generally supportive and limited to the use of mostly non-steroidal inflammatory agents orally or anti-coagulants in topical forms, with a focus on relief of pain and inflammation -9. OtherPMD is a rare and underreported condition that manifests as cord-like swelling on the dorsal surface of the penis. Attention should be given to secondary underlying causes, such as thrombophilia, malignancy, or sexually transmitted diseases. Ultrasonography with Doppler is the imaging of choice for diagnosis, and invasive or expensive investigations should be avoided. Management should be focused on reassurance about the condition and pain alleviation. Surgery should be reserved for only refractory cases."}
+{"text": "Streptomyces sp. strain BR123, isolated from rhizospheric soil that exhibited promising antimicrobial properties, was sequenced and assembled. Here, we report an 8,157,040-bp genome sequence with a G+C content of 72.63%. This genome sequence enlightens the genes responsible for the production of secondary metabolites and antimicrobial compounds by this strain.The genome of  Streptomyces sp. strain BR123, isolated from rhizospheric soil that exhibited promising antimicrobial properties, was sequenced and assembled. Here, we report an 8,157,040-bp genome sequence with a G+C content of 72.63%. This genome sequence enlightens the genes responsible for the production of secondary metabolites and antimicrobial compounds by this strain.The genome of  Streptomyces members are aerobic, Gram-positive filamentous bacteria belonging to the order Actinomycetales of Actinobacteria. Bacteria in this genus are well reported for their production of bioactive secondary metabolites that have great biofunctional diversity and different applications, such as antibacterial, antifungal, antiviral, anticancer, immunosuppressant, insecticidal, and herbicidal, which make them suitable for use as pharmaceuticals and in agricultural industries  medium (https://sourceforge.net/projects/samtools/files/samtools/1.9/), BEDtools 2.28 (https://bedtools.readthedocs.io/en/latest/), and BWA-MEM 7.12 (de novo assembly was performed (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/) was used to assemble metrics.Paired-end reads yielding 30\u00d7 coverage were checked for quality control, adapted reads were trimmed by using Trimmomatic 0.30 with a sliding window quality cutoff of Q15 , and quaMEM 7.12 . By the erformed , and annN50 value of 22,797\u2009bp, and the largest contig has 109,551\u2009bp. The genome of Streptomyces sp. strain BR123 contains 8,157,040 bp with a G+C content of 72.63%, a value highly similar to those reported in Streptomyces spp. (The genome assembly yielded 723 contigs with an ces spp. , 14. TheJACBGN000000000, BioProject number PRJNA643667, SRA number SRR12527047, and BioSample accession number SAMN15423153. The 16S rRNA gene sequence has been deposited at DDBJ/ENA/GenBank under the accession number MT799988.This whole-genome shotgun project has been deposited at GenBank under whole-genome sequencing project number"}
+{"text": "There is now good evidence that small group teaching provides a fruitful academic environment, which optimises learning, particularly in the healthcare setting, and especially when compared to lectures. An individual student\u2019s understanding of knowledge is increased when they are able to actively compare and build on their own understanding in conjunction with their peers. Small group teaching provides opportunities for learners to work collaboratively, and promotes team-building skills \u2013 skills that are essential to work within healthcare settings. The aim of this paper is to provide health professional students and early career health professionals involved in peer and near peer teaching, with an overview of approaches and tips to improve learner engagement when facilitating small groups. Health professional education occurs in a variety of contexts, including those within university, hospital, community-based and clinical settings. Curricula activities at the university target development of students\u2019 knowledge of the basic sciences of healthcare , which are then integrated into the clinical setting, thus contextualising this knowledge. The clinical setting also plays a crucial role in developing students\u2019 clinical skills, communication skills, and professionalism. The clinical application of the basic sciences also occurs in case scenario based small group teaching methods, such as problem based learning (PBL), Team-based learning (TBL), Case based learning (CBL), in the university setting \u20137; and cAn individual student\u2019s understanding of knowledge is increased when they are able to actively compare and build on their own understanding in conjunction with their peers \u201316. Smalactive participation, \u2018face-to-face\u2019 contact between participants, and purposeful activities. When implemented with all three elements in play, the small-group context offers many benefits, and enhances students\u2019 learning experiences in many ways. For example, small group learning has the potential to: . Af. Af31]. What went well during the lesson?What can be improved?Did the lesson cover the learning objectives I set at the start of the lesson?Was my questioning technique effective?How well did I engage learners?Where can I improve next time?Critical reflection is considered an essential step to effective education in healthcare, and should be practiced by facilitators . This in\u25e6 Be open to the feedback being given as it is intended to be helpful\u25e6 Avoid instantly dismissing feedback that does not match self-reflection\u25e6 Avoid becoming defensive - instead engage in constructive discussion\u25e6 Ask for specific examples to explain the feedback being given.Gaining feedback from learners can also assist in self-reflective practices . This caStudents being reluctant to engage in discussion with each otherStudents are not prepared for small group activitiesIndividual \u2018free riders\u2019 failing to contribute (may be shy or disinterested)Individual students dominating discussion or being disruptiveAttention being directed towards the facilitator, who is expected to provide answersFacilitator\u2019s questions don\u2019t go beyond the level of recallFacilitator\u2019s lack of attempt to get students to answer their own questionsFacilitators providing insufficient/poor feedbackFacilitators talking too much, lecturing rather than facilitating.Although small group teaching offers many advantages, it may pose some difficulties and limitations for the facilitator and students. Group problems commonly stem from , 26, 32:The facilitator should reflect on why the problem is occurring, what can be done differently to help overcome the problem, and how accountability for success can be shared with the student group . DependiIndividual dominant students: summarise points and divert the discussion to others; indicate time pressure; give the group specific tasks.Quiet students: give them time to respond; divide the group into pairs for a task; positively reinforce any contribution.Attention being directed towards the facilitator: build on students\u2019 responses to a limited extent by sharing clinical experiences or provide a clinical context where appropriate to heighten the relevance of the topic.Students receiving insufficient feedback: schedule a time for student feedback throughout the session, or afterward.Students attending unprepared for small group activities: ensure student accountability to their team members by including short tests at the beginning of class, which may also help to prevent late arrivals [arrivals .Small group teaching can be very rewarding, for both the learners and facilitators. However, successful small group facilitation requires appropriate facilitation methods to encourage active and purposeful participation, and enhance student learning. The facilitator\u2019s role is crucial in encouraging the learners to interact with the content, and with their peers. Self-reflective practices and the use of feedback provide a valuable means to improve small group facilitation skills."}
+{"text": "We discuss the impact of a Covid-19\u2013like shock on a simple model economy, described by the previously developed Mark-0 Agent-Based Model. We consider a mixed supply and demand shock, and show that depending on the shock parameters (amplitude and duration), our model economy can display V-shaped, U-shaped or W-shaped recoveries, and even an L-shaped output curve with permanent output loss. This is due to the economy getting trapped in a self-sustained \u201cbad\u201d state. We then discuss two policies that attempt to moderate the impact of the shock: giving easy credit to firms, and the so-called helicopter money, i.e. injecting new money into the households savings. We find that both policies are effective if strong enough. We highlight the potential danger of terminating these policies too early, although inflation is substantially increased by lax access to credit. Finally, we consider the impact of a second lockdown. While we only discuss a limited number of scenarios, our model is flexible and versatile enough to accommodate a wide variety of situations, thus serving as a useful exploratory tool for a qualitative, scenario-based understanding of post-Covid recovery. The corresponding code is available on-line. The Covid-19 pandemic has buffeted the world economy and induced one of the most abrupt drops in output ever recorded. What comes next? Will the economy recover quickly as lockdown measures are lifted, or will the damage inflicted by the massive waves of layoffs be more permanent? In pictorial terms, will the economic crisis be V-shaped (quick recovery), as commentators were initially hoping for, or U-shaped (prolonged drop followed by a quick recovery), or perhaps W-shaped, with a relapse due either to further lockdowns, or to premature lifting of the economic support to households and firms? The possibility of an L-shaped crisis, with a permanent loss of output, is also considered. Or else, maybe, a \u201cswoosh\u201d, with a rapid drop followed by an excruciatingly slow recovery?  express the sensitivity of the firm\u2019s target production to excess demand/supply. We posit that the coefficients i  are given by:x\u301b = 1 when x \u2265 1 and \u301ax\u301b = 0 when x \u2264 0. The factor \u0393 > 0 measures how the financial fragility of firms influences their hiring/firing policy, since a larger value of \u03a6i then leads to a faster downward adjustment of the workforce when the firm is over-producing, and a slower (more cautious) upward adjustment when the firm is under-producing. \u0393 itself depends on the inflation-adjusted interest rate and takes the following form:\u03b1\u0393 is a free parameter, similar to \u03b1c that captures the influence of the real interest rate.f thumb\u201d , 18. In Price update. Following the initial specification of the Mark series of models  random variables and \u03b3 is a parameter setting the relative magnitude of the price adjustment. The factor f models , prices Wage update. The wage update rule follows the choices made for price and production. Similarly to workforce adjustments, we posit that at each time step firm i updates the wage paid to its employees as:t, U random variable and g modulates how wages are indexed to the firms\u2019 inflation expectations. If i at time t with this amount of wages would have been negative, Wi(t + 1) is chosen to be exactly at the equilibrium point where i, is an increasing function of Wi. Hence, firms that want to produce more (hence hire more) do so by increasing Wi, as to attract more applicants.Note that within the current model the productivity of workers is not related to their wages. The only channel through which wages impact production is that the quantity The above rules are meant to capture the fact that deeply indebted firms seek to reduce wages more aggressively, whereas flourishing firms tend to increase wages more rapidly:If a firm makes a profit and it has a large demand for its good, it will increase the pay of its workers. The pay rise is expected to be large if the firm is financially healthy and/or if unemployment is low because pressure on salaries is high.Conversely, if the firm makes a loss and has a low demand for its good, it will attempt to reduce the wages. This reduction is more drastic if the company is close to bankruptcy, and/or if unemployment is high, because pressure on salaries is then low.In all other cases, wages are not updated.Profits and dividends. Finally, the profits of the firm \u03b4 of their cash balance \u03b8 is the Heaviside step-function. These dividends are then reduced from the firms\u2019 cash balance and added to the households savings in \u03c10 via a Taylor-like rule:\u03d5\u03c0 modulates the intensity of the central bank policy, \u03c1* is the baseline interest rate and \u03c0* is the inflation target for the central bank. The banking sector then sets interest rates for deposits \u03c1d (for households) and loans \u03c1l (for borrowing by firms). Defining f then determines how the impact of these defaults fall upon lenders and depositors\u2014f interpolates between these costs being borne completely by the borrowers (f = 1) or fully by the depositors (f = 0). The total amount of  money M in circulation is kept constant and the balance sheet of the banking sector reads:The banking sector in Mark-0 consists of one \u201crepresentative bank\u201d and a central bank which sets baseline interest rates. The central bank also has an inflation targeting mandate. The central bank sets the base interest rate R (hiring/firing ratio) and \u0398 (bankruptcy threshold for firms) are important Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0PartlyReviewer #3:\u00a0Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0YesReviewer #2:\u00a0N/AReviewer #3:\u00a0Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1:\u00a0YesReviewer #2:\u00a0NoReviewer #3:\u00a0Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The Results section should be presented at the end, after describing the model and all approximations. In this section, the authors describe the parameters of the model before describing the model itself.99-106 and table 1 shows the empirical parameters of the model. However, their significance is not substantiated.The Politics section offers different scenarios for overcoming the crisis, but without prior formalization of the mathematical model, it is impossible to understand these scenarios.ABM models - depend on random values. Therefore, the simulation results may be unstable. It is not clear from Figures 4 and 5 this is the result of the work of an ensemble of models or a single simulation.The authors do not provide an analysis of the sensitivity and stability of the model from changes in various factors.The Model section should be moved to the beginning of the article.In this section it is expedient to formalize the model in the form of the scheme of interaction of various components-blocks of systemIt would be expedient to justify the choice of values \u200b\u200bof empirical parameters of the model to the characteristics of a particular country.It would be interesting to compare the effectiveness of the strategies of different countries in comparison with the results of the modelReviewer #2:\u00a0The short title is better than long title that creates confusion.The study is interesting with the aim to suggest guidelines for economic recovery after COVID-19 pandemic crisis and shock of lockdowns.Introduction has to provide more theoretical background about COVID-19 and risk factors current and future in society to know the problem for health and economies (see suggested readings that have to be read and used in the text).The study is based on simulations and without empirical evidence. Of course, the recovery depends on countries. I suggest to clarify better research setting because countries are different \u2026a recovery in Italy is different from a recovery in Sweden because of different structural indicators\u2026. for instance, Italy has a high public debt and a similar shock generates different effect than other countries. Moreover, also the lockdown generates different effects according to the duration\u2026longer lockdown generates a higher deterioration of economic systems than short one (see new literature).How these aspects are considered in the model?The contraction of some economies is higher than others.In short, there are manifold variables to control and I suggest whenever possible to discriminate the design of the model for countries that have some similarities, such as Spain and Italy, Germany and France, Scandinavian countries etc. to be more consistent with the reality and reduce the heterogeenity between countries and provide more reliable policy implications.As you know the applications of policies generates different effects according to the countries and the structure of their economies\u2026 I would like to know how it is possible a match between these different shapes L, W, etc. of recovery and different countries, and policy implications\u2026. I know it is necessary a time consuming revision, but authors can provide more indications to provide a main contribution for political economy of growth post COVID-19.Conclusion has to present better manifold limitations of this study for the complexity of factors and heterogeneity of countries and in particular for the methods of inquiry based on simulations done in a computer lab.Suggested readings of relevant papers that have to be read and all inserted in the text and references to improve the study.Leach, M., MacGregor, H., Scoones, I., Wilkinson, A. 2021Post-pandemic transformations: How and why COVID-19 requires us to rethink development, World Development 138,105233Coccia M., 2020. National lockdown to cope with COVID-19 pandemic: effects (contradictory) on public health and (negative) on economic system, Working Paper CocciaLab n. 56D/2020, CNR -- National Research Council of Italy. Available at Research Square, DOI is: 10.21203/rs.3.rs-115665/v1Abuselidze, G., Slobodianyk, A. 2021 Pandeconomic crisis and its impact on small open economies: A case study of COVID-19 Advances in Intelligent Systems and Computing, 1258 AISC, pp. 718-728Coccia M. 2020. An index to quantify environmental risk of exposure to future epidemics of the COVID-19 and similar viral agents: Theory and Practice. Environmental Research, Article number 110155, DOI: 10.1016/j.envres.2020.110155Yoshino, N., Hendriyetty, N. 2020 The COVID-19 Crisis: Policy Recommendations for Japan. Economists' Voice , 17(1),20200017Iuga, I.C., Mihalciuc, A. 2020Major crises of the XXIst century and impact on economic growth. Sustainability (Switzerland), 12(22),9373, pp. 1-20Reviewer #3:\u00a0The article is well written and provides interesting insights about the response of the economy to the pandemic due to COVID-19. In general, the authors did a good job explaining the purpose of the study, and described properly the instruments they used to develop the study. I have some minor comments for the authors to be addressed before the study can be accepted for publication.1. I suggest including some literature in the introduction (Lines 40-44) about using ABM to understand the impacts of COVID. For example, please review the following article.https://doi.org/10.1016/j.ssci.2020.1050222. Line 204The authors could explain better why they selected 2 policies. Are there different policies that simulate the same effects from the policies that the authors used in the study in the literature? If so, explain why other policies are not selected.3. I suggest to the authors to include in the study some discussion about how the results were validated and verified. If not, please also state that clearly.4. I would also recommend to the authors to include the word economy in the title.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,\u00a0 4 Feb 2021We thank the reviewers for their comments which have helped to improve the manuscript. All points raised by the reviewers have been addressed in the Rebuttal letter attached with the resubmission.Attachmentrebuttal_letter.pdfSubmitted filename: Click here for additional data file. 15 Feb 2021V -, U -, L -\u00a0or W-shaped economic recovery after COVID-19: Insights from an Agent Based\u00a0ModelPONE-D-20-37904R1Dear Dr. Sharma,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. In this vein, there are required a couple of revisions regarding references.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Stefan Cristian Gherghina, PhD. Habil.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #1:\u00a0All comments have been addressedReviewer #2:\u00a0All comments have been addressedReviewer #3:\u00a0All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The author took into account the comments of the reviewer. Therefore, I believe that the article can be published in a journalReviewer #2:\u00a0V -, U -, L - or W-shaped economic recovery after COVID-19: Insights from an Agent Based ModelI have read thoroughly the revised version of paper. The authors have done considerable additional work, and addressed all concerns and criticisms in the revised manuscript, which I believe has improved substantially in the theoretical framework, study design and discussion of results.Finally, I suggest to revise some citations of papers to update them and correctly cite some articles in references, such as:https://ssrn.com/abstract=3560337--Fornaro, Luca and Wolf, Martin, 2020. Covid-19 Coronavirus and Macroeconomic Policy (March 2020). CEPR Discussion Paper No. DP14529, Available at SSRN: https://doi.org/10.1016/j.scitotenv.2021.145801--Coccia M. 2021. The relation between length of lockdown, numbers of infected people and deaths of Covid-19, and economic growth of countries: Lessons learned to cope with future pandemics similar to Covid-19. Science of The Total Environment, Available online 12 February 2021, 145801. --Eichenbaum M. S., Rebelo S., Trabandt M., 2020. The Macroeconomics of Epidemics, NBER Working Papers 26882, National Bureau of Economic Research, Inc. doi = 10.3386/w26882After that, the paper is OK.Reviewer #3:\u00a0Thank you for preparing a response to my comments. Since this is a very current and interesting topic, I recommended the article for publication.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1:\u00a0NoReviewer #2:\u00a0NoReviewer #3:\u00a0No 19 Feb 2021PONE-D-20-37904R1 V\u2013, U\u2013, L\u2013 or W\u2013shaped economic recovery after Covid-19: Insights from an Agent Based Model Dear Dr. Sharma:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Stefan Cristian Gherghina Academic EditorPLOS ONE"}
+{"text": "Thermally treated watermelon juice (TW) presents a strong unpleasant smell, resulting in poor consumer acceptance. It is necessary to identify the key off-flavor compounds in TW. Solid-phase microextraction (SPME) and solvent-assisted flavor evaporation (SAFE) coupled with gas chromatography\u2013olfactometry\u2013mass spectrometry (GC\u2013O\u2013MS) were applied to the extraction and analysis of the volatile compounds in TW. Five aroma-active compounds and seven off-flavor compounds were quantitatively analyzed by the standard curve method. Based on the flavor dilution factor (FD), odor attribute, odor activity value (OAV) of volatile compounds, and partial least-squares regression (PLSR) analysis, seven key off-flavor compounds were preliminarily identified as follows: (E)-2-heptenal, decanal, octanol, diisopropyl disulfide, hexanol, (E)-2-decenal, and (E)-2-octenol. Aroma recombination proved that these off-flavor compounds above had a negative impact on the overall flavor in TW. Omission experiments were taken to confirm them further. Finally, octanol, diisopropyl disulfide, and (E)-2-decenal were identified as the most potent off-flavor compounds in TW. In China, the annual planting area and yield are approximately 1.85 million hmHeating watermelon juice considerably affects its quality owing to its thermo-sensitive nature . HoweverTo identify the off-flavor compounds in thermally treated watermelon juice (TW), it is necessary to understand the mechanism of flavor change. Current studies have focused on the aroma of fresh watermelon (FW) or its juice. The flavor compounds in watermelon juice are mainly C6 and C9 aldehydes, ketones, and alcohols, such as nonanal, (E)-6-nonenol, -2,6-nonadienal, (Z)-3-nonenal, -2,6-nonadienal, and -3,6-nonadienal ,8. DevelIn the present study, the aroma profiles of FW and TW were compared to identify the off-flavor compounds. The key off-flavor compounds in TW were determined using gas chromatography\u2013olfactometry\u2013mass spectrometry (GC\u2013O\u2013MS), odor attributes, and partial least-squares regression (PLSR) analysis. Furthermore, aroma recombination and omission experiments were performed to verify the key off-flavor compounds.2SO4 used for extraction and separation of flavor substances were all analytical reagent and provided by Banxia Scientific Instruments Co. Ltd. .n-Alkanes (C7-C30) for the retention index (RI) calculation, 2-methyl-3-heptanone , -2,6-nonadienal (95%), (E)-2-heptenal (97%), decanal (\u226598%), octanol (\u226599%), -2,6-nonadienol (\u226595%), (E)-2-octenal (\u226595%), diisopropyl disulfide (\u226596%), hexanol (\u226599%), (E)-2-nonenal (97%), (E)-2-decenal (\u226595%), nonanol (98%), and (E)-2-octenol (97%) were purchased from Sigma-Aldrich . Hexane, diethyl ether, n-pentane, and anhydrous NaQilin is the main cultivar with a large yield in China. Twenty watermelons  were one-time selected and purchased from Beijing Yonghui Supermarket randomly on June 10th, 2018. The pulp was blended , and quickly filtered through a nylon mesh (200 mesh). FW was analyzed immediately. Batches of 100 mL juice were vacuum-packed with an odorless vacuum packaging bag with aluminum foil immediately and frozen using liquid nitrogen. All the packed juices (100 mL for each bag) were stored at \u221280 \u00b0C for further analysis. TW was processed using a water bath at 70 \u00b0C for 20 min according to the pasteurization method (the juice flavor changed greatly under this condition through the pre-experiments. A thermometer was inserted into the juice through the bag and timing began when the center temperature of juice reached 70 \u00b0C) .Volatile organic compounds of a 10 mL sample of watermelon juice with 1 \u03bcL internal standard  added in a 40 mL headspace vial  were extracted manually with SPME using a 2 cm, 50/30 \u03bcm divinylbenzene/carboxen/polydimethylsiloxane fiber . Samples were allowed to equilibrate for 20 min at 40 \u00b0C before collection for 40 min with continuous stirring at 100 rpm  .v/v) and stirred for 8 h. Fifty microliters of 2-methyl-3-heptanone (0.816 \u03bcg/\u03bcL) were added as the internal standard. Then, volatiles were extracted from the solvent extracts by distillation for 2 h at 10\u22124 torr. The solvent layer was concentrated to 2 mL with a Vigreux column  after being dried through an anhydrous Na2SO4 column. The volume further reduced to 0.2 mL under a flow of nitrogen [One hundred milliliters of watermelon juice was mixed with 150 mL diethyl ether-pentane mixture  with an olfactometer  was used to analyze the volatile compounds. DB-WAX and DB-5 chromatographic columns  were employed to separate these compounds .\u22121 scan rate. Compounds were identified according to NIST 14.0  mass spectra libraries.Mass spectra in electron ionization mode were recorded at 70 eV and a mass/charge range of 50\u2013350 amu at a 2.0 scan sGC\u2013O analysis was carried out on polar and non-polar columns by three well-trained panelists. Before analysis, the panelists were trained by smelling the odors of the model solutions of reference compounds at different concentrations. The aroma descriptor, intensity value, and retention time were recorded by the panelists during analysis . If two v/v). The process was ceased when aromas could not be smelled. FD factor could be expressed as the ratio of the initial and final concentration of the flavor compound of juice. The compounds with FD higher than 1 were identified as key flavor compounds.Two types of dilution analysis were used to identify key flavor compounds, including headspace dilution analysis (HDA) for SPME and aroma extraction dilution analysis (AEDA) for SAFE, as described by Zhang et al. . For SPMThe compound identification was carried out by NIST 14.0 mass spectrum database, the retention index (RI), and odor properties. The key flavor compounds were confirmed further compared with the standard compounds. RI was calculated using Equation (1) and compared with the references.and Cn+1 .Y-axis represented the peak area ratio of analyte to the internal standard, and the X-axis represented the concentration of reference standards of the analyte [C0 is the concentration of the compound before reference standard being added, C1 is the detected concentration after reference standard being added, and C2 is the reference standard concentration being added [GC conditions were the same as mentioned above, and a selected ion monitor (SIM) was selected as the mass acquisition mode. Both extract methods were used to quantify different ions. Reference standards with a series of concentrations were prepared. The mixed reference standards (1 \u03bcL) were added to the sample gathered with internal standard 1 \u03bcL, 0.816 \u03bcg/\u03bcL). The extraction procedures were the same as the methods of SPME and SAFE as described above. Standard curves were established based on the peak area and concentration of each compound. The  analyte . In orde \u03bcg/\u03bcL. TiC is a compound concentration and iOT is the odor threshold of this compound. Compounds with OAV \u22651 were considered to contribute to the juice flavor [The equation to calculate OAV was as below: e flavor .Twelve panelists  were recruited from the Molecule Sensory Laboratory of Beijing Technology and Business University. Members of the sensory panel were trained for 2 months to familiarize the watermelon aroma characteristics. Sensory evaluation was strictly in accordance with To verify the obtained result of off-flavor compounds, the aroma recombination system was prepared and compared with the actual watermelon juice flavor . A modelMixture models were produced by omitting one kind of key off-flavor compounds from the aroma recombination system. The same sensory panels evaluated the flavor similarity between the omission and recombination models in a triangle test. Every sensory evaluation was conducted in triplicate .Analysis of variance was carried out to determine the significance at a 95% confidence interval using SAS 9.3 software . Partial least-squares regression (PLSR) was implemented using SIMCA-P 11.5 software . All experiments were performed in triplicate.In order to extract and analyze the flavor compounds of watermelon juice comprehensively, the extract methods of SPME (non-solvent extract) and SAFE (solvent extract), the chromatographic columns of DB-Wax (strong polar) and DB-5 (weak polar)  were applied in this study. Thus, the aroma-active compounds and key off-flavor compounds could be identified accurately and thoroughly. Fifty-seven compounds in FW and TW were extracted and identified . Five grAmong the 17 compounds identified in FW and TW, aldehydes were the most prevalent and potent aroma compounds in watermelon or its juice . Of the Eleven alcohols, six of which  were identified with FD >1 in both FW and TW. Hexanol, octanol, (E)-2-octenol, nonanol, -3,6-nonadienol, and -2,6-nonadienol could be formed due to reduction of the corresponding aldehydes. All identified compounds were previously reported for watermelon aroma, and presented fatty, fruity, floral, green, or cucumber-like smell ,23. AmonAmong the seven identified ketones, geranyl acetone was found with FD >1 in both FW and TW. They were reported in the melon fruits with different rootstocks, seedless watermelon, and watermelon juice treated by high-intensity pulsed electric fields ,14,23. GFive sulfides, diisopropyl disulfide, and dipropyl trisulfide were identified with FD >1 both in FW and TW. Few reports have described sulfides in watermelon or its juice, although these compounds exist in Jiashi muskmelon . These tAs shown in Thermal treatment promoted the formation and release of flavor compounds with higher FDs in TW. After thermal treatment, watermelon juice produced a strong unpleasant flavor. As shown in As shown in As shown in p < 0.05). In summary, these seven volatile compounds were preliminarily identified as the key off-flavor compounds in TW.The odor attributes of \u201cfatty\u201d and \u201ccooking flavor\u201d concentrated on the left side of the loading plot, which presented significant positive correlations with compounds: (E)-2-heptenal, diisopropyl disulfide, (E)-2-decenal, decanal, (E)-2-octenol, octanol, and hexanol  was calculated to guarantee the accuracy of the quantitative results . The staOAV was another index that contributed aroma compounds to the overall flavor . As showp < 0.05) being observed. This indicated that the identification and quantitation experiments were accurate, and that the aroma-active and key off-flavor compounds were precisely identified [According to the quantitative results of TW, the aroma recombination was carried out to verify the contribution of the seven key off-flavor compounds to the overall flavor of TW. As shown in entified ,43. Therp \u2264 0.001). This result revealed that octanol played a very important role in the overall flavor in TW. Among them, the absence of diisopropyl disulfide and (E)-2-decenal showed significant differences (p \u2264 0.05 and p \u2264 0.01), which agreed with the higher FDs and OAVs, respectively. Therefore, these two compounds also had a significant influence on the overall flavor of TW. The omission experiments: hexanol and (E)-2-octenol (OAV \u22641 but higher FDs) also showed significance differences (p \u2264 0.01 and p \u2264 0.05). However, no significant differences were observed when (E)-2-heptenal and decanal were omitted from the recombination in spite of their higher FDs and OAVs. Comprehensive consideration of significance difference of omission experiment, FD, and OAV, octanol , diisopropyl disulfide , and (E)-2-decenal  were identified as the most potent off-flavor compounds in TW.In order to further verify and rank the contribution level among the seven key off-flavor compounds, omission experiments were divided into seven groups . When ocThe above three compounds were previously reported as off-flavor compounds. Octanol contributes to the off-flavor of whey protein concentrate during storage of 45 \u00b0C for 15 weeks, and it changes the organoleptic properties of packaged food ,45. In bIn conclusion, seven key off-flavor compounds in TW were preliminarily identified by concentration variation, odor attributes, and PLSR analysis. Five aroma-active compounds and seven key off-flavor compounds were quantified by the standard curve method. They were further confirmed by both OAV and FD. The aroma recombination was employed to verify the contribution of the seven key off-flavor compounds to the overall aroma profile. In addition, the omission experiment from the recombination system was carried out to confirm the results. Octanol, diisopropyl disulfide, and (E)-2-decenal were identified as the most potent off-flavor compounds in TW."}
+{"text": "The UK has set itself the ambitious target of zero new HIV transmissions by 2030. HIV stigma is a significant barrier to achieving this target. Media reporting plays an important role in shaping social representations of HIV and of stigma. Between 2016 and 2018, the media in the UK reported on the Daryll Rowe case \u2013 the first criminal prosecution for intentional transmission of HIV in the UK. This article examines the way that UK newspapers reported this case, which may have exacerbated HIV stigma. Using Nexis, 178 UK newspaper articles were extracted and subjected to qualitative thematic analysis through a social constructionist lens. Informed by social representations theory, the analysis yielded three discursive themes: (1) Representing the perpetrator through HIV-focussed metaphors; (2) Constructing volitional ambiguity; and (3) Anchoring the lived experience of HIV to misery and death. UK newspapers constructed an \u2018evil vs victimhood\u2019 dichotomy in relation to Rowe and the men infected with HIV, respectively. This article argues that news coverage of the Rowe story constructs HIV in ways that are inconsistent with public health messaging. Reporting failed to note innovations in HIV treatment and prevention but instead disseminated stigmatising social representations of HIV. This is important because stigma impedes effective HIV prevention, engagement with HIV care and ultimately our ability to achieve the zero-infections target. Between 2012 and 2018, HIV incidence in gay, bisexual and other men who have sex with men in the UK decreased by 71% . The UK  The epidemic is relatively small in the UK, with an estimated 103,800 people living with the condition, which disproportionately affects gay, bisexual and other men who have sex with men  in December 2016. On 15 November 2017, Daryll Rowe became the first person in England and Wales to be convicted of causing GBM under the Offences Against the Person Act 1861 for deliberately infecting, or attempting to infect, male sexual partners with HIV. He was sentenced to life imprisonment and will serve at least 12\u2009years in prison before being considered for parole.There have been a handful of criminal convictions for the sexual transmission of HIV in England and Wales and some of these cases, including that of Rowe, have been the subject of legal research . Given iThere has, so far, only been one article examining media reporting of this case as compared to another case in Italy . The autThe media constitute a key source of societal information regarding science, medicine and health . In thisAnchoring refers to the process of making something unfamiliar understandable by linking it to something that we already know about. HIV may be linked to imagery of death and destruction, which in turn leads people to think of it as a life-limiting condition.Objectification is the process whereby unfamiliar and abstract objects are transformed into concrete and \u2018objective\u2019 common-sense realities \u2013 most notably through the use of metaphor \u2013 allowing us to map aspects of more familiar knowledge  onto more unfamiliar knowledge  . For insAs an unprecedented legal case about a poorly understand health condition, the Daryll Rowe case is anchored to, and objectified in terms of, existing constructs in the popular media so that it resonates among readers and can become a topic for thought and discussion. Social representations theory enables the analyst to understand the origins, development and repercussions of emerging representations in this forum.This study contributes to a vibrant tradition of research into social representations of infectious diseases from The newspaper media may set the tone for public understanding about, and engagement with, such issues by influencing and reflecting policy agendas . They ar\u00ae news database, we conducted a search of all national and regional/local newspapers in the UK and extracted all articles published between 26 February 2016 (the first article to be published on this case) and 30 September 2019 (the last article to be published). Using the \u2018Hide Duplicate Results\u2019 function on Nexis\u00ae, which excluded duplicate stories, this process yielded a total of 178 newspaper articles, all of which were relevant and therefore included in the analysis. We decided not to include articles published in the lesbian, gay, bisexual and transgender (LGBT) media, such as Pink News (in which 22 articles were published on the case), which tend to be aimed at, and consumed by, LGBT people. We were interested in the mainstream press and its role in shaping public understanding of HIV .Using the keyword \u2018Daryll Rowe\u2019 on the NexisThe Daily Record and Sunday Mail (a Scottish tabloid newspaper based in Glasgow) and The Herald (a Scottish broadsheet newspaper). These outlets included in the analysis reflect a wide range of political perspectives (both right- and left-wing politics); feature both national and local newspapers; and are available both online and in hardcopy format. The first articles to mention the case were published in We analysed the corpus using a social constructionist variant of qualitative thematic analysis, which has been described as \u2018a method for identifying, analysing and reporting patterns (themes) within data\u2019 : 78. Thi\u00ae. We read and re-read the articles to familiarise ourselves with the broader themes that we subsequently discussed analytically. First, initial observations were made which captured the essential qualities of each article, the units of meaning, and dominant rhetorical techniques. Second, we discussed our respective initial codes, which included inter alia general tone, particular forms of language, comparisons, categorisations and emerging patterns in the data. Third, the initial codes were collated into preliminary themes, which reflected the content of the analysis, and subsequently arranged into a coherent structure that reflected the overall thematic analysis.Both the headline and the main body of each article was subjected to thematic analysis. Images were not included in the analysis, as they are not available on NexisIn addition to describing dominant themes in the corpus, we identified linguistic elements , which performed the functions of anchoring and objectification. The superordinate themes can be considered social representations because they \u2018assume a configuration where concepts and images can coexist without any attempt at uniformity, where uncertainty as well as misunderstandings are tolerated, so that discussion can go on and thoughts circulate\u2019 : 233. InIn this section, the following three discursive themes are described: (1) \u2018Representing the perpetrator through HIV-focussed metaphors\u2019; (2) \u2018Constructing volitional ambiguity\u2019; and (3) \u2018Anchoring the lived experience of HIV to misery and death\u2019. and \u2018HIV Scot\u2019  with the elaboration that he had been \u2018charged\u2019, that he had \u2018set out to infect more partners\u2019, that he had \u2018drunk his own urine\u2019 as a means of attempting to treat his infection, and that he had been \u2018charged\u2019 with a crime.In the headlines of articles reporting the case, Rowe\u2019s name was seldom used and was instead replaced by a variety of epithets which focused mainly on HIV. In some cases, he was simply referred to as \u2018HIV man\u2019 The epithet \u2018HIV hairdresser\u2019 was frequently juxtaposed with judicial metaphors, such as \u2018Key events that brought HIV hairdresser to justice\u2019 and \u2018HIV hairdresser freed on bail\u2019. Similarly, several headlines focused explicitly on his criminal status, as an \u2018HIV suspect\u2019, \u2018HIV trial man \u2018HIV fugitive\u2019 or as a \u2018 HIV attacker\u2019. There was also some use of medicalised epithets, such as \u2018HIV carrier\u2019 which were juxtaposed with judicial metaphors such as \u2018gets life\u2019. Some articles referred to Rowe as an \u2018HIV spreader\u2019, and coupled this with the observation that he had been \u2018jailed for life\u2019, thereby establishing a connection between HIV transmission and life imprisonment (discussed in more detail below).In some cases, his occupation as a hairdresser was foregrounded invariably in conjunction with \u2018HIV\u2019, which was again used adjectivally: \u2018HIV hairdresser\u2019. \u2018brute\u2019 and \u2018fiend\u2019. The animalistic metaphor \u2018fiend\u2019 was used as the subject of the sentence \u2018A fiend who infected lovers with HIV\u2019 and that of \u2018brute\u2019 was qualified with \u2018guilty of infecting sex dates\u2019. Moreover, in some articles, the animalistic metaphor that Rowe \u2018preyed\u2019 on men was used to describe his actions. When used in relation to a human being, animalistic metaphors such as \u2018fiend\u2019 and \u2018brute\u2019 construct the individual as evil, devious and immoral. Indeed, there were several instances in which Rowe was described explicitly as a \u2018devious and deceptive predator\u2019, often through the discourse of key stakeholders in his case, such as the detective inspector and prosecutor involved in his case.In most cases, however, \u2018HIV\u2019 was combined with animalistic metaphors, such as \u2018predator\u2019, thereby constructing a relationship between (gay) sex and Rowe\u2019s behaviour. In the same article, this sexualising epithet was used alongside the notion that Rowe had a \u2018porn secret\u2019, that is, that he had featured in the gay adult porn films. There was an element of stigma appended to gay sexuality with one article referring to \u2018unprotected romps\u2019 between Rowe and the men whom he infected, and to Rowe as \u2018lining up trysts\u2019. This constructed the act of condomless sex as necessarily irresponsible and the use of geospatial social networking applications, such as Grindr, to seek sexual partners as clandestine. More generally, in attempting to explore the possible motives underpinning Rowe\u2019s actions, one article noted that Rowe \u2018came out as gay at the age of 15, in the same year that he experienced his first sexual encounter\u2019, suggesting gay sexuality and early sexual debut as possible explanations for his behaviour.\u2018HIV\u2019 was sometimes used in conjunction with \u2018sex fiend\u2019, was also common in the corpus, as well as \u2018Grindr monster\u2019 and \u2018Date app fiend\u2019. These epithets were often juxtaposed with other animalistic metaphors, such as \u2018Virus brute caged in UK\u2019, and with criminal metaphors, such as \u2018HIV fiend extra jail\u2019. In some cases, these labels were associated with the utterances of key figures in authority, such as Detective Inspector Andy Wolstenholme, who reportedly \u2018blasted the monster\u2019. Although the detective inspector did not actually use this word himself, its use with the detective inspector as the subject of the sentence served to distance the epithet from the journalist and to associate it with the detective inspector, thereby bestowing on the label a degree of legitimacy. In a further exploration of others\u2019 responses to Rowe, the headline of an article focusing on other prisoners\u2019 hostility towards Rowe after he was incarcerated reported \u2018HIV fiend hounded from jail; lags target hated predator\u2019. This article served to extend the animalistic metaphor of Rowe as a predator to that of other prisoners as now preying on him, thereby positioning him in a metaphorical field of the animal kingdom. Furthermore, the representation that convicted criminals \u2018hate\u2019, \u2018hound\u2019 and \u2018target\u2019 Rowe further legitimised use of stigmatising terms to describe him.Yet, HIV was not the only component of the descriptions used for Rowe \u2013 \u2018virus brute\u2019 \u2018HIV GHB charges\u2019, \u2018HIV sex charges\u2019, \u2018HIV offences\u2019 (for which Rowe was reportedly \u2018charged\u2019), \u2018HIV violence\u2019 (for which he was reportedly \u2018jailed\u2019) and \u2018HIV attacks\u2019. These might be referred to as \u2018compound crimes\u2019 which in turn serve to anchor HIV transmission to criminal activity  and to judicial consequences . Similarly, articles which focused on the judicial case that followed Rowe\u2019s actions used HIV-focused compounds, such as \u2018HIV court case\u2019 and \u2018HIV life sentence\u2019, thereby reiterating the anchoring of HIV to judicial consequences and entrenching negative connotations of the illness. Throughout the corpus, there was, unsurprisingly, a focus on Rowe\u2019s crime but, in most newspaper headlines, the crime was poorly defined and the real focus shifted to HIV, equally ill-defined.\u2018HIV\u2019 was also used as part of a number of verbal compounds to describe Rowe\u2019s actions. Judicial metaphors were used alongside \u2018HIV\u2019 and included \u2018HIV rampage\u2019,volition explicitly, there was a tendency for ambiguity and many of the headlines, in particular, left open the possibility that HIV transmission was non-volitional, that is, unintended.Most article headlines in the corpus were characterised by \u2018volitional ambiguity\u2019 in relation to the crime, that is, ambiguity about the degree to which Rowe intentionally attempted to infect his sexual partners with HIV. Rather than stating  or a \u2018man charged over HIV\u2019, without elaborating on the nature of his crime or focusing on the notion of intentional HIV transmission. Here, unlike the HIV compounds discussed above, the focus was squarely on the illness or infection, framing it as a criminal tool, even weapon. In another article, the epithet \u2018HIV infection accused\u2019 which constructed his crime as the transmission of HIV to others, without clarifying the circumstances under which HIV transmission had occurred or, crucially, the degree to which the act of transmission had been volitional. In several articles, it was noted that Rowe  was \u2018accused of giving lovers HIV\u2019 and that he \u2018gets second jail term for giving partners HIV\u2019. The verb \u2018to give\u2019 in relation to HIV transmission was neutral and could include non-volitional HIV transmission, but, in this context, it rather came to mean \u2018inflicting\u2019. Similarly, in other articles, it was observed, sometimes through the discourse of the prosecutor, that Rowe \u2018was HIV positive\u2019 and that his offences involved \u2018him sleeping with his alleged victims\u2019. This served to construct the act of having sex with others  when living with HIV as a criminal offence, rather than the intentional transmission of HIV to unsuspecting sexual partners.Article headlines were often very ambiguous, referring simply to a \u2018man held over HIV infection\u2019 In the absence of more information about his case, this contrast between action (\u2018infecting victims\u2019) and self-presentation (as genuine) suggested that HIV transmission is necessarily indicative of disingenuity towards \u2018victims\u2019, that is, unsuspecting individuals. The headline foregrounded the social representation that HIV transmission is volitional and the constructed intentionality in HIV transmission extended beyond the specific case of Rowe. Thus, HIV transmission was anchored to disingenuity, thereby suggesting a sinister motive underpinning the transmission of HIV to others.In some articles, a contrast was drawn between Rowe \u2018who infected victims with HIV\u2019 and others\u2019 perception of him as \u2018a genuine person\u2019.charged over HIV infection\u2019, that he \u2018pleads guilty to infecting victims\u2019, and that he had been \u2018jailed for life for infecting men with HIV\u2019. Moreover, there was reference to a \u2018manhunt for a suspect\u2019 which culminated in \u2018cops making an arrest\u2019. The anchoring of HIV transmission to judicial metaphors served to construct HIV transmission invariably as a criminal act and to establish a discursive connection between the act of transmitting HIV and judicial retribution.In most article headlines, the act of HIV transmission was anchored to judicial metaphors. For instance, the specific observation was often made that Rowe had been \u2018 \u2018people\u2019, and \u2018Grindr dates\u2019 as the indirect object. Moreover, there was sometimes no reference to those who acquired HIV as a result of Rowe\u2019s actions \u2013 one headline reported, \u2018man jailed for spreading HIV\u2019.The article headlines referred to the indirect object, that is, the individuals who acquired HIV as a result of Rowe\u2019s actions, in a variety of ways. In some articles, they were referred to as \u2018victims\u2019, which suggested, but did not confirm, volitional HIV transmission. The reference to these individuals anchored HIV acquisition to victimhood, thereby suggesting that the perpetrator is invariably the person who is living with HIV and transmits it. Moreover, as discussed in the next subsection, the emphasis on victimhood constructed the experience of living with HIV in adverse terms. In other articles, the anchoring of HIV transmission to judicial metaphors appeared with \u2018men\u2019, which can be contrasted with the HIV-focussed epithets used to describe Rowe. Several articles used the judicial metaphor that the men were \u2018living with a life sentence\u2019, which referred to their HIV infection. Furthermore, in describing the men\u2019s experiences in the aftermath of their encounters with Rowe, the media focused on both the physical and psychological experience of living with HIV, both of which were represented negatively as being conducive to significant psychological distress. Articles referred to \u2018having to live with the \u201cdevastating consequences\u201d of contracting the virus\u2019, which constructed HIV invariably as being distressing.Although the majority of media reporting focused on Rowe, there was consistent reference to the health and wellbeing of the men who acquired HIV as a result of Rowe\u2019s actions. The men\u2019s reported experiences (mainly through direct quotes from the men themselves) were often represented as evidence of that Rowe was an evil person. A consistent tendency across the corpus was the emphasis of the men\u2019s victimhood and, accordingly, they were referred to as \u2018HIV victims\u2019, or a \u2018deadly sexual disease\u2019 which anchored HIV to imagery of death. This is consistent with dominant social representations of HIV in the pre-treatment era but inconsistent with modern developments in medical science which ensure a good prognosis and normal life expectancy if one is diagnosed and treated early. Furthermore, a quote from one of the men who acquired HIV was reproduced in order to illustrate the lived experience of HIV: \u2018I felt like I had been left with a poison inside me\u2019, which referred to his infection with HIV. Alongside anchoring to death imagery, use of the poison metaphor objectified HIV in terms of a life-limiting condition.Several articles referred to HIV as \u2018the deadly virus\u2019 His experience of GBH  was constructed as more serious, enduring and indeed life-limiting than other forms of GBH which reportedly might be more easily tolerated. Conversely, his acquisition of HIV was anchored to death imagery, which was qualified as \u2018horrible and painful\u2019. In other words, HIV was positioned as being even worse than death due to the suffering that it could cause before death. Similarly, another individual was quoted as saying \u2018it [HIV] was a lifelong sentence, which would eventually kill me off\u2019, further anchoring HIV to death imagery.Similarly, another man who acquired HIV contrasted his lived experience of HIV with that of victims of other forms of GBH: \u2018While for some cases of GBH it might be possible for victims to put the acts behind them, unfortunately this will never be the case for me.\u2004.\u2004. There is a virus inside me which will give me a horrible and painful death unless I take pills for the rest of my life\u2019.per se. A quote from one of men  constructed HIV infection as a rupture in his sense of continuity, that is, between past, present and future: \u2018the old me is no longer. The new me is constantly sad, thinking about how my life has changed\u2019. The construction of discontinuity is consistent with the aforementioned anchoring to death imagery since the \u2018old me\u2019 was said to be defunct and to be replaced by an undesirable \u2018me\u2019.Most articles focusing on the lived experience of HIV also emphasised the psychological trauma experienced by the men who had acquired HIV and constructed their overall experience of living with the condition as psychologically distressing. The psychological distress was attributed not to the specific circumstances of their acquisition of HIV  but rather to the experience of living with the condition  This quote foregrounded the social representation that individuals living with HIV are treated as social pariahs, which is questionable in the advent of decreasing HIV stigma and \u2018U=U\u2019. Furthermore, this quote served to construct the experience of living with HIV as socially isolating and, thus, the destruction metaphor \u2018ruined my life\u2019 was used to describe his experience with the condition. Similarly, another individual noted that \u2018Rowe claimed he was clean so we didn\u2019t use condoms. Then I caught HIV. He has ruined my life\u2019. In this quote, first, the individual used the term \u2018clean\u2019 to refer to being HIV-negative which implied that those living with HIV are, conversely, dirty. This clearly accentuated the stigma surrounding HIV. Second, Rowe was said to have transmitted HIV to him and, thus, \u2018ruined\u2019 his life, which anchored HIV to destruction beyond repair.An article included a quote from another individual, which constructed HIV as destructive to his life because of the stigma associated with his condition: \u2018I just felt like my life had been ruined, and that nobody would want to know me. There is still a huge stigma to HIV and I thought that no men would want to come near me\u2019. Another was quoted as referring to HIV as having a \u2018shattering effect\u2019 on his life. The experience of being diagnosed with HIV was constructed as being worse than death itself \u2013 indeed, an individual was quoted as saying \u2018I would rather he had murdered me than left me to live my life like this\u2019. This suggested that the experience of living with HIV rendered life unbearable, thereby representing death as preferable to living with HIV. Similarly, some of the men were said to be contemplating suicide: \u2018many told how they had considered suicide, having suffered physical and psychological damage, needing to take daily medication\u2019. One of the men was quoted as saying, \u2018I think about committing suicide most of the time. Daryll has destroyed my life. I lost a lot of weight and felt very ill for a long time. I can\u2019t see myself having a relationship ever again\u2019, while another reportedly said, \u2018I think about committing suicide most of the time. Daryll has destroyed my life.\u2019 The consistent anchoring of HIV to death and destruction imagery in the corpus contributed to the social representation that HIV is worse than death.In view of both the physical and psychological trauma to which HIV was anchored, articles also represented a proclivity for the men who acquired HIV to contemplate suicide. A victim was quoted as saying that his diagnosis felt like being \u2018hit by a bus\u2019 and that \u2018you feel like your life is over. You feel like you should go to a high place and jump\u2019. The article proceeded to invite readers to \u2018Imagine the horror these poor men lived through in court, only to have a smart QC claim their lives were going to carry on as before, belittling their situation\u2019.Most articles in the corpus focused on representing Rowe as malevolent through the use of negative epithets. This tendency appeared to be the discursive priority which superseded the discussion of innovations in medical science that have transformed HIV from a life-limiting into a life-changing illness. All but one of the articles in the corpus failed to mention innovations in medical science \u2013 the one article that did directly challenged the legitimacy of outlining these innovations in the context of Rowe\u2019s case: \u2018.\u2004.\u2004.the arguments advanced by his [Rowe\u2019s] defence team give cause for concern. Felicity Gerry QC claimed that HIV was \u201cnot a terminal illness\u201d, stating: \u201cThose that live with HIV have good and high life expectancies. There is a need for therapy and not incarceration.\u201d\u2019Thus, innovations in HIV medicine were constructed not only as irrelevant in this context but also as insult to the men who contracted HIV as a result of Rowe\u2019s actions. The focus was clearly on representing Rowe as evil, his compound crimes as malevolent and the men who acquired HIV as victims. The social representation that Rowe is evil overshadowed broader public health messages about HIV prevention and the reality of living with HIV in the era of antiretroviral therapy. Within this discursive context, the social representation that HIV is worse than death was able to thrive.In our study, we show that there is an overarching focus on communicating the evil of Daryll Rowe, the perpetrator of the crime, on the one hand, and on emphasising the victimhood and plight of those who acquired HIV as a result of his actions, on the other hand. There were three main social representations which were constructed and drawn upon in order to substantiate this \u2018evil vs victimhood\u2019 dichotomy in media reporting of the case: first, there was a social representation that HIV is a social, moral and legal flaw; second, HIV was socially represented as a criminal tool; and, third, the corpus constructed a social representation that HIV is a psychologically, emotionally and physically destructive disease. These three discursive themes demonstrated the social and linguistic mechanisms  which were used in order to construct and legitimise the three social representations. These included representing Daryll Rowe using negative epithets with HIV-focussed metaphors; ambiguity in the level of volition underlying his actions; and anchoring the lived experience of HIV to misery and death.There has been a focus on reducing HIV stigma in the UK with some success. The Indeed, the use of HIV-related metaphors and compounds to construct negative epithets, which replaced Daryll Rowe\u2019s name in article headlines, appended animalistic characteristics to Rowe, dehumanised him and constructed his brutality . Yet, HIHIV (rather than deliberate transmission) as a criminal tool, which was further bolstered by the animalistic, judicial and war metaphors that were frequently used in conjunction with HIV in reporting. Yet, it is noteworthy that most infections occur because people are actually unaware of their HIV status and, thus, transmit HIV when they are untreated and have a high viral load  remains a key comorbidity of HIV infection .In his study, An analysis of media reporting of HIV  found thHaving said this, one has to acknowledge that journalists find themselves grappling with a dilemma, namely writing sensationalised and headline-grabbing stories that, intentionally or not, amplify stigma, or counterbalancing such reporting with messages that attenuate this trend which runs counter to public health messages. This is not easy, but we suggest that journalists should at least be made aware of the dilemma that they face in HIV reporting.Pink News. It would be advantageous to examine how the case was reported on these media platforms, which tend to take a more critical view of HIV stigma, because HIV stigma also remains a significant challenge in the gay community (There are some limitations, which should be addressed in future research. First, our study focused on media reporting in the mainstream national and regional press, though it is acknowledged that the case was also covered in the LGBT press, such as in ommunity . Second,This article argues that news coverage of the Rowe story constructed HIV in ways that are inconsistent with public health messaging. Reporting reinforced dominant social representations of HIV in the pre-treatment era but did not acknowledge, or explain, modern developments in HIV science which ensure a good prognosis and normal life expectancy if one is diagnosed and treated early. None of the articles noted the advent of antiretroviral therapy, pre-exposure prophylaxis or \u2018U=U\u2019, all of which play a key role in reducing HIV stigma and in promoting effective HIV prevention and wellbeing. Media reporting of the Rowe case understandably focused on the events, the motivations of Rowe, and the voices of those he infected. However, social stigma is a recurring motif which may have significant implications for how people think, talk and behave in relation to HIV and, consequently, for our ambition to prevent new HIV transmissions by 2030."}
+{"text": "What are the self-reported practices of frontline dispensary staff who interact with customers purchasing cannabis for medical purposes?In this survey study of responses from 434 staff from 351 unique dispensaries, recommendations were most often based on experiences of the respondent, other customers, and other staff recommendations. Higher medicalization scores were associated with physician or clinician input as a basis for recommendation.The results of this survey study suggest a need for clinicians to be aware of dispensary staff practices and to engage in discussions about the benefits and harms of cannabis use with their patients. Over the last decade, cannabis has become more accessible through the proliferation of dispensaries in states that have legalized its use. Most patients using cannabis for medical purposes report getting advice from dispensaries, yet there has been little exploration of frontline dispensary staff practices.To describe the practices of frontline dispensary workers who interact with customers purchasing cannabis for medical purposes and assess whether dispensary practices are associated with medicalization of state cannabis laws (degree to which they resemble regulation of prescription or over-the-counter drugs) and statewide adult use.This nationwide cross-sectional survey study was conducted from February 13, 2020, to October 2, 2020, using an online survey tool. Potential respondents were eligible if they reported working in a dispensary that sells tetrahydrocannabinol-containing products and interacting with customers about cannabis purchases.Participant responses to questions about formulating customer recommendations and talking to customers about risks.The 434 survey responses from 351 unique dispensaries were most often completed by individuals who identified as budtenders (40%), managers (32%), and pharmacists (13%). Most respondents reported basing customer recommendations on the customer\u2019s medical condition (74%), the experiences of other customers (70%), the customer\u2019s prior experience with cannabis (67%), and the respondent\u2019s personal experience (63%); fewer respondents relied on clinician input (40%), cost (45%), or inventory (12%). Most respondents routinely advised customers about safe storage and common adverse effects, but few counseled customers about cannabis use disorder, withdrawal, motor vehicle collision risk, or psychotic reactions. A higher state medicalization score was significantly associated with using employer training  and physician or clinician input  as a basis for recommendation. Medicalization score was not associated with counseling about cannabis risks.This survey study provides insight into how frontline dispensary staff base cannabis recommendations and counsel about risks. The findings may have utility for clinicians to counsel patients who purchase cannabis, customers who want to be prepared for a dispensary visit, and policy makers whose decisions affect cannabis laws. This survey study describes the practices of frontline dispensary staff who interact with customers purchasing cannabis for medical purposes and assesses whether dispensary practices vary according to state cannabis law medicalization. In this context, clinicians may encounter increasing numbers of patients with questions about using cannabis for medical purposes. It is not known what proportion of cannabis used is obtained from dispensaries; a 2020 study1 suggests that individuals obtain cannabis from dispensaries as well as from alternative sources . However, dispensary sales are increasing, underscoring their importance in the US cannabis market.2 Moreover, most patients who use cannabis for medical purposes report receiving specific advice about cannabis formulations and use patterns directly from dispensaries rather than from clinicians.3Cannabis access has substantially increased over the past decade in the US,4 describe the \u201cvoid in clinician counseling of cannabis use,\u201d which they propose is due to federal laws that prohibit physicians from prescribing cannabis and lack of evidence-based recommendations about benefits and harms. The health care clinician\u2019s role is relegated to assessing whether the patient has a state-sanctioned qualifying condition, resulting in a phenomenon the authors describe as \u201ccannabis dispensary workers as proxy clinicians.\u201d There has been little exploration of frontline dispensary staff practices.Understanding how dispensary staff interact with consumers is key to knowing how consumers make cannabis purchasing decisions. Calcaterra et al5 of all states with legal medical cannabis identified substantial variability in requirements across several domains, including manufacturing or testing, product labeling, and types of products permissible for sale, as well as limits on the supply or dose that can be dispensed. The degree to which these regulations resemble regulation of prescription and over-the-counter drugs has been termed medicalization.5One factor that complicates understanding dispensary practices is state law variability. A recent studyThis study describes the practices of frontline dispensary staff who interact with customers purchasing cannabis for medical purposes. We assessed whether dispensary practices varied according to state cannabis law medicalization and whether the state allowed adult use. We hypothesized that respondents from states with more medicalized programs vs less medicalized programs would rely on traditional sources of information  and would talk with customers more about risks.10 and finding no detailed survey querying staff practices, our team developed a survey specifically to query dispensary staff practices. The eMethods in the AAPOR) reporting guideline for survey studies. This study was classified as exempt from review and informed patient consent by the University of Pittsburgh Institutional Review Board because of the study type.After reviewing relevant literature11 but since dispensaries open and close frequently, the true denominator of dispensaries cannot be definitively determined.To identify US dispensaries, we purchased a list of 4715 dispensaries across 34 states from a marketing company. We identified additional dispensaries through internet searches of state databases and websites such as Leafly.com and WeedMaps.com. This is an accepted method of locating dispensaries that are operating at a given time,We initially planned to recruit respondents with telephone calls to a representative random sample of all identified US dispensaries. We generated random samples of 1000 dispensaries stratified by state, and research staff called these dispensaries using a standard script. The script involved asking for a manager who could send the survey to frontline staff. We found that dispensary managers were often difficult to reach, and managers and higher-level administrators expressed concern about the potentially proprietary nature of the information we wished to gather  and our intentions regarding speaking directly to staff . This recruitment method led to few completed surveys.To increase responses, we mailed hard copies of the survey to all identified US dispensaries including instructions for online completion, and used a snowball sampling approach by sending an electronic link to leaders at 2 large national dispensary chains and a cannabis retailers association. Although not specifically encouraged, the electronic link could be forwarded so that survey responses were not limited to the dispensaries we directly recruited. To calculate the response rate for telephone calls and mailers, responses were attributed to the most recent method of contact.Participants were eligible to complete the survey if they reported working in a dispensary and interacting with customers about cannabis product purchases. To be considered a cannabis dispensary, the dispensary had to sell tetrahydrocannabinol (THC)\u2013containing products, whether for medical or adult-use purposes or both; we excluded stores that sold only cannabidiol products. We excluded employees under the age of 18 years and those who had been in their current position for less than 3 months.All surveys were delivered online via Qualtrics. The survey was developed and then piloted from May 2019 to January 2020. It opened for completion by respondents on February 13, 2020, and closed on October 2, 2020. Respondents were given the choice of receiving a $10 payment card immediately on completion of the survey or the option of being randomly selected to receive a $250 payment card at the end of the study.5 This score includes 7 domain scores  and a summary score for each state that had enacted medical cannabis laws as of July 2019. Herein, we used the summary scores, which range from 23  to 86 . The statewide adult use variable was whether the state had legal adult use as of July 2019.A state cannabis medicalization score was developed based on a review of cannabis laws.Continuous variables are presented as mean (SD); categorical variables are presented as frequencies and percentages. Multivariable regression analyses were used to estimate associations between state regulations and the 2 practices of interest: basis of recommendations and talking to customers about risks. A separate regression was performed with each basis of recommendation and risk as the outcome. For each regression, the 2 independent variables of interest were state medicalization score  and statewide adult use (dichotomous). All models included the following prespecified covariates: age, role , years working in the cannabis industry, receipt of sales commission, and level of education.P\u2009<\u2009.05.Logistic regression analyses were conducted for the basis-of-recommendation outcome variables; results are reported as odds ratios (ORs) with 95% CIs. The talking-to-customers-about-risk outcome variables were modeled using ordinary least-squares regression; results are presented as regression coefficients and standard errors. All statistical analyses were performed using R version 3.6.0 . Statistical significance was set at 2-sided 11 or whose website did not confirm sales of THC-containing products.Our survey was mailed and emailed widely and could have been forwarded to unanticipated respondents, and came with a small financial incentive. To ensure that our analyses included only responses that could be legitimate, the primary analytic set included surveys that were at least 95% complete, from states in which sales of THC-containing products are legal, and from respondents not affiliated with major chain pharmacies or grocery stores where THC-containing products are not sold. We conducted a sensitivity analysis in which we removed dispensaries that were not on our list to contact that did not have a website Of 1988 dispensaries who received at least 1 telephone call attempt, 127 (6%) returned at least 1 completed survey. Of the 4733 identified US dispensaries who received a mailer, 352 (7.4%) returned at least 1 completed survey. We received 735 total responses, of which 222 were more than 95% complete, 38 were from states in which sales of THC-containing products are not legal, and 41 were from chain pharmacies or grocery stores in which cannabis is not sold, leaving 434 eligible for the primary analysis (from 351 unique dispensaries). These 351 responding dispensaries were from states with mean (SD) medicalization scores of 46 (5) vs 40 (10) from dispensaries that did not respond, and were less often from adult use states (42%) than dispensaries that did not respond (50%). Of the 434 surveys eligible for the primary analysis, 43 did not have a website confirming THC-containing product sales, leaving 391 eligible for the sensitivity analysis (from 308 unique dispensaries). The results of the sensitivity analyses were similar to the primary analyses and are presented as The largest number of surveys were from New York, Oregon, California, and Florida  and physician or clinician input  as a basis for recommendation, and was negatively associated with using product appearance , the respondent\u2019s personal experience , or what needs to get moved out of inventory  as a basis for customer recommendations. Statewide adult use was associated with using trade literature , app or website , experience of friends or colleagues , product appearance  and product smell  as a basis for customer recommendations.Regression analyses were conducted to assess the association between state medicalization score  and statewide adult use (yes) and respondent\u2019s use of a given basis for recommendations to customers . A higheP\u2009=\u2009.03). Otherwise state medicalization score and adult use were not associated with counseling about cannabis risks.In this national survey study, most dispensary staff had worked in the cannabis industry for 1 or more years, were college-educated, and many used cannabis for medical or adult-use purposes. Staff often relied on personal and coworker experience to make recommendations. While most staff reported routinely counseling customers about safe storage of cannabis and routine cannabis adverse effects such as sleepiness, few reported routinely counseling customers about cannabis-related risks such as psychosis, motor vehicle collisions, cannabis withdrawal syndrome, or cannabis use disorder.State medicalization and adult use were associated with how respondents based their recommendations. Being in a state with a higher medicalization score was associated with an increased likelihood of using employer training and physician or clinician input as a basis for clinical recommendations. This finding may indicate that medicalization is associated with an environment where physician or clinician input is more likely to be incorporated. Additionally, respondents who lived in states with legalized adult and medical use were more likely to endorse using personal experience and cannabis product smell or appearance as a basis for recommendations, perhaps indicating more product familiarity. However, state medicalization and adult use were generally not associated with counseling about cannabis-related risks. Although cannabis risks have been well-characterized, our findings suggest that state regulations have not been associated with dispensaries where such risks are emphasized.12 To our knowledge, no current research clearly outlines the balance of cannabis benefits and harms. Despite customers\u2019 potential reliance on dispensaries for health-related information about cannabis,4 it may not be reasonable to expect dispensaries, which are retail and not medical establishments, to bear primary responsibility for such counseling, much as alcohol retailers may not provide counseling about alcohol-related harms.It could be expected that dispensary workers do not routinely counsel customers about risks. A survey of the US general population found that less than half of respondents who had reported any past year cannabis use were concerned about risks such as cannabis use disorder, and that perceived risk of regular cannabis use has decreased over time.13 including reviewing cannabis-related harms, and to counsel patients about potential ways to mitigate these harms, such as previously-published guidance for low-risk cannabis use.14Clinicians may not be aware of dispensary staff practices, and engagement in discussions about the benefits and harms of cannabis use with their patients is warranted. For example, clinicians might alert patients that dispensary staff purchasing recommendations may be based on nonprofessional anecdotal experience and they should not expect counseling about harms. This approach could be an opportunity for the health care clinician to have evidence-based discussions with their patients about cannabis5 has shown, there is substantial variability in states\u2019 degree of medicalization. Therefore, we suggest that dispensary environments are highly variable and cannot be assumed to be medical environments. Certifying clinicians, particularly in less medicalized states, should be aware that the decision-making that occurs at a dispensary is often different from that which occurs in a medical environment such as a pharmacy.Although a higher degree of medicalization was associated with using health care clinician input and information from training, legal statewide adult use was associated with using personal experiences of friends or colleagues. Additionally, as research15 and the uncertainties in the evidence base, leading to a low comfort level related to recommending medical cannabis.8There is likely a gap between the way cannabis is perceived by dispensary staff and the way it is perceived by clinicians. Our findings suggest that dispensary staff are comfortable giving advice from an experiential standpoint. Conversely, clinicians may view cannabis through a traditional pharmacotherapeutic lens and be troubled at the lack of standardized dosing, regulatory oversight,6 reported results from a smaller sample of dispensary workers in 2 cities but focused on comparing characteristics and practice among workers who had and had not received training and on their online behaviors. The procedural revisions we made to our initial recruitment approach could help advance knowledge in the field about dispensary staff research recruitment methods. We attempted to call a large number of randomly selected dispensaries from a comprehensive list. However, dispensary lists may be obsolete due to dispensaries opening and closing, such that a denominator for response rates cannot be determined. Given the importance of research in the industry, we recommend that metrics assess the use and success of a best possible approach. Such metrics could include reach of a survey across states in which cannabis is legal and the absolute number of responses. In this case, we had hundreds of responses from most states in which cannabis is legal, suggesting that this approach is viable for addressing frontline dispensary practices.This study has strengths. To our knowledge, this is the first national study to examine self-reported dispensary staff practices. A prior studyThis study also has limitations. Responses are based on self-report; actual staff practices are unknown. Additionally, the degree to which the respondents are representative of dispensary workers nationally, or the responses are representative of dispensary practices nationally, is not known. Respondents may be fundamentally different from nonrespondents in unmeasured ways that could confound findings or limit generalizability. Our sample size is fairly modest, limiting statistical power to detect small effects. Finally, the field is dynamic: dispensaries open and close and laws and policies change.This survey study provides insight into frontline dispensary staff cannabis recommendations and counsel about risks. Our findings may have utility for clinicians to counsel patients who purchase cannabis from dispensaries, customers who want to be prepared for a dispensary visit, and policy makers whose decisions affect state cannabis laws."}
+{"text": "To face the rapidly growing world human population, an increase in agricultural productivity and production is necessary to overcome the enhanced food demand. This can be achieved either by increasing the cultivated area or by deploying improved crop plants and yield using next-generation plant breeding. The application of advanced technologies in plants has accelerated the generation of multi-omic data at various levels, such as the genome, proteome, transcriptome, epigenome, and metabolome levels. To take full advantage of this, integrative approaches using mathematical or relational models are needed to sequentially or parallelly combine the available multi-omics data to understand molecular interactions and physiological mechanisms at an organism-wide level ,2. In otRecent improvements in high-throughput genotyping methods have fueled the development of analytical methods for QTL identification. Macko-Podg\u00f3rni et al.  provide Advances in genomics also illustrate the high complexity of quantitative host\u2013pathogen interactions. Thus, in the current issue, Miedaner et al.  review tPodwyszy\u0144ska et al.  have estFurthermore, Kibe et al.  have conConsidering that extrinsic and intrinsic sources of stress can damage DNA, plants have developed highly conserved DNA damage\u2013response pathways to protect them from this harmful effect. Jaskowiak et al.  analyzed\u2013phenotype relationship prediction with GS and ML is becoming a powerful approach in plant genetic and breeding.G\u00f3ralska et al.  successfTaken together, it is not only the sequence of plant DNA that matters: how do some genes get activated, and why are others silenced? How can genomics facilitate the study of complex traits in plant breeding? In this regard, Salgotra and Stewart  review tFurthermore, RNA sequencing has emerged as a powerful tool for analyzing transcriptomes to identify genes that show differential expression between unstressed and various stress conditions. However, the transcripts obtained from RNA-Seq require reference genome or transcriptome sequences for read-mapping and annotation. In this context, Schaarschmidt et al.  used PacThe advances in next-generation genome sequencing and the rapid development of cost-effective and easy-to-use genome engineering editing methods have facilitated researchers\u2019 use of these tools for the functional characterization of many genes that are useful for crop improvement. In this regard, genome-editing tools such as CRISPR/Cas9, which can be exploited to explore the genetic resources for improving the various economic traits of crop plants in terms of stress tolerance and nutritional quality, are reviewed by Salava et al. . This reFinally, Kloc et al.  show thaDue to the current demand for nutritional security, the obtained data can be used as a resource to address the need to generate resilience to climate change in crop plants by targeting several traits of interest ,11. TherWe wish to thank all contributors to this Special Issue and hope that it will raise interest in and understanding of how genomics and related technologies can drive the next generation of plant breeding. Nevertheless, the current Special Issue can only cover a small part of our scope, and since the subject is of the utmost importance, we are proud to announce the opening of the second part of this Special Issue, \u201cFunctional Genomics for Plant Breeding 2.0\u201d and invite our readers to follow and contribute to it."}
+{"text": "Like arboviruses, chronic neurodegenerative diseases, like Alzheimer\u2019s and Parkinson\u2019s disease, are found wherever there are humans. Significant differences in baseline gene and protein expression have been determined between human-induced pluripotent stem cell lines derived from non-Parkinson\u2019s disease individuals and from individuals with Parkinson\u2019s disease. It was hypothesized that these inherent differences could impact cerebral organoid responses to viral infection. (2) Methods: In this study, cerebral organoids from a non-Parkinson\u2019s and Parkinson\u2019s patient were infected with Chikungunya virus and observed for two weeks. (3) Results: Parkinson\u2019s organoids lost mass and exhibited a differential antiviral response different from non-Parkinson\u2019s organoids. Neurotransmission data from both infected non-Parkinson\u2019s and Parkinson\u2019s organoids had dysregulation of IL-1, IL-10, and IL-6. These cytokines are associated with mood and could be contributing to persistent depression seen in patients following CHIKV infection. Both organoid types had increased expression of CXCL10, which is linked to demyelination. (4) Conclusions: The differential antiviral response of Parkinson\u2019s organoids compared with non-Parkinson\u2019s organoids highlights the need for more research in neurotropic infections in a neurologically compromised host. There are over 100 medically relevant arboviruses recognized, and anyone who experiences an insect bite is at risk for exposure. Medically significant arboviruses are commonly found in the Togaviridae, Bunyaviridae, and Flaviviridae families. These families contain viruses that cause encephalitis or hemorrhagic fever, and vaccines or treatments are not available for most infections. Death rates are roughly 20,000\u201350,000 per year for any given arbovirus ,4,5. OftInfected individuals present with a spectrum of disease ranging from subclinical to death. The role of chronic diseases on intrinsic and innate immune defense is emerging as a significant player in a patient\u2019s ability to respond to viral infections ,13,14,15Viral parkinsonism has been documented for a variety of human pathogens though there are few studies that evaluate the effects of viral infection on degenerative neurological diseases . Post-viHow viruses cause parkinsonism is not known. Animal models of neurological infections do not translate to nor mimic changes in the human cerebral cortex documented in postmortem reports and imaging studies. Evaluation of central spinal fluid in human and rodent studies indicate that an inappropriate neuroimmune response is responsible . This beThe use of human-induced pluripotent stem cells (hiPSC) in disease research is increasing not only because relevant data can be generated but because these cells can be differentiated systems that directly translate to a human model. Research on different hiPSCs and organoids has shown that products derived from patients with a disease state exhibit different morphology and gene expression compared with products derived from a normal patient ,29,30,31Since significant differences in gene and protein expression exist between hiPSC lines derived from individuals with and without PD, we hypothesized that these differences could impact response to viral infection . This st2) to the 12th day post infection (p.i.) (6.5338 mm2) (p = 0.2855) (2 (p = 0.0149), while all days of the experiment, PD and non-PD organoids did not differ in size (p = 0.0792\u20130.492) (The size of non-PD organoids did not significantly differ between the day of inoculation (6.2454 mm 0.2855) A. Over t2\u20130.492) B,C. Ther2\u20130.492) C.p = 0.0347) (p = 5.531 \u00d7 10\u22127 (non-PD), p = 2.046 \u00d7 10\u22126(PD)) (p = 0.0002) (p = 0.00257 (PD), p = 0.02076 (non-PD)) (p = 0.1213) (Immunofluorescence for morphology markers showed that both PD and non-PD organoids expressed sex-determining region Y-box 2 (SOX2), beta-III tubulin (Tuj1), neurofilament, and glial fibrillary acidic protein (GFAP). SOX2 is expressed in proliferating neural progenitors, and Tuj1 is a neuron-specific \u03b2-Tubulin. Both markers had increased fluorescence on the outer margins of the organoids, indicating growth of new neurons in both infected and control organoids A. GFAP i 0.0347) A,B. When0\u22126(PD)) A,B). Cas0\u22126(PD)) A,B. Sign 0.0002) A,B. PD onon-PD)) A,B. When 0.1213) A,B.p = 2.883 \u00d7 10\u22127)  A. The vi \u00d7 10\u22127) B. RT-PCR \u00d7 10\u22127) C.\u2206\u2206Ct comparison of Parkinson\u2019s and Non-PD organoids with a non-infected non-PD control showed unique expression patterns for each organoid type. Of 208 genes with significant changes in expression, both organoid types had similar patterns of expression  for 143 targets. A total of 65 genes displayed opposite patterns of differential expression for both organoid types.\u2206\u2206Ct comparison of PD and non-PD organoids with their non-infected control showed that global expression of neurotransmitters was down-regulated in PD organoids in response to CHIKV . The datSince endogenous differences in expression exist for PD and non-PD cells ,38,39, wOverall, markers associated with cholinergic neurotransmission displayed decreased expression in non-PD organoids, while PD organoids tended to have no expression or increased expression of the same targets . Of noteFourteen GABA receptors displayed significant differential expression for non-PD organoids, and 13 receptors were differentially expressed in PD organoids . Twelve Four glycine receptors  showed increased expression for both PD organoids when compared with the non-PD non-infected control. Reduced expression was observed in non-PD organoids both in relation to housekeeping gene and in relation to PD organoids . Both orSix glutamate receptors were evaluated, and PD organoids exhibited decreased expression of all targets over non-PD organoids when comparing their \u2206\u2206Ct values to their respective non-infected controls . Of noteEight genes associated with serotonin neurotransmission were examined. Here, PD organoids displayed increased expression of HTR2A, HTR3A, and HTR3B and decreased expression of HTR7, MAOA, and TPH1 . In non-Thirty-six targets representing a spectrum of transporters involved in neurotransmission were examined. \u2206\u2206Ct values showed that non-PD organoids exhibited decreased expression of all targets except SLC6A16, SLC32A1, and SLC6A7 . \u2206\u2206Ct vap = 0.00081, p = 0.000011)  (p = 2.976 \u00d7 10\u22125) but similar fluorescence with the mock-infected control (p = 0.17156)  (Immunofluorescence for neurotransmission markers for glutamate receptors NMDA1 and NMDAR2c indicated that PD organoids had stronger fluorescence of both markers compared to non-PD organoids (.000011) A,B. STX1 \u00d7 10\u22129) A,B. Non-0.17156) A,B. PD o \u00d7 10\u22125) A,B.Both PD and non-PD organoids were evaluated for immune response to CHIKV infection. Markers included surface receptors, stress response, oxidoreductases, cytokines including multiple chemokine receptors, and markers for cell lysis. Overall, PD organoids exhibited increased expression of all markers evaluated on the array when compared with their non-infected control A. \u2206\u2206Ct aMarkers for stress response exhibited a similar pattern of expression for both organoids types though non-PD organoids were down-regulated \u22123.2 logs for AGTR2, while PD organoids were up-regulated 4.99 logs . FurtherEight chemokine receptors were evaluated, and here, too, both organoid types had a similar expression pattern of up-regulation B. Of notTwenty-three surface receptors comprising a variety of interleukins, tumor necrosis factors, and chemokine ligands were evaluated. CCL2, CCL3, IL-6, IL-10, and TBX21 were significantly up-regulated in expression in PD organoids but down-regulated in non-PD organoids . IL-1A, p = 2.115 \u00d7 10\u22126, p = 9.004 \u00d7 10\u22125)   (p = 0.00822) but significantly increased in PD organoids (p = 2.376 \u00d7 10\u22127)  A,B. SELE \u00d7 10\u22126) A,B. CYP4 \u00d7 10\u22126) . ICAM fl \u00d7 10\u22127) .p = 0.3789), while PD organoids had increased expression of Iba1 (p = 0.0004) (p = 0.002), GBA (p = 0.0014), and CRYM (p = 0.0002) (p = 0.0281) (p = 0.019)  . CRYM, T 0.0002) . DRD1 wa 0.0281) . CCR5, I= 0.019) . PD orga= 0.019) . For the= 0.019) .Research of viral encephalitis and other viral infections of the CNS are crippled by necessary ethical restraints. This field relies on autopsy findings, which are then typically applied to rodent models. Rodents do not present with symptoms of CNS pathologies unless they are genetically modified to be immune-deficient or have large quantities of virus administered via intracerebral injection or injection into other parts of the CNS. This has provided useful insights into the pathogenesis of these viruses but unfortunately has not translated to treatment or prevention of human disease.While animals are valid and useful models, organoids could serve as a preliminary platform for screening that can better inform the design of animal studies and choice of genetic background. Ongoing advances with stem-cell research have provided a platform for producing specific cell types or organoids from human stem cells. Within the last few years, significant advances in human health have been made using stem cells and organoids ,47,48,49+ T-cell response to establish persistent infections [+ T-cell response [When infected with CHIKV, non-PD and PD organoids produced similar amounts of virus for the same period, but after two weeks post-infection, PD organoids started to shrink. Immunofluorescence showed unique virus distribution patterns for non-PD and PD organoids. While non-PD organoids displayed uniform distribution of the CHIKV E2 protein, PD organoids exhibited local accumulation of CHIKV E2. This matches postmortem and necropsy data showing focal distribution of West Nile virus in brain tissue ,53. Unfofections . Gene exresponse . It coulThe distribution and density of astrocytes in non-infected organoids is comparable to other studies that document organoid morphology ,58. GFAPSOX2 and Tuj1 were used to observe neuron proliferation in response to CHIKV. When compared to non-infected organoids, SOX2 fluorescence was similar in CHIKV-infected non-PD organoids but was reduced in CHIKV-infected PD organoids. SOX2 is a transcription factor that regulates pluripotency and neurogenesis and is integral to the growth and repair of neurons . The red\u2206\u2206Ct was used to analyze changes in gene expression. When infected organoids were compared to their respective non-infected controls, PD organoids showed a pattern of up-regulation, while non-PD organoids showed a pattern of down-regulation. While this is interesting in and of itself, this analysis did not provide much insight as to whether PD organoids were mounting an antiviral response that reflected the response of non-PD organoids. Previous work has documented that endogenous expression of most genes is different for PD and non-PD cells and patients ,38. ThusThis study showed an overall pattern of excitation of GABA, glycine, glutamate, and serotonin receptors in PD organoids when infected with CHIKV, while non-PD organoids exhibited a decreased pattern of expression for the same markers. While it is well documented that CHIKV can cause long-term or permanent depressive sequelae, there are no studies describing changes in neurotransmission. Two studies have reported the potential antiviral activity of serotonergic drugs on CHIKV replication though viral inhibition assays though impacts on serotonin neurotransmission are not described ,63. GABRWhile the gene expression data shows that the response to CHIKV involves changes in neurotransmission and immune response, clinical trials have shown that dysregulation of the inflammatory response can remodel neurotransmission, leading the changes in mood and cognition ,65,66. TThe data show that both PD organoids have increased expression of NFKB2, a transcription control protein that functions in the innate antiviral response . In PD, Oxidoreductases determine MHC class I surface exposure and influence the activation of inflammation cascades. When found on the plasma membrane, oxidoreductases signal intracellular stress status to the immune system . In partCytokines direct the innate immune response and play an important role in regulating the adaptive immune response. Specific cytokines can serve as biomarkers for viral infections . Our datChemokines are a subset of cytokines that are activated in response to tissue damage as well as foreign proteins and antigens. Overproduction of chemokines is associated with a variety of autoimmune diseases. Most chemokines we examined were expressed at greater levels in PD than non-PD organoids at 14 days post infection. This state of inflammation could potentially cause complications for responding to viral infections. CCL19 is a chemokine that binds to the CCR7 receptor and acts to recruit dendritic cells. CCL19 was up-regulated in PD and non-PD organoids. CCR7 was down-regulated in non-PD organoids but up-regulated in PD organoids. The expression profiles of PD organoids reflect expression profiles documented from cerebrospinal fluid from patients infected with Varicella\u2013Zoster virus . Also, sCCL3 interacts with CCR4 and CCR5 during the acute inflammatory response and functions to recruit monocytes, which can have an impact on neuroimmunity . The incThe complement system is a part of the innate immune response that can lyse cells, activate inflammation, target virus to phagocytic cells, and clear non-cytopathic viruses from the circulatory system. Here, we evaluated the expression of C3 as it functions in both classical and alternative complement activation pathways, and deficiency of C3 can make humans more susceptible to viral and bacterial infections ,92. In oThe use of only two cell lines  is a limitation of this study due to the extensive genetic variation of PD. There are nearly 400 hiPSC cell lines derived from PD patients available for research . While tCercopithecus aethiops kidney cell line Vero E6 (ATCC CRL-1586) were grown in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) with 10% FBS, supplemented with penicillin/streptomycin, 1\u00d7 non-essential amino acids, 1\u00d7 Glutamax, and 1 mM HEPES. All cell lines were incubated at 37 \u00b0C/5% CO2. CHIKV (181/25) was obtained from BEI Resources (NR-50345) and expanded once in Vero E6 cells in a biosafety level 2 laboratory. This specific strain of CHIKV, which is a live attenuated strain derived from a human isolate in Asia, was selected based on recent peer-reviewed reports for CHIKV persistence in tissues and use in stem-cell models [Two cell lines were cultured: human-induced pluripotent stem cells  and human-induced pluripotent stem cells (hiPSC) with Parkinson\u2019s disease , which has mutations at the marker for tyrosine hydroxylase 1 . The expl models ,104,105.l models . Infecti+ surges with glutamate release, which can be affected when a virus is present [For this preliminary, observational study, cerebral organoids were utilized because they have cortical neurons that contain functional synapses that produce Ca present . Cerebra present . While m present . Cerebra present , brain d present , and a h present . Briefly present ,111.At 53 days, organoids were transferred to ultra-low attachment 24-well plates at 1 organoid per well. Organoids were infected with 100,000 (~MOI 0.001) PFU per well. Controls included mock-infected cerebral organoids. Supernatant was taken at 48 h, 4 days, 7 days, 10 days, and 14 days post infection and pooled amongst similar treatments. Samples of cerebral organoid tissue were also taken at 48 h, 3 days, 7 days, and 14 days post infection, and preserved in 4% paraformaldehyde solution in PBS (ThermoScientific Cat# J19943-K2) at 4 \u00b0C.Plaque assays were performed using the pooled supernatant samples from each treatment at each time point taken during the experiment, following methods described elsewhere , and cDNA was generated using Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems #4368814). Gene expression studies were then conducted using TaqMan Array Human Neurotransmitters (Applied Biosystems Cat #4414094), TaqMan Array Human Immune Response (Applied Biosystems #4414204), and TaqMan Array Human Alzheimer\u2019s Disease (Applied Biosystems Cat #4414070) with Applied Biosystems TaqMan Gene Expression Master Mix (Applied Biosystems Cat #4369016). Results were analyzed using the \u2206\u2206CT method.Additional RT-PCR was performed to validate gene expression data and to measure expression of astrocytes, microglia, and markers associated with Parkinson\u2019s disease. RNA and cDNA were obtained as described above. RT-PCR was performed using PowerTrack SYBR Green Master Mix (ThermoFisher Cat #A46012), per manufacturer\u2019s instructions. Primers were designed in Primerquest (IDT SciTools) from human transcripts obtained NCBI Nucleotide. RT-PCR primers were designed for glial fibrillary acidic protein (GFAP), ionized calcium binding adaptor molecule 1 (Iba1), chemokine receptor 5 (CCR5), dopamine receptor D1 (DRD1), tyrosine hydroxylase (TH), crystallin mu (Crym), and glucosylceramidase beta (GBA) . DRD1 ist-test was used to identify significance between organoids size pre-inoculation and at 13 days post infection.Organoids were imaged using ImageQuant LAS 4000 with the bright field filter under high resolution with automatic exposure. Organoid size was determined by using ImageJ . The scale of the program was set to 13.9327 pixels/mm, and the area of each organoid was recorded. Results are expressed as an average between at least 12 organoids per treatment. ANOVA was performed to determine significance between PD and non-PD organoids. A Student\u2019s t-test was used to perform pairwise comparisons of the fluorescence of non-PD organoids and PD organoids.Immunofluorescence was used to validate gene expression and PCR data. Organoids were fixed in 4% paraformaldehyde in PBS (ThermoScientific Cat# J19943-K2) overnight at 4 \u00b0C and then cryoprotected in 30% sucrose prior to sectioning. After freezing samples at \u221280 \u00b0C, organoid sections of 18 micrometers thick were produced using a cryomicrotome . Afterwards, organoid sections were blocked in 5% fetal sheep serum, and primary staining was conducted overnight at 4 \u00b0C . SecondaThe data show that neurophysiology is dramatically different between a non-PD and PD organoids and the response to viral infection is altered in PD organoids. This is of significant concern given the rising numbers of persons with neurodegenerative/neurological disease. Could viral infection with a neurotropic virus cause or exacerbate the development of neurological disease in persons predisposed for such conditions? The differential antiviral response of PD organoids highlights the need for more research in neurotropic infections in a neurologically compromised host."}
+{"text": "Implementation Science and Implementation Science Communications. We remain most interested in rigorous empirical studies of the implementation of evidence-based healthcare practices  and the de-implementation of practices that are demonstrated to be of low or no benefit. Implementation strategies  are of central interest to the journals. We see the field as large and complex, with a wide literature that is published in many venues. We urge people for whom it is new to spend some time reading the existing literature, and learning the scope of the work that has already been done, and published, in our journals and in an increasing number of other journals in the field.This editorial provides a comprehensive consolidated overview of the scope and expectations of  Implementation Science declared that it was the journal\u2019s mission to publish scientific contributions to implementation science in health, which they defined as \u201cthe scientific study of methods to promote the systematic uptake of research findings and other evidence-based practices into routine practice, and, hence, to improve the quality and effectiveness of health services\u201d [Implementation Science receives over 800 submissions annually and publishes around 120 papers. The number of downloads was 2.3 million last year. These numbers demonstrate the continued high interest among researchers, practitioners, managers, policymakers, and research funders.In 2006, the founding editors of ervices\u201d . ImplemeImplementation Science. We felt that these studies deserve to be published in an implementation science journal. In 2020, we launched the companion journal Implementation Science Communications to serve the same mission, but to enable consideration of a broader range of submissions [Implementation Science Communications published more than 100 papers in its first year, more than we had anticipated. It is on target to receive about 400 submissions in 2021. The success of the journals has reinforced the need to reflect on the journals\u2019 scope and expectations, a desire that was further enhanced by developments in healthcare and society\u2014not least the global COVID-19 pandemic [Despite being an open-access journal, we inevitably have limited editorial and reviewer resources. A substantial proportion of the rejected manuscripts are in scope, scientifically sound, but do not meet the expectations of pandemic .. Implementation strategies, or implementation interventions, are of central interest: interventions that aim to enhance the uptake, sustainment, and scale-up of practices and those that aim to remove, reduce, replace, or restrict the use of ineffective interventions or practices. Our focus on health is broad and includes physical and mental health conditions, including addiction and other behavioral health areas. We consider clinical practice, preventive care, and health promotion interventions delivered in healthcare and other settings . The effectiveness of these interventions has been the topic of public health research, while other interventions have been examined in clinical or health services research.This editorial does not represent a change in scope compared to our latest comprehensive editorial . We remaImplementation Science is particularly interested in studies that substantially advance the field by providing innovative, analytical, and generalizable insights (including non-mainstream approaches). Implementation Science Communications is interested in methodologically sound studies that contribute to new knowledge, which extend existing concepts and methods. Contributions may have a narrow focus in terms of clinical topic, patient population, or setting. In addition to empirical research, both journals publish systematic reviews and other types of contributions that we describe in the next section.We are open to a variety of methodological approaches, including qualitative, quantitative, and mixed-methods approaches. Our focus on implementation strategies means that we focus on approaches to enhance the uptake of evidence-based practices . Given the absence of widely accepted definitions of implementation strategies, we are open to various terms, including quality improvement, knowledge translation, and knowledge transfer. For instance, quality improvement is in scope, if the focus is clearly on the implementation of evidence-based practices. However, we do not publish on the implementation of research procedures . We recognize that implementation strategies are frequently applied in combination with clinical interventions, but we only consider studies that focus on the effects, processes, costs, and contextual factors associated with the implementation of these interventions. We focus on implementation strategies targeted at \u201cagents of implementation,\u201d such as health workers, patient navigators , school teachers, and policymakers. This excludes most interventions to inform patients or populations about healthcare improvements or interventions to involve patients more actively in their healthcare . Such interventions are covered by other fields . The implementation of these interventions may be in scope, if the evidence for the interventions is sufficiently robust, and the primary research question is associated with strategies to incorporate these into everyday clinical practice. The implementation of broadly phrased recommendations on desirable states in healthcare  is out of scope, if specific strategies to achieve these states are not specified.Outcomes of most interesting to us are observable aspects of healthcare delivery and healthcare providers\u2019 behaviors, such as adoption, fidelity, penetration, and sustainability of changes in practice. These aspects may actually impact the health of patients and populations. We are less interested in studies that consider perceptions or cognitions of targeted individuals  as primary outcomes, but we do consider these in the context of process evaluations. We also are less inclined to publish studies that focus exclusively or primarily on clinical outcomes or patient-reported health.evidence-based practices implies that we do not consider effectiveness trials of clinical and public health interventions, or other studies of interventions with uncertain effectiveness. Interventions for which evidence of effectiveness comes from a single study are borderline in scope. We prefer interventions that are supported by consolidated and synthesized research evidence, such as a systematic review or a systematically developed clinical guideline. We realize that emerging data-driven, individualized medical treatments  may complicate the distinction between the generation and implementation of research evidence, and we will consider submissions in this domain on a case-by-case basis. We note that the use of an established framework that derives from implementation research  does not equate to the study of implementation. Frameworks are increasingly used, particularly in the evaluation of novel interventions, but such use does not necessarily imply a contribution to the knowledge in implementation science .The focus on Better implementation of evidence-based practices frequently contributes to a reduction of disparities in health and greater equity in societies, given the unequal distribution of access to healthcare across subgroups in the population, as well as across nations. Health equity has become high on the societal agenda in recent years, which has also reinforced the attention for the topic in implementation science . A substhttps://www.nice.org.uk/corporate/ecd7). We are interested in the implementation of evidence-based digital interventions, but not in the technical implementation of infrastructures .Given the rise of submissions on the implementation of digital technologies in health settings , we will elaborate on the journal scope in this domain. We will examine submissions in this field on a case-by-case basis. Given our focus on evidence-based practice, we would expect to see any technology underpinned by recognized evidence standards for effectiveness regarding the improvements on health and population outcomes , if the focus in the manuscript is clearly on consequences for implementation into practice. Mere analyses of change in healthcare practice  are not considered. We also do not publish studies that merely document gaps between recommended and actual healthcare (\u201cevidence-practice gaps\u201d), but we do consider studies of factors associated with these gaps  and studies that develop implementation interventions. Given the descriptive nature of many of the latter two categories (barrier identification and intervention development), many of these studies will be transferred to added value to the field of implementation science. Manuscripts with a strong analytical approach  and a study design that facilitates generalizability  are likely to be reviewed at Implementation Science. Replication of previously conducted studies may also be considered, if the rationale is convincingly presented. Some manuscripts relate to implementation science, but they mainly have added value for a particular field of application . Others provide new insights or in-depth analyses, but have limited generalizability, given the study design, methods, or narrow focus. Implementation Science Communications welcomes such manuscripts, while Implementation Science is less likely to publish these. The boundary between relatively simple reports of lessons learned, with little to no analytic or theoretical insight, and small studies that provide thoughtful analyses, is not always easy to distinguish. At both journals, we receive large numbers of manuscripts in which little analytic insight is applied to the findings, and increasingly, these are being rejected at both journals.Obviously, one of our priorities is to publish studies (and other types of articles) that have This section provides further information on the boundaries of scope in relation to specific types of articles. The main considerations are summarized in Table Many theories and frameworks for implementation science are available. We are careful about adding new ones to the literature, as we feel that the field is better served by empirical testing of available theories and frameworks. Before we consider publication, the rationale for a new concept, theory, or framework (including extensions of published ones) needs to be convincingly presented. We welcome manuscripts adopting a critical approach to existing theories and frameworks, acknowledging their limitations  and building on previous conceptual knowledge to develop new generalizable insights from empirical data. However, it is essential that a comprehensive  review of existing theories and frameworks is included in any critical reflection or proposal for a new theory. When deploying existing theories and frameworks in studies, the authors should ensure that these are not applied in a superficial, tokenistic fashion. Instead, we recommend an in-depth engagement with selected theories and frameworks throughout the manuscript. Both journals are increasingly reluctant to publish studies that categorize data according to a framework without offering interpretations that relate to the underlying theory. Theories and frameworks should explicitly inform research aims and objectives, guide data collection and data analysis, shape the presentation of findings, and provide a basis for articulating the study\u2019s contribution in the discussion section.We publish various types of reviews of published research, including traditional (\u201cCochrane style\u201d) systematic reviews, scoping reviews, syntheses of qualitative research, and rapid reviews. At a minimum, the literature search needs to be transparent and generally exhaustive, cover at least three databases, and not be more than two years old at the date of submission.Implementation Science expects a study design that optimizes internal validity, such as a (cluster) randomized trial. We regard pragmatic trial designs as important in implementation research. The journal also considers rigorous non-randomized controlled studies, difference-in-differences, and interrupted time-series designs, if a clear rationale for the choice of study design is provided. Implementation Science Communications considers a broader range of designs for outcome evaluation, including uncontrolled before-after comparisons when well justified. However, weak designs and poorly conducted studies will likely be rejected by both journals.Outcome evaluations are studies that aim to determine the effectiveness of, or changes associated with, implementation interventions. We consider hybrid designs that place emphasis on the evaluation of implementation outcomes rather than clinical or population health effects. Evaluations of novel or existing interventions with limited or no evidence are not considered. We welcome outcome evaluations that show that implementation strategies had little or no impacts, provided that the rationale for the chosen strategies was plausible. As outcomes, we prefer validated measures of observable aspects of healthcare delivery or professional performance, which have clear relevance for clinical and population health. Studies reporting only self-report outcomes are unlikely to be considered. Studies that exclusively examine the effects of interventions on participants\u2019 knowledge, cognitions, or perceptions are unlikely to be considered as outcome evaluations, but we may consider these as process evaluations. For outcome evaluations, Implementation Science prefers the use of a theory or framework for the guidance of the process evaluation and a study design that facilitates generalization. Implementation Science Communications also considers process evaluations that are more pragmatic or less generalizable. Within these parameters, both journals welcome submissions of process evaluations that help to explain null results of trials as well as those with positive findings.Process evaluations of implementation interventions examine the degree of uptake of implementation interventions, processes, and factors associated with changes in implementation outcomes (or absence of it), side effects of the interventions, and/or factors that influence the sustainability and scalability of the implementation interventions. Both journals expect that the focus is on implementation interventions of known effectiveness, so that the findings of the process evaluation explain and contextualize the findings of the main study. We reject a large proportion of submissions because the effects of the implementation interventions of interest are not reported (elsewhere or in the same manuscript). Process evaluations of complex clinical or public health interventions, which have a strong implementation science component and are part of large multi-center cluster randomized trials, are considered on a case-by-case basis. Implementation Science Communications.We welcome economic evaluations of implementation interventions, preferably those that analyze both effects and costs. Cost-benefit and cost-effectiveness evaluations of implementation interventions essentially need to meet the same requirements as outcome evaluations. Manuscripts that describe cost analyses (ignoring effects) are only considered if the effectiveness of the implementation interventions is reported . Cost analyses without examinations of outcomes are usually transferred to Implementation Science Communications welcomes manuscripts on the development, pilot testing, and feasibility of implementation interventions. Few if any of these will be published in Implementation Science. To be considered, intervention development and testing should be based on systematic methods and, at least to some extent, empirical research in healthcare settings. They must be focused on implementation interventions, not other types of intervention . We prefer comprehensive reports of intervention development and testing over manuscripts that focus on specific aspects of the intervention development.Implementation Science prefers studies that relate to existing theories, frameworks, or concepts, seeking to yield new theoretical insights applicable to a wide range of settings and useful for guiding future empirical enquiry [Implementation Science Communication also considers more pragmatic studies, although lack of any theoretical insights is increasingly resulting in rejection, often with a recommendation to revise and provide stronger analytical and theoretical insights.We welcome qualitative and mixed-methods studies but have rejected many because of poor methodological design, conduct, and reporting. Like in all research papers, the methods need to be specified and described in sufficient detail to be able to understand what has been done. We are open to a variety of qualitative methods, but it is important that the chosen method matches the research question and field of application. For instance, it may be hard to justify a purely inductive analysis in a study of barriers for implementation, given the large number of available conceptual frameworks that could be used to categorize the items.  enquiry . ImplemeImplementation Science, but they may be considered by Implementation Science Communications. In all studies, we prefer that the study protocol and data analysis plan are pre-specified. We are increasingly inclined to reject studies that pick a framework and mechanically present measures or data categorized by its constructs, yielding little to no further analysis.We welcome theory-guided, quantitative observational studies, which use up-to-date or advanced analysis methods to explore or confirm determinants, processes, costs, and mechanisms of implementation. Quantitative observational studies may use concepts and methods from a variety of sciences, such as psychology, economics, and sociology. Some of these studies are (quantitative) process evaluations of implementation interventions. Quantitative observational studies that are outcome evaluations  are usually not considered by Implementation Science is particularly interested in the validation of new measures of implementation constructs, except if many similar measures have already been validated. Implementation Science Communications also considers manuscripts on the adaptation and validation of existing measures for specific healthcare settings, countries, or languages. In both journals, we prefer comprehensive reports rather than a series of incremental reports about parts of the validation study.An increasing number of submitted manuscript report on the development and validation of measures of implementation constructs. This is an important type of research, because valid measures are required for scientific progress. Implementation Science Communications, but Implementation Science remains interested in study protocols of large and innovative research projects or programs of research that are submitted within 12 months of ethical approval. We require prospective registration of all studies meeting the WHO definition of clinical trials , and note that we include behavior change, on the part of providers as well as patients, to constitute outcomes under the WHO definition. We continue to reflect on our policies regarding study protocols, with a particular view on countries that offer little opportunity to get funding from recognized research agencies.We publish  protocols that have been through competitive peer review to receive funding from a nationally or internationally recognized research agency and that have received ethical approval or exemption. We will publish these increasingly in The field of implementation science needs well-trained researchers and practitioners. Therefore, we also consider manuscripts concerning education and building capacity in implementation science, provided that they include empirical research\u2014ideally, longitudinal evaluation of a program . We prioBoth journals welcome contributions on research methods, which are relevant for implementation science . We prefer that the use of these methods is demonstrated through concrete examples, which relate to implementation science rather than other fields and are generally in scope for both journals. Both journals usually reject descriptive accounts of largely established methods without any associated novel methodological insights.Commentaries are considered if they convey new or important ideas on implementation science. We carefully consider whether the contribution is sufficiently embedded in the implementation science literature. Some commentaries have been invited, because the journal editors feel that specific themes need to be highlighted. We also receive unsolicited commentaries, and have published a few, but we often feel that the added value is limited and inadequately contextualized in the existing literature. We therefore strongly recommend making an enquiry with the senior editors before making any submission. The journals will continue to invite commentaries on specific topics, which are internally prioritized.We receive numerous debate manuscripts at both journals. We welcome debate and discussion in the field and review these carefully. However, many submissions provide only highly selective reviews of the literature, often setting up arguments that are easy to critique. We commonly reject these. As the field is evolving, we consider it essential to ground arguments in a full view of an increasingly large and complex literature, so we strongly recommend that the authors conduct a careful and comprehensive  review of the literature before drafting a debate manuscript.We encourage the use of letters to the editor, noting that they should be short (800 words or less), and focus on a critique of a specific published paper in the journal to which they are submitted. Letters that address issues from multiple manuscripts without a clear focus on a specific paper will likely not be accepted as letters. As with all publications, letters to the editors will be peer reviewed.Implementation Science Communications encourages the use of short reports as well as the use of online additional files that provide added information.Manuscripts on empirical studies can also be submitted as short reports, provided that they are sufficiently comprehensive despite their format. Given its focus on sound research with moderate added value, http://www.icmje.org/recommendations/browse/roles-and-responsibilities/defining-the-role-of-authors-and-contributors.html). For manuscripts with large numbers of authors , we increasingly ask that a writing group is named that then appears in the article byline. We appreciate that authors may be concerned that their contribution will not be recognized, but MEDLINE indexing can handle group authorship. We also recognize a growing trend towards the designation of more than one \u201cfirst\u201d or \u201clast\u201d author. We do not explicitly make these designations as part of our publications. Regarding submissions from low- and middle-income countries, we recommend to include at least one author from the countries of interest; these authors should obviously meet the ICJME criteria for authorship. We expect ethics approval (or a waiver/exemption) by a recognized, independent ethics committee for all empirical studies. Authors of all empirical studies and systematic reviews are requested to use an appropriate reporting guideline, most of which can be found online (www.equator-network.org). We require prospective registration in a recognized trial register for all studies that meet the criteria for clinical trials according to WHO or NIH . For other studies, particularly studies that are designed to test hypotheses and systematic reviews of intervention studies, prospective registration is strongly encouraged and may become obligatory in the future.Both journals have a number of expectations regarding the reporting of studies, which are summarized here. We expect that authors adhere to the principles of scientific integrity, including those that apply to the authorship of scientific papers. We perceive a trend towards increasing number of authors on manuscripts, but it is not feasible for journal editors to examine whether all fulfill the requirements for authorship. We therefore expect those designated as authors to demonstrate that they meet all four ICJME criteria for authorship (Empirical studies should obviously be scientifically sound, which implies that objective, methods, results, and conclusions are adequate and logically linked. As journals, we remain committed to improving the quality of intervention description; implementation strategies are often inconsistently labeled and poorly described. Without sufficient detail, it can be difficult for readers to determine what was actually implemented or for researchers to use or replicate a strategy in other studies. TIDieR  is most Implementation science is a fast-moving field, and we expect all manuscripts to be well-embedded in the contemporary literature. For empirical papers, this requirement relates both to the literature review presented in the front end of the manuscript and to the discussion section, which should aim to interpret the findings in light of relevant scientific literature rather than merely describe empirical results. We receive many submissions across both journals which do not fulfill this criterion and that are either sent back to authors or deemed to be of insufficient priority for review. Both journals are international in scope, which implies that authors are expected to relate their study to the global scientific literature or fully justify why orientation to a single jurisdiction is necessary. In addition, sufficient contextual information is essential for readers from other jurisdictions. We also encourage the authors to acknowledge the limitations of their studies, articulate their implications for policy and practice, and outline directions for future research. Finally, we require that each manuscript includes 2\u20133 statements on what this study adds to knowledge and understanding in implementation science. Authors will be increasingly requested to revise these statements, if they do not provide pertinent information. These should not be duplicative of the abstract.For a regular research paper, we allow a maximum of 5500 words but encourage authors to use fewer words as we believe that many readers prefer concise papers. The number and size of supplements to a manuscript are unlimited, and these can be used to provide additional information.Implementation Science Communications was to support publishing a broader variety of papers reporting on aspects of the science of implementation. We are still working out clear principles for transfer, many of which are articulated below. It is important to note that transfer from Implementation Science to Implementation Science Communications does not imply that a manuscript will be accepted for publication, although the likelihood of review is higher than average. We make independent editorial decisions at both journals and may deem a manuscript out of scope when assessed at Implementation Science Communications even when it is transferred from Implementation Science. In addition, although we make every effort to streamline processes for transferred manuscripts, if comments were provided before transfer, and particularly if the manuscript was reviewed, we expect that appropriate revisions will be made prior to transferring to Implementation Science Communications.Our primary purpose in launching Implementation Science Communications is committed to sound science [. While the identity of Implementation Science Communications as a new journal is still emerging, our intention is to promote the development of its distinct scope, contributing to advancing the field in the following directions. First, in its openness to publish smaller-scale empirical contributions, pilot studies, and alternative viewpoints, the journal aims to increase the diversity within the field, helping new ideas enter the mainstream. Second, by inviting research conducted outside healthcare settings, Implementation Science Communications aspires to facilitate dialog among implementation scholars working in a wider range of settings, such as education, social services, and public policy, and thus better understand sector-specific contextual influences on implementation. Finally, Implementation Science Communications encourages submissions that engage with theory, either through theoretically informed or theoretically informative approaches [It is also worth reiterating that  science . While tproaches , but do Implementation Science and now also publishing Implementation Science Communications, we see the field as large and complex, with a wide literature that is published in many venues, and urge people for whom it is new to spend some time reading the existing literature, and learning the scope of the work that has already been done, and published, in our journals and in an increasing number of other journals in the field.As we note, implementation research is a rapidly growing field, with growing efforts to dedicate funding in some jurisdictions. This draws new entrants into the field, many of whom come from other health research backgrounds, and for whom implementation science seems novel and highly emergent. With over 15 years of publishing Implementation Science and Implementation Science Communications and highlighted some differences between the journals. We intend to support authors in their consideration on whether to submit to the journals, and hope we will continue to receive many high-quality submissions in the coming years.This editorial described the mission, scope, and expectations of"}
+{"text": "Implementation science, defined by NIH as \u201cthe scientific study of the use of strategies to adopt and integrate evidence-based health interventions,\u201d continues to grow within research, education, and practice-based settings. Building on principles from organizational psychology, intervention science, health economics, and health services research, implementation science aims to explore how, and under what conditions, evidence-based interventions are successfully implemented and sustained in real-world settings. Applying implementation science to aging programs and settings may help to accelerate the translation of effective programs and policies into practice. This interdisciplinary symposium will provide an introduction to key principles and applications of implementation science. The first three presentations will focus on largescale spread of interventions while the last two presentations will focus on broader applications of implementation science. The first two presentations will focus on adapting interventions from delivery in one setting or population to another. The third presentation will discuss the role of implementation strategies in scaling an intervention from a controlled research setting into a large integrated healthcare system. The third presentation will focus on the intersection of implementation science and policy. The final presentation will discuss the role of implementation science in alleviating health disparities and advancing health equity. Each presentation will utilize examples from ongoing research studies to demonstrate principles. The session will close with an interactive discussion on the role of implementation science within aging, including challenges and considerations for aging programs, policies, and populations as well as opportunities for further training and education."}
+{"text": "The Spanish Civil War (1936\u20131939) is an example of a historic event involving nurses, with the participation of professional and volunteer nurses from Spain and other countries. In this context, nurses were trained over short periods of time and recruited to work at hospitals serving the two warring camps.To identify the characteristics of the training received by volunteer nurses on both sides in the Spanish Civil War and compare it with previous experiences in the history.Historical research.ABC and La Vanguardia. The following variables were analysed: duration, entry requirements, and theoretical content of the training courses. Consolidated Criteria for Reporting Qualitative Research (COREQ) has been used.Heuristic and hermeneutical analysis of nurse training manuals and news articles from 1936 to 1939. Spanish primary sources were consulted at the Red Cross Documentation Centre Archive in Madrid, the General Military Archive in \u00c1vila, the Municipal Newspaper Archive in Madrid, and the archives of Spanish daily newspapers Both sides in the conflict offered a varied training programme, which was supported by official institutions and private initiatives. The courses lasted between one week and two months. Entry requirements were influenced by education level, age, moral conduct, health status, and social and political background. Training content focused on the techniques needed in conflict settings and covered specific moral values.Despite the different social and political characteristics of the two warring factions, the variety of training programmes on offer, the entry requirements, and the theoretical content of volunteer nurse training were similar on both sides. At the end of the Spanish Civil War, volunteer nurses on the Republican side suffered reprisals or had to go into exile. We now know that some countries involved in World War II provided training courses for volunteer nurses. It would therefore be interesting to ascertain whether Spanish volunteer nurses contributed their experience to these courses. Like any other social or health emergency, armed conflict has shaped the role played by nurses in healthcare delivery , forgingThroughout the 20th century, nurses participated in a number of armed conflicts around the world, tackling physical, psychological, and clinical problems. Training for civilian nurses differs in a number of ways from specific training allowing nurses to work in military hospitals, where specific preparation for ethical and care dilemmas is vital . ResearcNurse training is crucial in preparing nurses for the challenges of working in a conflict setting, where their theoretical knowledge, ethical commitment, and social responsibility are put to the test as they provide care in hostile, unpredictable, and challenging environments . Public Public health emergencies have shaped the development of the nursing profession, imbuing it with the social, political, and economic values of each historical context and influencing and modifying its image over time. It is important to study these emergencies and their incorporation into training programmes in order to understand nurses\u2019 influence and contribution to society .practicante (practitioner). The nurse was a professional related to the woman-caregiver-salaried duality and the practitioner with the male-healer-autonomous duality  and translated from the French by Mar\u00eda de Corral, a nurse with the Spanish Red Cross, was available  despite the fact that the rules state that the minimum age is 20 years old and she is 16. It\u2019s nonsense that these girls hang around in hospitals instead of school, so they can\u2019t be given the [training] card! Not being mobilised or on night shift, we cannot be indulgent until 18, but not less in any way. I don\u2019t know what these parents are thinking about!\u201d .Continuing with the Nationalist zone, applicants were asked to submit a certificate of good conduct, their birth certificate, and their academic qualifications with their application . In otheThe conflict situation led to a change in training content see , which fAs well as new training manuals focusing on knowledge relating to the conflict situation, some existing manuals also added sections for this purpose, including nursing, chemical warfare, and military administration .Theory lessons covered the spirit of sacrifice, sense of duty, caution, patience, and charity . VolunteTraining manuals also addressed matters relating to public perceptions of nurses, encouraging specific moral qualities. Their humanitarian work was expected to be characterised by kindness, gentleness, and altruism, as well as warmth, which nurses were required to display to all patients while remaining discreet and respecting patient confidentiality .In the training provided for nurses on the Nationalist side of the conflict, a sense of vocation and Christian charity  were presented as the model of perfection that nurses should strive to attain, with no expectation of professional recognition beyond that of God. Religious indoctrination became more entrenched as the war went on and nurses were asked to profess their love of God \u201cby practising a Christian life of meditation and piety\u201d that would allow them to care for the sick .The contribution of volunteer nurses during World War I is widely recognised , raisingAlthough dependence on the medical profession waned during World War I , doctorsIn World War II, the countries involved were already equipped with professional nurses, albeit in insufficient numbers. Nurses had to be mobilised from different countries , 69, stuDuring the Spanish Civil War, training courses lasted for a short period of time as their purpose was to provide intensive training for volunteers, including World War II volunteer nurses from Brazil  and FinlWe have identified that although the courses provided by both sides in the Spanish Civil War varied in terms of the training provided, their duration, and their entry requirements, the theoretical content was similar in all of them. The aim was to prepare volunteer nurses to provide healthcare in conflict settings. In such settings, knowledge and professional skills are extremely important. Studies such as Firouzkouhi et al. provide evidence of this, focusing on the need to include specific training in triage to ensure adequate healthcare .As we have seen, comprehensive, targeted training was needed during the Spanish Civil War to meet healthcare needs. The volunteers\u2019 lack of experience and training were compensated by their eagerness to help and their good intentions, but these were not always sufficient to enable them to cope with the emotional distress caused by working in a war zone. None of the lessons in the manuals referred to the pain and suffering that the nurses would witness. They were asked to be kind, pious, and caring, but they were not prepared for the mental fatigue that they would face when providing compassionate care . These pThe training provided by the Spanish Civil War courses contributed to the public image of what a nurse should be, emphasising the moral qualities that nurses were expected to possess in particular. As well shaping their image, training courses also helped to foment a sense of belonging to a group and served as a way of homogenising attitudes and practices, as well as highlighting gender issues in this context . In thisWe have not found any cases of training courses for male nurses (practitioners) during the Spanish Civil War. This is related to the sexualisation of care that was present in the early 20th century . It woulAs well as theory lessons on providing healthcare and assistance to the wounded, the training manuals also contained recommendations on rest, diet, and exercise for nurses to ensure that they remained healthy and able to carry out their work to the best of their abilities. Recent studies have demonstrated the importance of self-care among nurses. Clinical training, rest, recuperation, exercise, and diet are all important factors in reducing psychological stress in war zones .During the Spanish Civil War, volunteers nurses were trained in the ethics of care, twinned with vocation and humanitarianism, which are essential in any conflict setting . Both siThe outcome of the Spanish Civil War and the first few decades of the Franco dictatorship (1939\u20131975) shaped the development of the nursing profession in Spain. The dictatorship revoked nursing diplomas issued by the Republican side , retaliaLey de Sanidad), nursing, midwifery, and nursing assistant studies were unified in Spain in 1953 under a new qualification named ayudante t\u00e9cnico sanitario  [Under the 1944 Spanish Health Law (sistant) . For decsistant)  and focusistant) .The health emergency caused by the Spanish Civil War gave rise to a need for greater numbers of nurses, leading to the emergence of courses aiming to train volunteers on both sides in a pattern that would be repeated in World War II years later. The variety of training programmes, entry requirements, and theoretical content were similar in both factions, although they were shaped by different sociopolitical contexts. The outcome of the war was pivotal in the lives of the volunteers. Volunteer nurses on the Republican side went into exile, so it is difficult to know how their experiences in the Spanish Civil War may have influenced volunteer nurse training systems in World War II or their integration into the workforce in their destination countries. The volunteer nurses on the winning Nationalist side helped to consolidate the nursing profession and nurses\u2019 professional identity, which were shaped by the values of the Franco dictatorship."}
+{"text": "Older adults are particularly at risk from infectious diseases, including serve complications, hospitalization, and death.This study aimed to explore the drivers of vaccine hesitancy among older adults based on the \u201c3Cs\u201d  framework, where socioeconomic status and vaccination history played the role of moderators.A cross-sectional questionnaire survey was conducted in Jiangsu Province, China, between June 1 and July 20, 2021. Older adults (aged \u226560 years) were recruited using a stratified sampling method. Vaccine hesitancy was influenced by the 3Cs in the model. Socioeconomic status and vaccination history processed through the item parceling method were used to moderate associations between the 3Cs and hesitancy. Hierarchical regression analyses and structural equation modeling were used to test the validity of the new framework. We performed 5000 trials of bootstrapping to calculate the 95% CI of the pathway\u2019s coefficients.P=.002), convenience , and less complacency  were positively associated with less vaccine hesitancy. Socioeconomic status weakened the positive effect of low complacency  on low vaccine hesitancy. COVID-19 vaccination history negatively moderated the positive association between confidence  and lower vaccine hesitancy.A total of 1341 older adults participated. The mean age was 71.3 (SD 5.4) years, and 44.7% (599/1341) of participants were men. Confidence (b=0.967; 95% CI 0.759-1.201; Our study identified that confidence was the more influential dimension in reducing vaccine hesitancy among older adults. COVID-19 vaccination history, as well as confidence, had a positive association with less vaccine hesitancy and could weaken the role of confidence in vaccine hesitancy. Socioeconomic status had a substitution relationship with less complacency, which suggested a competitive positive association between them on less vaccine hesitancy. Older adults are at risk of illness, serious complications, hospitalization, and death from vaccine-preventable diseases owing to declining immunity associated with aging ,2. Vacci\u201cVaccine hesitancy,\u201d proposed by the Strategic Advisory Group of Immunologists working group, could considerably hinder vaccinations. This term was defined as \u201cdelayed acceptance or refusal of vaccines despite their availability\u201d . SeveralThe causes of vaccine hesitancy among older adults were summarized by the following factors, including misinformation, perception of good health, perception of vaccine ineffectiveness, side effects, and distrust of the health care system . Many stAs China is the most populous country in the world, the number of adults aged \u226560 years (267 million in 2022) is larger than in any other country . This stA cross-sectional questionnaire survey was conducted between June 1 and July 20, 2021, using a stratified sampling method. We assessed vaccine hesitancy among adults aged \u226560 years. After stratifying by economic level, the cities of Changzhou and Yancheng were selected as the survey areas for this study, among 13 cities in Jiangsu province, China, using the random number method. The gross domestic product per capita in Jiangsu province in 2019  was used as the cutoff between high  and low  economic levels . Then, wOlder adults who visited designated medical checkup clinics for annual physical examinations, which were government-organized checkups for all eligible residents within that residence, were recruited as participants. Participation was voluntary. All adults aged older than 60 years were encouraged to participate. The one-on-one interview survey method was adopted, and all investigators were trained uniformly. Those with cognitive impairment were excluded, as were those who had a relative complete the survey for them. The calculation of the sample size required for this study was shown in Material S1 in The questionnaire consisted of 3 sections. The first section was demographic , SES, and health information. Individual SES was evaluated by survey area (low or high economic level), education (primary or lower or secondary school or higher), medical career background (yes or no), occupation , and monthly revenue  \u201cThe poor quality of service at the vaccine clinic would make me not want to go for vaccination\u201d; (2) \u201cIt was easy and took me a short time to get the vaccination\u201d; (3) \u201cI can get the vaccine that I want\u201d; and (4) \u201cI can afford the vaccine.\u201d Complacency was measured using four items: (1) \u201cIf I do not get vaccinated, I will not get the disease\u201d; (2) \u201cNatural immunity is better than that produced by vaccination\u201d; (3) \u201cThe probability of getting a disease is low, so I do not need to get vaccinated\u201d; and (4) \u201cEven if I get infected with a disease I can resist it, so I do not need to be vaccinated.\u201d For structural consistency, we shifted all items in the complacency dimension.In addition, a vaccine hesitancy scale was included in the third section, which included three items: (1) \u201cHow likely would you go for a COVID-19 vaccine?\u201d (2) \u201cWould you get an in\ufb02uenza shot this year?\u201d and (3) \u201cIf you could now get an influenza vaccine at your own expense, what is your choice?\u201d Each item in the second and third sections was assessed using a 5-point Likert scale. A higher score chosen for each item in the second section means higher confidence, a greater level of complacency, and a higher level of convenience in the vaccine. A higher score chosen for the third section (vaccine hesitancy) means less vaccine hesitancy. The detailed information and the original questionnaire are presented in Material S2 in We proposed 5 hypotheses according to our purpose:Hypothesis 1: More confidence is positively associated with less vaccine hesitancy.Hypothesis 2: More convenience is positively associated with less vaccine hesitancy.Hypothesis 3: Less complacency is positively associated with less vaccine hesitancy. .Hypothesis 4: Vaccination history moderates the strength of the association between confidence (Hypothesis 4a), convenience (Hypothesis 4b), and complacency (Hypothesis 4c) with less vaccine hesitancy.Hypothesis 5: SES moderates the strength of the association between confidence (Hypothesis 5a), convenience (Hypothesis 5b), and complacency (Hypothesis 5c) with less vaccine hesitancy.t values, as well as factor loadings and average variance extracted (AVE) [A confirmatory factor analysis was conducted to assess the reliability and validity of each scale. Reliability was evaluated by calculating squared multiple correlations and composite reliability ,34. Thised (AVE) . ValiditAccording to Gerbing et al , the gooMICE package in R software  was used to complete this imputation. Sensitivity analyses were also conducted for the original data and where the missing data were deleted. Participants\u2019 characteristics were described with mean  and frequencies . The variables of SES and vaccination history were processed using the item parceling method [P<.05.To minimize bias from the exclusion of any missing data for the older adults, we used multivariate multiple imputations to fill in missing values. The g method . All iteg method . Hierarcg method . Controlg method . HierarcBefore completing the questionnaire, it was mandatory for all participants to provide verbal informed consent. They were informed that this was an anonymous survey and that all data would remain confidential. Participants would receive US $2 worth of gifts in return for completing the survey. The Ethics Committee of the Wuxi Center for Disease Control approved this study (2020No10).Initially, 1384 participants  took part in this survey. Based on the inclusion and exclusion criteria, a total of 1341 participants were included finally . Of thesOf the specific vaccines, the percentages of older adults who self-reported having received herpes zoster vaccine, influenza vaccine (last year), and pneumonia vaccine were 0.8% (n=11), 1.5% (n=20), and 0.1% (n=2), respectively. A total of 30.9% (n=415) of the participants had received at least one dose of the COVID-19 vaccine. Additionally, 21.4% (n=287), 3.7% (n=50), and 29.9% (n=401) of the participants self-reported choosing uncertainty, postponing, and refusing vaccination, respectively. Further, 45% (n=603) of the participants were willing to receive vaccines.The items of each scale were censored based on the factor loadings, followed by the modification indices. Items with factor loadings below 0.5 were not retained, principally. The modified questionnaire is presented in Table S1 in P=.003), whereas single participants  were more hesitant. Age, sex, chronic diseases, self-assessment of health status, and self-assessment of the financial situation were not associated with vaccine hesitancy .Married participants were more willing to be vaccinated than their counterparts . Of these, confidence , convenience , SES , and vaccination history  were all significantly and positively associated with less vaccine hesitancy. Complacency was not significantly associated with vaccine hesitancy (P=.82). In block 3, the added moderators, SES and vaccination history, also had significant changes, compared with block 2 . SES moderated the impact between confidence  and complacency  with vaccine hesitancy. Convenience  was not statistically significant. Therefore, SES can positively moderate the positive effects of confidence on reducing vaccine hesitancy. In other words, the higher SES in people with higher confidence scores was associated with less vaccine hesitancy. SES negatively moderated the positive effect of less complacency on less vaccine hesitancy, which meant that lower SES weakened this positive effect between complacency and vaccine hesitancy. In addition, SES had a substitution effect with complacency. The positive effect of less complacency on vaccine acceptance was more pronounced when the degree of SES was low; however, as the degree of SES increased, the positive effect of less complacency decreased.Compared with the model in block 1, where control variables were presented, significant changes were made in block 2 (\u2206P=.06), less complacency (P=.14), and convenience (P=.16) with lower vaccine hesitancy. Hence, hypotheses 4a, 4b, 4c, and 5b were rejected, and hypotheses 5a and 5c were accepted. All detailed information about hierarchical moderator regression analysis is presented in Vaccination history had no moderating effect on the association between confidence (P=.02). COVID-19 vaccination history had a substitution effect with confidence. The positive effect of COVID-19 vaccination history on less vaccine hesitancy was more pronounced when the degree of confidence was low. Complacency and convenience were not moderated by COVID-19 vaccination history. In the herpes zoster, influenza, and pneumonia vaccination history subgroup  and convenience  exhibited a significant positive association with less vaccine hesitancy, thus confirming hypotheses 1 and 2. Unlike the hierarchical regression analysis, in the structural equation model, less complacency had a statistically significant effect on less vaccine hesitancy . Since the structural equation model considered the potential of multicollinearity, whereas the regression analysis did not, the result in the complacency dimension is the more reliable one. Detailed information is shown in To avoid the effects of multicollinearity, we calculated the effects of each of the 3 dimensions of confidence, convenience, and complacency on older adults\u2019 vaccine hesitancy separately. Then, a full model was constructed, where the effects were split equally to solve multicollinearity ,42. As wThe confidence dimension was assigned 2 components: participants\u2019 confidence in the vaccines, and participants\u2019 confidence in the manufacturer who provided the vaccine and in the health care workers who administered it to them. The results for the second-order constructs showed that the direct effects (unstandardized estimate) between confidence in the vaccine confidence and confidence in health care workers and vaccine manufacturers confidence were 1.000 and 0.810, respectively.In this study, a 3Cs model moderated by SES and vaccination history was developed in the group of older adults. The explanation power of the new framework was examined, and the findings supported that confidence, complacency, and convenience all played an important role in older adults\u2019 vaccine hesitancy. Additionally, SES was a significant moderator of confidence and complacency that scales with vaccine hesitancy. COVID-19 vaccination history moderated the association between confidence and vaccine hesitancy. The dual testing of the hierarchical moderator regression and structural equation modeling analyses made the results more reliable and robust.Analysis of the 3Cs model revealed several factors that influenced older adults' vaccination perceptions. Our findings in the confidence dimension among Chinese older adults were consistent with studies conducted in other countries, such as Peru ,44, EuroThe positive moderating effect between SES and the confidence dimension suggested that older adults with high SES had less vaccine hesitancy owing to better educational attainment and higher income than their counterparts. The positive effect of lower complacency on reducing vaccine hesitancy was more pronounced at lower SES. In other words, as SES increased, the positive effect of low complacency gradually decreased. This suggested a substitution effect between low complacency and SES on vaccine hesitancy among older adults. For disadvantaged older adults, complacency was exacerbated by low SES because of low knowledge level and social media occlusion . In the The difference in the moderating effect of COVID-19 vaccination history and the other vaccines indicated that the promotion of getting COVID-19 vaccination was a success among the older adults in China. The findings presented the substitution relationship between COVID-19 vaccination history and confidence, which was not presented in the other vaccines history group. Although vaccination history (either COVID-19 or other) did not reduce older adults\u2019 complacency, their hesitancy would be decreased after COVID-19 vaccination by influencing confidence, which was a more powerful indicator of vaccine hesitancy in this study.Marital status was the significant demographic factor that could influence vaccine hesitancy among older adults. As summarized in a large review, positive perceptions and encouragement of vaccination by older adults\u2019 family members and social communities were important for increasing vaccination rates . TherefoThis study provided evidence for practical applications in reducing vaccine hesitancy among older adults. Our findings suggested that increasing the vaccination willingness of older adults was strongly dependent on increasing confidence, both in the vaccine and in health care workers. Future interventions should focus on increasing vaccine recommendations by health care workers, especially those in primary care, on which older adults rely heavily . MoreoveFurther, since some older adults do not use social media and are distrustful of pharmaceutical interventions, future interventions among older adults should focus on instilling information about the safety, importance, and necessity of vaccines. The convenience dimension was a variable that required special attention in the older adult population compared with other groups. Increasing the convenience of vaccination, such as opening convenient vaccination sites, increasing reimbursement rate by health insurance, and increasing transportation convenience measures, can also reduce vaccine hesitancy among older adults .Finally, awareness of the severity of vaccine-preventable diseases and infection risk should be increased, especially among older adults with low SES. Complacency was a substitute factor for SES, and our findings provided evidence that reducing complacency could eliminate the large impact of SES\u2014a variable that was essentially immutable in older populations\u2014on vaccination intentions.Selection bias is one possible limitation. Although all older adults were required to participate in this free annual physical examination where we recruited participants, it was not mandatory. Therefore, the individuals who participated in this survey might have been more concerned about health care than their counterparts. However, we controlled for demographic factors in our analysis. In addition, the AVE of convenience for the questionnaire was slightly lower than the related correlation with complacency; however, the overall model fit was ideal. This questionnaire should be improved in future studies. All the data were self-reported, and realistic data such as vaccine efficacy and safety were not included in this study. This study was conducted in Jiangsu Province, an economically developed province in China; hence, extrapolation need to be considered with great caution, especially in less economically developed areas. In these areas, for example, the association between convenience and older adults\u2019 vaccine hesitancy may increase positively.This study developed a model based on the 3C theory including SES and vaccination history as moderating variables to explore the drivers of vaccine hesitancy among older adults. Confidence was the more influential factor in reducing vaccine hesitancy among older adults. Our findings also highlighted that COVID-19 vaccination history, which had a positive influence on less vaccine hesitancy, weakened the role of confidence in vaccine hesitancy. The substitution relationship between SES and complacency suggested a competitive positive effect of SES and less complacency on less vaccine hesitancy. These associations could provide potential strategies that can be used to mitigate vaccine hesitancy for older adults with different reasons for SES, confidence, and complacency."}
+{"text": "Ankle foot orthoses are mainly applied to provide stability in the stance phase and adequate foot clearance in the swing phase; however, they do not sufficiently assist during the entire gait cycle. On the other hand, robotic-controlled orthoses can provide mechanical assistance throughout the phases of the gait cycle. This study investigated the effect of ankle control throughout the gait cycle using an ankle joint walking assistive device under five different robotic assistance conditions: uncontrolled, dorsiflexion, and plantar flexion controlled at high and low speeds in the initial loading phase. Compared with the no-control condition, the plantar flexion condition enhanced knee extension and delayed the timing of ankle dorsiflexion in the stance phase; however, the opposite effect occurred under the dorsiflexion condition. Significant differences in the trailing limb angle and minimum toe clearance were also observed, although the same assistance was applied from the mid-stance phase to the initial swing phase. Ankle assistance in the initial loading phase affected the knee extension and ankle dorsiflexion angle during the stance phase. The smooth weight shift obtained might have a positive effect on lifting the limb during the swing phase. Robotic ankle control may provide appropriate assistance throughout the gait cycle according to individual gait ability. Smooth walking requires proper movement of the ankle joint. The rocker function plays an important role as a rotation axis for shock absorption in the early stance and in generating an anterior propulsive force in the late stance and pre-swing phases  tuned for individual walking disabilities compensates for gait disturbance by adequately controlling the motion of the tibia during the stance phase  provides ankle plantar flexion and dorsiflexion in the stance and swing phases at the preferred timing, which is based on angular velocity. In previous studies, we focused on the device's assistance in forefoot rocker function and revealed its positive effects on gait function and spinal cord excitability  without lower limb orthopedic disease participated in this study. This study was approved in advance by the Ethics Committee of Hiroshima University in accordance with the Declaration of Helsinki, and written consent was obtained from all participants.RE-Gait is a close-fitting assistive walking device that consists of a controller  and an ankle foot orthotic equipped with a motor and two sensors on the toe and heel (weight 1 kg) . Only thParticipants were asked to walk on a treadmill  at a comfortable speed (2.0\u20132.8 km/h) under five conditions, wearing RE-Gait on their right leg. The comfortable speed for each participant was determined by measurements before the experiment.Walking was performed for 2 min, with the first 1 min serving as a warm-up period, and the last 1 min as the assessed period. Furthermore, the participants walked for another 30 s with the device attached but not controlled by the motor to avoid the aftereffects of each walking condition .Five conditions were set depending on the initial control method from the heel contact to toe contact period . The setSubsequently, the control between the mid-stance and swing phase was set to be constant except for the OFF condition as follows: plantar flexion assistance (55\u00b0/s) for 10% of the gait cycle period after the heel off, and dorsiflexion assistance (55\u00b0/s) for 10% of the gait cycle period were applied immediately after the plantar flexion assistance, based on previous studies  and assessed the joint angles of the knee and ankle, trailing limb angle (TLA), and minimum toe clearance (MTC) using markers attached to body feature points at the greater trochanter, lateral epicondyle of the knee, lateral malleolus, fifth metatarsal head of the right leg, and heel on the right leg. The marker of the lateral malleolus was placed perpendicular to the lateral malleolus on the device.The joint angle was calculated by capturing the video and tracking the coordinates of the markers of each landmark using MATLAB R2020a Image Processing Toolbox and Computer Vision System Toolbox . If the marker was not detected, linear interpolation was applied.The knee joint angle was calculated as the angle between the thigh vector joining the greater trochanter with the lateral epicondyle and the lower leg vector joining the lateral epicondyle with the lateral malleolus. The ankle joint angle was calculated as the angle between the lower leg vector and the foot vector with the fifth metatarsal head and the heel. Changes in each gait cycle were analyzed by identifying two consecutive heel contacts. To analyze the angular changes, five periods were identified based on the toe and heel foot pressures: heel-contact, toe-contact, middle-stance, heel-off and toe-off. The middle-stance was defined as the intermediate period between toe-contact and heel-off. The gait cycle was divided into five phases according to the following five periods: loading response, mid-stance, terminal stance, pre-swing, and swing phases. Angular changes were calculated from peak-to-peak values in each phase.The TLA was defined as the peak angle between the vertical axis and the vector joining the greater trochanter with the fifth metatarsal head , and the electrodes were placed on the tibialis anterior (TA), soleus (SOL), medial head of the gastrocnemius (MG), rectus femoris (RF), vastus medialis (VM), and biceps femoris (BF) of the right leg. After proper cleaning of the skin, EMG electrodes were placed over the belly of each muscle. The EMG signals were amplified ( \u00d7 100) using an EMG amplifier system  and digitized at 1000 Hz using Power Lab system .The EMG data were band-pass filtered at 10\u2013200 Hz and rectified for offline analysis using LabChart v.8.1.12 (AD Instruments). They were then time-normalized and rectified into 100 data points for each gait cycle. The EMG amplitudes were normalized by the average amplitude of each muscle over the gait cycle for comparison of each condition .The angular changes and, TLA, MTC, and EMG amplitudes in each phase were compared under the five different conditions  using a one-way repeated measures analysis of variance. Before the analysis, we confirmed that the data were normality distributed using the Mauchly's sphericity test. If the significant differences were appeared, paired  = 12.19, P < 0.01). Moreover, the post-hoc test revealed that it was significantly increased under the PFh condition compared with that under the OFF condition.The average changes in the knee and ankle joint angles throughout the gait cycle are shown in  = 18.44, P < 0.01), terminal stance phase  = 7.69, P < 0.01), and swing phase  = 7.64, P < 0.01). Post-hoc tests showed that the dorsiflexion angle in the mid-stance phase was significantly lower under the PFs (7.79 \u00b1 3.54\u00b0) and PFh (6.19 \u00b1 3.90\u00b0) conditions than under the OFF (10.36 \u00b1 2.31\u00b0) condition, and that in the terminal stance phase was significantly smaller under the DFs (7.12 \u00b1 2.21\u00b0), DFh (7.70 \u00b1 2.79\u00b0), and PFs (8.32 \u00b1 2.31\u00b0) conditions than under the OFF condition. During the swing phase, the dorsiflexion angle in the DFh (14.15 \u00b1 6.41\u00b0) condition was significantly higher than the OFF (7.21 \u00b1 3.42\u00b0) condition.The ankle dorsiflexion angle also differed among the conditions; the timing of dorsiflexion was earlier under the DF conditions, and later under the PF conditions. Statistical analysis revealed significant differences in the dorsiflexion angle in the mid-stance phase  = 3.64, P < 0.05, MTC: F = 6.06, P < 0.01). Post-hoc tests revealed that the TLA was significantly lower under the OFF condition than under the DFs, DFh, and PFh conditions. The MTC under the DFh condition was significantly higher than that under the OFF condition.The averaged results of the TLA and MTC under each condition are also shown in  = 3.21, P < 0.05, SOL: F = 5.41, P < 0.01). The VM activation under the PFh condition was significantly higher in the loading response phase  = 3.52, P < 0.05) and significantly lower in the pre-swing phase  = 3.38, P < 0.05).In this study, the effects of ankle control using an ankle joint walking assistive device were assessed. The advantages of robotic ankle control are as follows: (1) ankle dorsiflexion and plantar flexion assistance in the initial loading phase control knee extension and ankle dorsiflexion in the stance phase, (2) assistance for the forefoot rocker function enhances TLA, and (3) obtaining smooth heel rocker function may have a positive effect on the lifting of the limb in the swing phase.To improve the weight shift, appropriate rocker functions are needed , and funded by Space Bio Laboratories Co., Ltd. Part of this work was supported by the Center for Integrated Medical Research, at Hiroshima University Hospital.The authors declare that this study received funding from Space Bio-Laboratories Co., Ltd. The funder had the following involvement in the study: Provision of equipment. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. Author LY is employed by Space Bio-Laboratories Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "ObjectivesOsteoradionecrosis is one of the most severe complications in patients with head and neck cancer, which is characterized by persistent exposed and devitalized bone without proper healing after radiation. The extent to which mandibulotomy and marginal mandibulectomy influence the occurrence of osteoradionecrosis remains unclear. This study evaluated the incidence and risk factors for developing osteoradionecrosis of the mandible after oral cancer treatments.MethodsA retrospective study was performed to analyze medical records of patients who underwent surgery and postoperative radiotherapy for oral cancers from 2009 to 2019 at a tertiary care hospital. Patient characteristics, incidence, and risk factors for developing osteoradionecrosis were reviewed. Comparisons between continuous and categorical data were performed using t-test and Chi-squared test. Cox regression analysis was used to assess the association between factors and the development of osteoradionecrosis.ResultsAmong the 61 patients included in the study, osteoradionecrosis of the mandible occurred in 9 of 32 (28.1%) patients who underwent mandibular surgery during oral cancer resection  and 2 of 29 (6.9%) patients without mandibular surgery.The development of osteoradionecrosis was significantly associated with performing mandibular surgery  and HIV infection . In the subgroup analysis of mandibular surgery, the development of osteoradionecrosis significantly increased in patients undergoing mandibulotomy  but not in patients undergoing marginal mandibulectomy .The analysis also showed that concurrent chemoradiation, radiation doses \u2265 60 Gy, and smoking were potential risk factors for the development of osteoradionecrosis, but none of these factors were statistically significant.ConclusionOur findings suggest that mandibular surgery is a significant risk factor for the development of osteoradionecrosis in patients with oral cancer. Further studies including larger population sizes are required to verify these findings. Oral cancer is a common type of head and neck cancer, which is reported to be the fifth most common cancer among countries in Southeast Asia and ranks among the top ten dominant cancers in Thailand .While surgical treatment and postoperative radiotherapy are the mainstay treatments for oral cancer, surgical management of these cancers is often more challenging because of the small area of the mouth and adjacent bony structures, which limit surgical exposure. Adequate surgical exposure and en-bloc resection are crucial to ensure oncological safety .Mandibulotomy is an approach that divides the mandible to gain surgical access to the oral cavity and oropharynx. Although this technique requires repair of the mandible afterwards, it provides excellent exposure to the submental area and posterior part of the mouth . ComplicMandibulectomy is a procedure used to eradicate a disease that involves the mandible. Marginal mandibulectomy involves resecting a portion of the lingual cortex or the alveolar ridge of the mandible as part of the surgical margin. This technique is indicated for oral cancers that abut or minimally erode the mandible without gross invasion. A decrease in mandibular bone thickness after resection may increase the risk of developing fracture and osteoradionecrosis .Several risk factors, such as poor dental health, post-radiation dental extractions, and radiation doses, have been reported to be associated with the development of osteoradionecrosis . HoweverThe aim of this retrospective study was to provide objective data on the role of mandibular surgery as a risk factor for the development of osteoradionecrosis in patients with oral cancer. The characteristics, incidence, and risk factors for developing osteoradionecrosis after oral cancer treatments were analyzed.This retrospective study reviewed surgical cases performed in the Department of Otolaryngology at the Chonburi Cancer Hospital, Thailand. Data regarding surgical techniques, clinical course, and complications were obtained from patients\u2019 medical records.The inclusion criteria were: new diagnosis of oral cancers with at least three-year survival and receiving postoperative radiotherapy. The exclusion criteria were: receiving radiotherapy prior to surgery; premature discontinuation of treatment; performing segmental mandibulectomy with or without free flap reconstruction; or requiring revision surgery due to major surgical complications.\u00a0Patients with a daily frequency of smoking were classified as smokers, regardless of duration.Surgical techniquePatients were divided into two groups based on mandibular involvement. The first group was composed of patients who underwent oral cancer surgery without mandibular surgery, while the second group was composed of patients who underwent mandibular surgery . All patients in the mandibulotomy group underwent straight midline mandibulotomy and repair with double mini-plates Figure .Marginal mandibulectomies were either performed vertically or horizontally depending on the location of the tumor. Figure Statistical analysisComparisons between continuous and categorical data were performed using t-test and Chi-squared test. Cox proportional hazard model was used to assess the association between these factors and the occurrence of osteoradionecrosis. Statistical significance was set at p \u2264 0.05.Ethical approvalThe Chonburi Cancer Hospital ethics committee approved this study and permitted a waiver of informed consent .Between 2009 and 2019, from a total of 421 patients with operable oral cancer, 61 patients were included in this study. The reasons for exclusion were surviving less than three years in 154 patients, receiving surgery alone in 112 patients, and loss to follow-up in 39 patients.The follow-up period was 3-10 years. Since osteoradionecrosis usually occurs late after radiation, only patients who survived for at least three years were included in the analysis. All patients had started dental treatment during surgery and dental problems, such as retained root, extensive periapical lesion, and periodontitis, were completely corrected prior to postoperative radiotherapy.Postoperative radiotherapy was administered to patients with stage III-IV disease or inadequate margins. Chemotherapy was also used to treat patients with positive margins or adverse features, such as lymphovascular invasion. Concurrent chemotherapy was cisplatin-based and was administered every week at a dose of 40 mg/m2. Chemotherapy was administered for four to six cycles depending on the patient tolerance. The characteristics of the patients included in this study are shown in Table In our institute, the transoral approach is used for small oral lesions (T1), whereas resection of T4 tumors is usually performed using the mandibulotomy approach. For intermediate oral lesions (T2-3), selection of the approach technique depends on the location of the tumor and the surgeon\u2019s preference and experience. The mandibulotomy approach is usually performed for larger tumors that are difficult to access. This may have led to higher stages of disease in patients who underwent mandibular surgery in this study.In patients without mandibular surgery, the proportion of patients with lip and buccal cancer was higher. Most patients in this group were elderly women with a history of betel quid chewing. These factors explained the difference in sex and smoking between the groups in Table Of the 32 patients who underwent mandibular surgery, 11, 15, and six underwent marginal mandibulectomy, mandibulotomy, as well as combined mandibulotomy and marginal mandibulectomy, respectively. Among the 17 patients who underwent marginal mandibulectomy, none experienced postoperative fracture or osteomyelitis.Among the 21 patients who underwent mandibulotomy, three had mandibulotomy-related complications, one of which had an immediate postoperative wound fistula. The fistula resolved with conservative treatment within three weeks after surgery. The other two patients had plate exposure. These complications were corrected prior to administering radiotherapy. Malunion, non-union, malocclusion, and dental complications were not observed.The radiotherapy protocol for each patient was conventional two-dimensional radiation with cobalt or a linear accelerator. Among these patients, eleven developed osteoradionecrosis of the mandible. Table Table In subgroup analysis, marginal mandibulectomy was nonsignificantly associated with the development of osteoradionecrosis. Moreover, other factors, such as tumor concurrent chemoradiation, radiation dose \u2265 60Gy, and smoking, showed hazard ratios of two or more but none of these factors were statistically significant.Table Osteoradionecrosis is one of the most serious oral complications following head and neck cancer treatment. It is characterized by bone tissue necrosis and failure to heal for at least three months . AlthougSeveral factors, such as poor dental health, post-radiation dental extractions, radiation doses, smoking and alcohol use, tumor locations , proximity of the tumor to the bone, and nutritional status, have been implicated as risk factors for the development of osteoradionecrosis ,10-12.While previous studies suggested that mandibular surgery during oral cancer operation might increase the risk of osteoradionecrosis, to date, few studies have demonstrated a statistically significant association between mandibular surgery and osteoradionecrosis occurrence ,12,13. TThe mandibulotomy approach is associated with increased complications, such as wound infection, plate exposure, and osteoradionecrosis . PreviouThe overall incidence rate of osteoradionecrosis in this study was 18.0%. This was similar to the rates from other studies using the same two-dimensional radiotherapy technique, which ranged from 5% to 20% . Recent The extent to which mandibular surgery influences the occurrence of osteoradionecrosis remains unclear. The analysis of risk factors for osteoradionecrosis may have various results, depending on population selection, types and stages of tumors, radiotherapy protocol, and duration of study. The existence of confounding variables in previous studies makes it difficult to establish a clear causal link between mandibular surgery and osteoradionecrosis occurrence ,7-12.In this study, confounding factors from the site of tumor, oral hygiene, and nutrition were minimized because only patients with oral cancer were included. In addition, the hospital treatment protocol required all patients to correct their oral hygiene and nutrition prior to surgery and radiotherapy. Unlike previous studies that might include various protocols of radiotherapy and conditions of patients, the treatment in this study was surgery and postoperative radiotherapy with curative intent for every patient ,12,18.The univariate and multivariate analyses revealed that mandibular surgery and mandibulotomy were significantly related to the occurrence of osteoradionecrosis. However, the study is underpowered to clarify the role of marginal mandibulectomy as an independent risk factor for developing osteoradionecrosis.Our study also found that patients with HIV infection showed a significant increase in the occurrence of osteoradionecrosis. HIV-infected patients are at a higher risk of developing osteonecrosis disease due to vascular compromise. This may be related to the HIV disease itself or its therapy . To our The primary limitation of this study is the inherent selection bias and confounders associated with the retrospective nature of this study. Analysis of risk factors for osteoradionecrosis may have limited the validity. Osteoradionecrosis is a late complication and many patients died or were lost to follow-up prior to the occurrence of osteoradionecrosis. The incidence rate of osteoradionecrosis varies depending on population selection. Although patient factors were minimized, as only patients with oral cancer were included, the relatively small number of cases included in this study limited the statistical significance of several parameters.The findings of this study suggest that mandibular surgery, especially in patients undergoing mandibulotomy, was significantly associated with the occurrence of osteoradionecrosis. Preventive therapies, such as the maintenance of meticulous oral hygiene and frequent visits to the dentist, should be the key focus for these patients. However, further studies including larger population sizes are required to verify these findings."}
+{"text": "In summary, our data revealed that the mechanism of dFdC resistance may be that c-Myc overexpression contributed to increased PD-L1 expression, and ARTS could overcome dFdC-resistant pancreatic cancer by inhibiting c-Myc and PD-L1. Our findings not only suggest c-Myc and PD-L1 as novel prognostic biomarkers in dFdC-resistant pancreatic cancer, but also provide ARTS as a promising candidate for overcoming dFdC resistance.Pancreatic cancer ranks fourth among cancer-related deaths, with a 5-years overall survival rate being below 10%. Gemcitabine (dFdC) has been considered the first-line drug for patients with pancreatic cancer. However, the clinical effectiveness is less than 20% due to drug resistance. Most importantly, overwhelming evidence suggested c-Myc and PD-L1 were generally highly expressed in pancreatic cancer patients. However, whether dFdC-resistant pancreatic cancer is associated with c-Myc and PD-L1 has not been elucidated. In our present study, we found that the expression of c-Myc and PD-L1 was markedly increased in pancreatic tumor tissues compared with adjacent tissues. Similarly, c-Myc and PD-L1 expression were also remarkably elevated in dFdC-resistant Panc-1 cells compared with parental cells. In addition, dFdC sensitivity was enhanced by the combination of dFdC and c-Myc inhibitors in Panc-1 cells. Interestingly, its sensitivity was reduced when c-Myc was overexpressed. Moreover, PD-L1 protein expression was dramatically down-regulated when treated with c-Myc inhibitors. Furthermore, artesunate (ARTS) screened from 18 compounds could reverse dFdC resistance in combination with dFdC in dFdC-resistant Panc-1 cells  Howevers. 4.8%) . Thus, tvia NT to be active. Studies have shown that the exact dFdC resistance mechanism is a decrease of human equilibrative nucleoside transporter 1 (hENT1) expression, which results in restricted intracellular uptake  Nucleoside transporters (NT): dFdC must cross the plasma membrane r uptake ; 2) Nuclr uptake ; 3) Tumor uptake ; 4) Epitr uptake . In addir uptake . HoweverRecent studies demonstrate that programmed death ligand 1 (PD-L1) and receptor molecule PD-1 are important targets in immune checkpoint blocking therapy. PD-L1 molecules highly expressed on the tumor cells surface specifically bind to PD-1 on the T cell membrane surface to inhibit the activity and function of T cells, thereby promoting tumor immune escape and drug resistance . PD-L1 eHerein, our findings revealed the effect of c-Myc and PD-L1 on dFdC resistant pancreatic cancer: 1) c-Myc and PD-L1 expression markedly raised in clinical cases of pancreatic cancer, and c-Myc expression correlated with PD-L1 expression in pancreatic cancer and may serve as prognostic predictors clinically. By constructing dFdC-resistant pancreatic cancer Panc-1 cells, our data found that the expression of c-Myc and PD-L1 was significantly elevated, suggesting c-Myc and PD-L1 may be novel prognostic biomarkers in pancreatic cancer, and inhibiting c-Myc and PD-L1 may be a potential strategy to rescue dFdC resistance in pancreatic cancer therapy. 2) By assessing the effect of natural compounds alone or in combination with dFdC in dFdC-resistant Panc-1 cells, our study revealed that ARTS in combination with dFdC could overcome dFdC resistance and inhibit DMBA-induced pancreatic cancer, implying that ARTS wound be a promising candidate to overcome dFdC resistance.Homo c-Myc plasmid was obtained from Gene Pharma . Primary antibodies against c-Myc and PD-L1 were purchased from Cell Signaling Technology ; \u03b2-actin was purchased from Santa Cruz Biotechnology . All secondary antibodies were purchased from Cell Signaling Technology .c-Myc inhibitors  were obtained from Selleckchem . Artemisinin (ARTS), Dihydroartemisinin (DHA), Shikonin, Oridonin, Andrographolide, Baicalein, Sulforaphane and Luteolin (purity \u226598%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. . RPMI-1640, Dulbecco\u2019s Modified Eagle\u2019s medium (DMEM), Opti-MEM and fetal bovine serum (FBS) were purchased from Gibco/BRL . Sulforhodamine B (SRB) and DMSO were purchased from Sigma-Aldrich . 2 cell incubator preserved by international institute for translational Chinese medicine, Guangzhou University of Chinese Medicine .Panc-1 and Mia-paca cells were obtained from Guangzhou Cellcook Biotech Co., Ltd. . All cells were culture in a 37\u00b0C, 5% COPanc-1 and Mia-paca cells were grown to 60\u201380% confluence in 6-well plates. Plasmids and Lipofectamine\u2122 2000 (Invitrogen) were transfected with c-Myc overexpression in Opti-MEM medium, respectively, and incubated for 20\u00a0min at room temperature. Their mixture was then added to the cells.3 cells were seeded in 96-well plates (100\u00a0\u03bcL per well), and treated with different compounds for 72\u00a0h. After that, cells were fixed with 10% trichloroacetic acid and then stained with 0.057% SRB, and crystals were solubilized with 10\u00a0mM Tris-HCl. Measure the absorbance of each well at 540\u00a0nm in a microplate reader (Bio-Rad Laboratories).Cell viability was assessed by SRB assays. 3 \u00d7 10Whether dFdC with 10,058-F4 and 10,074-G5 had synergistic, additive or antagonistic effect was analyzed by combination index (CI) method . CI = CAPanc-1 cells were treated with 15\u00a0nM dFdC and passaged after reaching 80% confluence. One-third of the cells continued to be maintained in culture with 15\u00a0nM dFdC and counted as R-P (passage number); one-third of the cells were tested for dFdC sensitivity; one-third were analyzed and cryopreserved.\u2212\u0394\u0394ct method. The sequences of PCR primers MYC is: 5\u2032-GCC\u200bACG\u200bTCT\u200bCCA\u200bCAC\u200bATC\u200bAG-3\u2032 5\u2032-TCT\u200bTGG\u200bCAG\u200bCAG\u200bGAT\u200bAGT\u200bCCT\u200bT-3\u2019; sequences of PCR primers CD274 is: 5\u2032-AAA\u200bTGG\u200bAAC\u200bCTG\u200bGCG\u200bAAA\u200bGC-3\u2032 5\u2032-GAT\u200bGAG\u200bCCC\u200bCTC\u200bAGG\u200bCAT\u200bTT-3\u2019.Total mRNA was extracted using Trizol reagent  and cDNA was synthesized by the PrimeScriptTM RT reagent Kit . PCR was performed with SYBR Green real-time PCR using an ABI 7500 system . Relative gene expression was normalized to GAPDH. All the primers were designed and obtained from Beijing Genomics Institute . The data were analyzed by 2The pancreatic cancer cells were collected and lysed on ice for 30\u00a0min, centrifuged, and quantified by the BCA protein quantification kit . Then protein samples were subjected to gel electrophoresis and transferred to PVDF membranes. The proteins were incubated overnight at 4\u00b0C with primary antibodies against c-Myc, PD-L1 and \u03b2-actin diluted in 2.5% BSA, and were detected with ECL luminescent solution (Thermo Fisher Scientific).Male C57BL/6J mice were purchased from Guangdong Medical Laboratory Animal Center , and maintained in the animal facility in the SPF animal laboratory (License number: SYXK (GZ) 2019\u20130,144) with 12\u00a0h light/dark cycle, 21 \u00b1 2\u00b0C, humidity 50 \u00b1 10% at International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine . All the animal experiments were approved by the Animal Experiment Committee of Guangzhou University of Chinese medicine .n = 10) and then administered intragastrically with 0.9% saline, dFdC (100\u00a0mg/kg), ARTS (100\u00a0mg/kg) or dFdC combined with ARTS for 5\u00a0weeks. Ultrasound and MRI were performed to visualize pancreatic cancer development. After the end of experiment, the mice were sacrificed and the pancreatic tissues and tumors of each mouse were dissected.After anesthesia by pentobarbital injection into the abdominal cavity, the pancreas was exposed by laparotomy in mice, and 10\u00a0mg 7,12-dimethyl-1,2-benzanthracene (DMBA) dissolved in 0.1% trioctane triglyceride was once injected into the pancreas . In addiMouse pancreatic tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5\u00a0\u00b5m). Sections were deparaffinized with xylene and rehydrated with graded ethanol. The slices were stained with hematoxylin for 5\u00a0min, then eosin for 10\u00a0s, and covered with neutral gum. The images were taken with a microscope.Pancreatic cancer tissue microarray (HPan-Ade120Sur-01) was obtained and analyzed from Shanghai Outdo Biotechnology Co., Ltd. There was a total of 63 cases on the tissue microarray, of which 58 cases detected paired tumor- adjacent tissue.Human pancreatic tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4\u00a0\u00b5m). Sections were deparaffinized with xylene and rehydrated, followed by antigen retrieval. The samples were incubated with anti-c-Myc and PD-L1 antibodies overnight at 4\u00b0C, followed by incubation with secondary antibodies for 1\u00a0h. Sections were stained to brownish-yellow with diaminobenzidine (DAB). The images were taken with a microscope.p < 0.05, **p < 0.01 and ***p < 0.001. Survival analysis of the patients was compared by the Kaplan-Meier method.All statistical analyses were conducted using GraphPad Prism 7.0. Significant differences were analyzed by one-way ANOVA between the samples and their respective controls. Differences were considered statistically significant at *p = 0.042; p = 0.022) and PD-L1 (p < 0.001) expression were associated with poor prognosis of patients  and 3-fold (p < 0.01), respectively (p < 0.05) and 4-fold (p < 0.001), respectively . And the CI of dFdC with 10,058-F4 and 10,074-G5 were 0.47 and 0.39, respectively, suggesting that c-Myc inhibitor combined with dFdC improved drug sensitivity. Additionally, similar results were also observed in Mia-Paca cells. When Mia-Paca cells were treated by dFdC, 10,058-F4 and 10,074-G5 alone for 72\u00a0h, the IC50 values were 15.59\u00a0nM, 63.09 and 60.76\u00a0\u03bcM, respectively , we initially examined the cell viability in pancreatic cancer Panc-1 and Mia-Paca cells. When Panc-1 cells were treated by dFdC, 10,058-F4 and 10,074-G5 with different concentrations for 72 h, the ICectively . To furtectively . But wheectively . Taken tp < 0.05; Some of evidences demonstrated that PD-L1 binds to PD-1 expressed on immune T cells, thereby promoting tumor immune escape and drug resistance . PD-L1 ep < 0.05; p < 0.001; 50 of dFdC was markedly elevated in Mia-Paca cells with overexpression of c-Myc , the c-Myc protein expression was enhanced by 1.4-fold and 1.5-fold in the c-Myc over-expressed group in Mia-Paca and Panc-1cells, respectively , and dFdC in combination with ARTS markedly decreased tumor weight/volume by 65.5 and 50.3%  and PD-L1 (p < 0.001) in pancreatic tumor tissues predicted a poor prognosis  contains numerous bioactive components, some of which exert anti-pancreatic cancer effects by reversing drug resistance, providing a new drug candidate for the treatment of pancreatic cancer . HoweverMyc axis . Meanwhiignaling . Consistt cancer . Considell death . Howeverll death . Consistll death , implyinIn conclusion, our present data revealed that c-Myc expression correlated with PD-L1 expression in pancreatic cancer and may serve as prognostic predictors clinically, indicating that restrainting c-Myc and PD-L1 overexpression in pancreatic cancer may provide a window to overcome dFdC resistance. Moreover, ARTS in combination with dFdC inhibited DMBA-induced pancreatic cancer, suggesting that ARTS may become an adjuvant dFdC-resistant agent for patients who suffer from pancreatic cancer. In addition, mechanistic studies on natural compounds such as ARTS have contributed to developing safer and more effective chemotherapeutic agents. This research provided a theoretical basis for the mechanism of dFdC resistance in pancreatic cancer, and provided potential targets and ideas for the treatment of pancreatic cancer and the exploitation of natural compounds."}
+{"text": "Material and Methods. Model cells for AS were created by inducing oxidized low-density lipoprotein (Ox-LDL) in human umbilical vein endothelial cells (HUVECs). After processing with AF and interleukin-1 receptor-associated kinase 1- (IRAK1-) overexpressed plasmid, cell viability was assessed by CCK-8; cholesterol efflux was tested using liquid scintillation counter; IL-6 and TNF-\u03b1 levels were determined with ELISA kits; ROS and apoptosis were confirmed using Flow cytometry. Besides, IRAK1-TAK1 pathway and apoptosis- and mitochondrial fusion-related proteins were monitored with western blotting analysis.  Atherosclerosis (AS) is a chronic inflammatory disease characterized by lipid metabolism disorder and vascular endothelial damage. Albiflorin (AF) has been certified to be effective in the therapy of certain inflammatory diseases, while the therapeutic effect and mechanism of AF on AS have not been fully elucidated.  Our results verified that AF could not only dramatically accelerate viability and cholesterol efflux but also attenuate inflammation, ROS production, and apoptosis in Ox-LDL-induced HUVECs. Meanwhile, AF could prominently prevent the activation of IRAK1-TAK1 pathway, downregulate apoptosis-related proteins, and upregulate mitochondrial fusion-related proteins in Ox-LDL-induced HUVECs. Moreover, we testified that IRAK1 overexpression memorably could reverse suppression of AF on inflammation, apoptosis, and IRAK1-TAK1 pathway and enhancement of AF on viability, cholesterol efflux, and mitochondrial fusion in Ox-LDL-induced HUVECs.  By blocking the IRAK1/TAK1 pathway, AF can significantly slow the course of AS, suggesting that it could be a viable therapeutic option for AS. Cardiovascular disease (CVD) is the leading cause of death worldwide, causing about 17.3 million deaths each year . AS is aRadix Paeoniae Rubra (RPR) is a traditional Chinese medicine derived from the dried root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch . RPR hasIn the current study, we used oxidized low-density lipoprotein (Ox-LDL) to stimulate human umbilical vein endothelial cells to create a cellular model of AS (HUVECs). Then, we explored the influences of AF on the related biological functions  of AS model cells. Moreover, we revealed the possible mechanisms of AF in alleviating HUVEC injury induced by Ox-LDL. Thus, AF might provide a new therapeutic opportunity for AS treatment.2.HUVECs were provided by American Type Culture Collection  and maintained in DMEM medium  with 10% fetal bovine serum  at 37\u00b0C with 5% CO\u03bcM AF for 24\u2009h, respectively. Vector (control) and IRAK1-overexpressed plasmids were purchased from Origene . Ox-LDL-induced HUVECs were evenly placed into a 6-well plate and cultured for 12\u2009h at 37\u00b0C. Then, the cells were transfected with the vector and IRAK1-overexpressing plasmid with Lipofectamine 3000  on the basis of the instructions.The cultured HUVECs were stimulated using 100\u2009mg/L Ox-LDL  in endothelial basal medium for 24\u2009h, and Ox-LDL-induced HUVECs were treated with 0, 4, 20, 100, 200, and 300\u20094/well. The HUVECs were treated based on the experimental purpose, respectively, and each group had 6 duplicate holes. After routine culture for 0, 24, 48, and 72\u2009h, HUVECs in each well were added with 10\u2009\u03bcL CCK-8 reagent . After 3\u2009h of culture, the OD value at 450\u2009nm was tested with a microplate analyzer.HUVECs were inoculated uniformly into 96-well plates at a rate of 1 \u00d7 10As described in previous literature , HUVECs \u03b1 levels were verified using IL-6 ELISA kit  and TNF-\u03b1 ELISA kit , respectively [On the basis of the instructions, IL-6 and TNF-ectively .5/well. After processing in line with different groups, HUVECs were washed 3 times with precooled PBS. After centrifugation and resuspended, HUVECs were dyed with propidium iodide (PI) and Annexin V-FITC based on the instructions of Annexin V-FITC/PI Apoptosis Detection Kit . Cell apoptosis was tested and analyzed using flow cytometry (Becton-Dickinson FACScan).The HUVECs were evenly inoculated into 6-well plates at a rate of 5 \u00d7 106 cells/well) in each group were inoculated into a 6-well plate and addressed with 2.5\u2009mmol/L DCFH-DA (Cat. No. d6883) at 37\u00b0C for 25\u2009min. After washing and staining, the fluorescence intensity values were detected by flow cytometry.The harvested HUVECs (1 \u00d7 10\u03bcg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Roche). PVDF membranes were blocked with 5% skimmed milk powder at room temperature for 2\u2009h and incubated with primary antibody (dilution ratio: 1\u22361000) overnight at 4\u00b0C and secondary antibody (dilution ratio: 1\u22362000) at 37\u00b0C for 2\u2009h. The membranes were addressed with ECL solution (Thermo Fisher Scientific), and the results were acquired with a gel imaging system. p-IRAK1 is referenced by IRAK1, p-TAK1 is referenced by TAK1, and GAPDH was applied as the reference for the other proteins. Anti-interleukin-1 receptor-associated kinase 1 (IRAK1), anti-p-IRAK1, anti-TGF-beta-activated kinase 1 (TAK1), anti-p-TAK1, anti-Bax, anti-caspase 3, anti-mitofusion1 (MFN1), anti-mitofusion2 (MFN2) and anti-opticatrophy1 (OPA1) were all purchased from Abcam (USA) [Total proteins were extracted with RIPA lysate from the collected HUVECs in each group, and protein concentration was tested by BCA method . Protein samples (40\u2009am (USA) .5 cells/well) in each group were inoculated into sterile cover glasses in a 6-well plate, respectively. After incubation overnight, HUVECs were fixed by 4% paraformaldehyde  and permeated by 0.5%Triton X-100. After sealing with normal goat serum, the HUVECs were exposed with anti-p-TAK1  overnight at 4\u00b0C and fluorescent secondary antibody  at 37\u00b0C for 1\u2009h. After DAPI  nucleation, the fluorescence intensity was observed using a fluorescence microscope  [The collected HUVECs  .P < 0.05 was for statistically significant.Statistical analysis was conducted using SPSS 21.0 ; statistical graphs was made using GraphPad Prism 8 software. The experimental data was represented as mean \u00b1 SD. One-way analysis of variance (ANOVA) was adopted for data comparison among multiple groups, and \u03bcM) to stimulate HUVECs. As displayed in \u03bcM AF group. Then, cellular model of AS was established with HUVECs through stimulation of 100\u2009mg/L Ox-LDL, and model cells were processed with 4\u2009\u03bcM and 100\u2009\u03bcM AF, which were selected based on the CCK-8 results above. Subsequently, the CCK-8 data signified that cell viability was observably attenuated in Ox-LDL group relative to that in control group, while cell viability was signally enhanced in Ox-LDL+100\u2009\u03bcM AF group versus that in Ox-LDL group  on cell viability in Ox-LDL-induced HUVECs , and a range of inflammatory cytokines [IRAK family is a family of protein kinases involved in TLR signal transduction . IRAK1, ytokines , 47. Andytokines . Therefoytokines \u201351. TAK1ytokines . And IRAytokines , 54. In Finally, our findings show that AF can halt the course of AS by inhibiting apoptosis and inducing mitochondrial fusion via IRAK1. As a result, we hypothesized that AF could be a molecular medication for the treatment of AS."}
+{"text": "Parental support plays an important role in children's schoolwork motivation and may have been even more important during the first UK COVID\u201019 pandemic lockdown because all schoolwork was completed at home. When examining the effect of parental support on children's schoolwork motivation, research has typically focused on comparing families with each other . In reality, however, the effect unfolds as a transactional, bidirectional process between parents and children over time . This research trend can result in imprecise conclusions about the association between parental support and schoolwork motivation.We examined bidirectional effects of parental schoolwork support and children's schoolwork motivation at both the between\u2010family and within\u2010family level.N\u00a0=\u00a098) in Years 7\u20139.This study reports findings from a weekly\u2010diary study conducted during the first UK COVID\u201019 school lockdown. Cross\u2010lagged within and between multilevel modelling was used to analyse data from UK secondary school students  predicted more support from parents. However, in contrast with predictions, weekly levels of parental support did not predict children's weekly fluctuations in motivation.Within\u2010family results were not consistent with between\u2010family results. This study is novel in showing that child\u2010driven effects appear to be important in eliciting parental support within families over time. Schoolwork motivation is an important predictor of academic achievement  and within\u2010person (state) processes, thereby shedding light on the complex interplay of schoolwork motivation and parental support. We proposed the following hypotheses:Based on research focused at the between\u2010person level such as Heatly and Votruba\u2010Drzal\u00a0, SilinskAt the within\u2010person level, we hypothesized that when children received higher parental support in one week, they would report higher schoolwork motivation in the subsequent week in comparison to a week where they received lower parental support. We also expected that individuals with higher schoolwork motivation in one week would receive more parental support in the subsequent week.Mage\u00a0=\u00a012.70\u2009years, SD\u00a0=\u00a01.04\u2009years). Among the students, 47% were in Year 7 (first year of secondary school), 22% were in Year 8, 29% were in Year 9. Most children in the sample were of white ethnicity  and one of their parents. All participants were recruited using email through schools or online advertisements. For the children's questionnaire, responses were obtained from 48 children at week 1 (51% missing), 72 children at week 2 (24% missing), 78 children at week 3 (20% missing), 76 children at week 4 (22% missing), and 65 children at week 5 (34% missing). Most parental responses were from mothers (90.59%). There were 28 participants who had five data points, 33 participants who had four data points, nine participants who had three data points, 11 participants who had two data points, five participants who had one data point, and 12 who did not answer the schoolwork motivation questions. Students' ages ranged from 11\u201315\u2009years  set up during the pandemic to recruit participants for studies. We included the researcher's email address and sign\u2010up link in our information. The sign\u2010up form asked the parent to provide email addresses so we could send a link to the weekly questionnaires. Data were collected through online questionnaires via Qualtrics. Data collection started on 19th June 2020 (week 13 of the UK lockdown) and was completed on 20th July 2020 . During five consecutive weeks, students and parents received an invitation email including the questionnaire link at 10\u00a0am every Friday. The weekly questionnaires covered the children's weekly schoolwork experience during the COVID\u201019 pandemic, etc. In addition, demographic questions were included at the start of the study (the first questionnaire that participants completed). Therefore, the total duration for the children's weekly questionnaire was around 20\u2009min for the first time and 15\u2009min for all subsequent weeks. For the parents' weekly questionnaire, the total duration was about 15\u2009min for the first questionnaire and 10\u00a0min for all subsequent weeks. To increase the responses to the questionnaires, families who completed half the weekly questionnaires received \u00a310 in vouchers, and families who completed all weekly questionnaires received \u00a320 in vouchers. In the current study, we only include demographic information from parents.We used two methods of recruitment. First, we emailed schools asking them to send out an advertisement about our study to parents in their newsletters. Six schools agreed to include our information in school newsletters. The six schools were in southeast England and are broadly representative of English secondary schools. Second, we placed advertisements about the study on social media (Twitter) and a website .Students' schoolwork motivation was measured by self\u2010report every week and was formed of two subscales assessing expectancy and value beliefs. Expectancy and value beliefs were assessed for two core school subjects, mathematics and English. Expectancy beliefs were assessed with eight items adapted from a measure used in Trautwein et al.\u00a0. Four itEnglish\u00a0=\u00a0.82, \u03b1Maths\u00a0=\u00a0.83). All items utilized a 4\u2010point Likert scale from 1 (Completely disagree) to 4 (Completely agree). High scores on these measures indicate high expectancy beliefs and high value beliefs. The schoolwork motivation  items are reported in the Table\u00a0To measure the value component, we adopted eight items from the Task Value Scale . In weekly studies, where participants have to complete the same questionnaire multiple times, it is common practice to use fewer items  to 4 (Completely agree). High scores on these items indicate more parental schoolwork support. Table\u00a0Two items were adapted from Dumont et al.\u00a0 to assesChildren's race , free school meal eligibility, and school types were reported by parents when they completed the first questionnaire.between, Supportbetween). To obtain the within\u2010person component, a person's mean was subtracted from the individual's value for each week , representing weekly variations from a person's mean. Furthermore, we computed a lag variable for each within\u2010person component . This variable allows us to predict how expectancy and support in week t  predicts expectancy in the subsequent week t\u2009+\u20091 (Expectancywithin t+1). Thus, this approach allows us to compute auto\u2010regressive effects (expectancy in week t affects expectancy in the following week t\u2009+\u20091) as well as cross\u2010lag effects (expectancy in week t affects support in the subsequent week t\u2009+\u20091).The main aim was to disentangle the reciprocal relationship between children's schoolwork motivation  and their perceived parental schoolwork support at the between\u2010person and within\u2010person level. Cross\u2010lagged between\u2010 and within\u2010level models were used to analyse the nested data . This strategy requires an analytical strategy involving dummy codes (0/1) and the use of interaction terms with the dummy codes to compute the effects for both dependent variables Expectancywithin_t+1 or with Supportwithin_t+1 , i indexes individual, and t indexes week. \u03b300 and \u03b303 indicate the two fixed intercepts. \u03b301, \u03b302, \u03b304, and \u03b305 represent between\u2010person level fixed effect. \u03b310, \u03b320, \u03b330 and \u03b330 represent the within\u2010person level fixed effect. up0i and uc0i indicate person\u2010specific random intercepts. up1i, up2i, uc1i and uc2i represent the random slopes of Expectancywithin_t and Supportwithin_t respectively. \u03b5it stands for the regression residual for person and week.In this double intercept multilevel model, Y represents the dependent variables  and parental schoolwork support were high. Table\u00a0We first assessed the variability of children's schoolwork motivation over time. This variability can be examined at the level of raw data, within\u2010person changes (how much someone changes from wave to wave) and between\u2010person differences . Figure\u00a0Intraclass correlations (ICC) were computed to decompose the variance in motivation into between\u2010person and within\u2010person components. Table\u00a0To test our hypotheses, we examined the bidirectional associations between schoolwork motivation and parental schoolwork support at the between\u2010person and the (cross\u2010) lagged within\u2010person levels.B\u00a0=\u00a00.03; English expectancy as the predictor, B\u00a0=\u00a0\u22120.03) or Maths . Average levels of parental support and average levels of value beliefs did not significantly influence each other in English  or Maths .The between\u2010person analyses assessed whether average levels of parental support were associated with average levels of motivation (see Tables\u00a0B's range from \u22120.07 to \u2013 0.04). The time effects on expectancy beliefs and value showed an overall decrease over time (B's range from \u22120.01 to \u2013 0.006) except for the Maths value. Results for the Maths value beliefs indicated a slightly increasing time trend. However, these changes did not differ at a statistically significant level (B\u00a0=\u00a00.002).The results revealed that there was a negative effect of time on parental support  or English . Parental schoolwork support also did not predict subsequent maths schoolwork value  or English schoolwork value . Thus, the results did not indicate that parental support in one week predicted children's motivational beliefs in the subsequent week.Parental schoolwork support did not predict subsequent expectancy beliefs in either Maths . However, weekly variations in English expectancy beliefs were not significantly related to subsequent perceived parental support in English . Both value for English  and maths  did not predict subsequent levels of parent support.There was some evidence that children's motivational beliefs in one week predicted perceived parental support in the subsequent week. Specifically, when children reported higher maths expectancy beliefs than usual, they reported greater perceived parental support in maths in the subsequent week . Specifically, children tended to report lower levels of support in the subsequent weeks following a week in which they reported a high level of perceived support.Additionally, perceived parent support in one week significantly negatively predicted subsequent levels of parent support  was due to the difference between individuals . The second aim of this study was to examine the bidirectional effects between parental support and schoolwork motivation. We found that both individual differences between children and within\u2010person fluctuations in children's perception of parental support did not predict children's schoolwork motivation. Another finding was that higher maths expectancies predicted higher parental support for maths schoolwork at the within\u2010person level. Overall, these results shed new light on the association between perceived parental support and children's schoolwork motivation at the between\u2010person and within\u2010person levels.When splitting variance in children's schoolwork expectancies and value, there is a substantial trait\u2010like proportion and a smaller within person time\u2010variant proportion. Most of the variance in students' expectancies and value were located at the between\u2010person level, which is in accordance with findings that there are large individual differences in children's motivation . Children lower in motivation and those without good internet access are likely less represented in our sample. However, the consistently high level of motivation in this sample points to high levels of resilience among these children. In the academic context, resilience is viewed as the student's ability to successfully adjust to adverse life events Martin,\u00a0. StudentOn the question of the association between parental support and schoolwork motivation, our study found that parental support did not predict students' expectancy or value beliefs at the between\u2010person level. This finding is contrary to our expectations and previous cross\u2010sectional and traditional longitudinal studies (with longer time lags) that have suggested that parental support seems to facilitate students' motivation  typically showed low relations (Gaspard et al.,\u00a0We found no evidence of within\u2010person associations between schoolwork values and parental support. The lack of association may have occurred because motivational values are more stable than expectancy beliefs (Gaspard et al.,\u00a0Furthermore, children perceived less parental support and had lower expectancy and value over time. Children and parents may have been overwhelmed with home learning, parents felt unable to help, or parents had limited understanding of the schoolwork material. There was also a negative association between parent support in one week and the subsequent week, which may have resulted from parents believing that their children were able to complete schoolwork independently after parents previously provided a high level of support. Thus, parents might have provided less support with schoolwork subsequently. Our findings illustrate the importance of schoolwork motivation for subsequent parental support. One practical implication is that parents should be advised to pay special attention to children who have low maths schoolwork motivation because these children are less likely to receive parental support.The weekly diary study design allowed us to examine the association between parental support and schoolwork motivation at between\u2010person and within\u2010person levels. Nonetheless, some limitations should be noted. First, the generalisability of these results is subject to certain limitations because our results are based on an opportunity sample from years 7 to 9. The extent to which our findings generalize to other ages is unknown. Second, this study's duration was fairly brief at only five weeks and took part in a very specific context of the first UK lockdown when children were forced to study at home rather than in school. Findings may differ under typical circumstances where children attend school in person and parental support is limited to homework outside school. It is difficult to know if the findings would generalize when children learn in school rather than at home. We suspect that parents would continue to be affected by their children even when children are in school. There is much past work suggesting that parents often help children with homework (for a review, see Wilder,\u00a0In conclusion, at the between\u2010person level, there was no evidence of bidirectional effects between motivation and support; at the within\u2010person level, more parental support did not predict subsequent increases in motivation. However, there was some evidence supporting the presence of child\u2010driven effects on parental support. The present study adds to the growing body of research exploring schoolwork motivation and its bidirectional relations with parental help. This study goes beyond previous research by using an intensive weekly diary approach to assess schoolwork motivation and parental support. Taken together, the results of this study highlighted the predictive power of schoolwork expectancy on parental support. The findings suggest that children influence parents. Future research needs to incorporate an understanding of children as actors to better understand the development of academic motivation.Yao Wu: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; software; visualization; writing \u2013 original draft; writing \u2013 review and editing. Peter Hilpert: Formal analysis; software; visualization; writing \u2013 review and editing. Harriet Tenenbaum: Conceptualization; methodology; writing \u2013 review and editing. Terry Ng\u2010Knight: Conceptualization; data curation; formal analysis; investigation; methodology; software; visualization; writing \u2013 review and editing.The authors do not have a conflict of interest.Appendix S1Click here for additional data file."}
+{"text": "There is limited knowledge on durability of neutralization capacity and antibody affinity maturation generated following two versus three doses of SARS-CoV-2 mRNA vaccines in na\u00efve versus convalescent individuals (hybrid immunity) against the highly transmissible Omicron BA.1, BA.2 and BA.3 subvariants. Virus neutralization titers against the vaccine-homologous strain (WA1) and Omicron sublineages are measured in a pseudovirus neutralization\u00a0assay (PsVNA). In addition, antibody binding and antibody affinity against spike proteins from WA1, BA.1, and BA.2 is determined using surface plasmon resonance (SPR). The convalescent individuals who after SARS-CoV-2 infection got vaccinated develop hybrid immunity that shows broader neutralization activity and cross-reactive antibody affinity maturation against the Omicron BA.1 and BA.2 after either second or third vaccination compared with na\u00efve individuals. Neutralization activity correlates with antibody affinity against Omicron subvariants BA.1 and BA.2 spikes. Importantly, at four months post-third vaccination the neutralization activity and antibody affinity against the Omicron subvariants is maintained and trended higher for the individuals with hybrid immunity compared with na\u00efve adults. These findings about hybrid immunity resulting in superior immune kinetics, breadth, and durable high affinity antibodies support the need for booster vaccinations to provide effective protection from emerging SARS-CoV-2 variants like the rapidly spreading Omicron subvariants. Here the authors show that a third SARS-CoV-2 vaccination significantly boosts neutralizing antibodies against Omicron subvariants and that hybrid immunity (infection and vaccination) results in broader neutralization activity and cross-reactive antibody affinity maturation. Viral spike protein of the Omicron variant contains higher number (>30) of mutations compared to other variants, raising concerns that Omicron is resistant to neutralizing antibodies generated following SARS-CoV-2 vaccination or infection, and a third vaccine dose was recommended to boost immunity.The emergence of Omicron (B.1.1.529/BA.1) variant of SARS-CoV-2 in November 2021, and its rapid spread across the globe, resulted in designation of Omicron as a variant of concern (VOC)2. Thus far, the BA.2 demonstrates higher transmissibility compared with BA.1 and has reached dominance in several countries in Europe and Africa3. However, limited knowledge exists regarding vaccine-induced neutralizing and antibody affinity following two doses vs. three doses of mRNA vaccination in na\u00efve vs. COVID-19 recovered individuals against SARS-CoV-2 Omicron BA.2 and BA.3 lineages and the durability of the vaccine-induced immunity. Therefore, it\u00a0is critical to know the persistence of cross-reactive immunity generated following SARS-CoV-2 vaccination to neutralize the SARS-CoV-2 omicron subvariants BA.1, BA.2 and BA.3.Since then, several subvariants of Omicron emerged (BA.2 and BA.3), each with its own set of mutations  vaccination in na\u00efve versus convalescent individuals (infection before vaccination) both at one-month after the second and third vaccinations and at 4 months post-3rd vaccination against vaccine-homologous WA1 and Omicron BA.1, BA.2 and BA.3 subvariants.N\u2009=\u200950) or SARS-CoV-2 convalescent  individuals both at peak (1 month) immune response after the second and third vaccination, as well as at 4 months post-3rd vaccination, against vaccine-homologous WA1 and Omicron BA.1, BA.2 and BA.3 subvariants. There were no significant differences for age, sex, race, ethnicity, body mass index (BMI) or vaccine type between the two cohorts  vaccination in a cohort of 81 adults: either na\u00efve  against the SARS-CoV-2 vaccine homologous WA1, as well as Omicron BA.1, BA.2 and BA.3 subvariants that were previously shown to correlate well with neutralization titers measured with authentic SARS-CoV-2 in plaque reduction neutralization testsNinety-four percent (94%) of previously na\u00efve individuals had positive WA1-neutralizing antibody titers (>1:60) at 1 month after the second vaccination with geometric mean titers (GMT) of 396. Following a third vaccine dose, 100% of na\u00efve adults were seropositive with 5.9-fold average increase in neutralization titers (GMT\u2009=\u20092333) compared with the second vaccination. However, by four months after the third vaccine dose, the neutralization titers were 3.2-fold lower (GMT\u2009=\u2009729) with 96% na\u00efve individuals with PsVNA50\u2009>\u20091:60 Fig.\u00a0. In compNeutralization titers were significantly reduced against all three Omicron subvariants compared with WA1 Fig.\u00a0. After trd vaccine dose  but not at 4 months after the third vaccine dose , and at 1 month (p\u2009=\u20090.0005) as well as 4 months (p\u2009=\u20090.0005) after the third vaccine dose  on the chip such as to measure monovalent interactions independent of the antibody isotype, as described before11.Surface Plasmon Resonance (SPR) was used to measure total antibody binding against recombinant spike proteins derived from vaccine homologous WA1 and currently predominant circulating Omicron BA.1 and BA.2 subvariants as determined by Resonance Units (RU) Fig.\u00a07. The pup\u2009<\u20090.0001) and BA.2 (p\u2009=\u20090.0211) spikes, and against BA.1 spike at 4 months after the third vaccine dose (p\u2009=\u20090.0068)  Fig.\u00a0.14. Previously, we showed that antibody kinetics measured under optimized SPR conditions primarily represent the monovalent interactions between the antibody-antigen complex, as antigen-antibody binding off-rates of the IgG and Fab interaction with protein antigens were similar14.To determine qualitative difference in antibody avidity maturation against SARS-CoV-2 spike proteins induced following mRNA vaccination, antibody dissociation kinetics (off-rate constants), which describe the stability of the antigen-antibody complex, i.e., the fraction of complexes that decay per second in the dissociation phase, that are independent of antibody concentration were used as a surrogate for overall average avidity of polyclonal antibody were determined directly from the human polyclonal sample interaction with recombinant purified SARS-CoV-2 spike proteins of vaccine homologous WA1 as well as Omicron BA.1 and BA.2 using SPR in the dissociation phase only for the sensorgrams with Max RU in the range of 10\u2013150 RU, as described beforeAntibody binding dissociation rates against WA1 spike were slower indicating higher binding affinity after the third vaccination compared with the second vaccination against the vaccine spike protein Fig.\u00a0. The antnd dose , one month after the 3rd dose  and at four months after the 3rd dose (p\u2009=\u20090.0024 for BA.2 spike)  was observed one month after the 3rd dose compared with post-2nd vaccine dose that was maintained at four months post-3rd vaccination  Fig.\u00a0 and S7.The antibody evolution after mRNA vaccinations and their decay followed parallel trajectories with good correlation between PsVNA50 neutralizing antibody titers and spike antibody binding  are determined by multiple factors including immune escape due to specific amino acid mutations in the spike, the levels (specificity and affinity) of cross-reactive polyclonal antibodies elicited by prior infection and/or vaccination, and waning immunity18, as well as BA.3. But in our study, convalescent individuals with hybrid immunity showed better antibody response against the rapidly spreading Omicron BA.1, BA.2 and BA.3 compared with vaccinated and boosted individuals with no history of prior infection. Our data and other studies suggest that Omicron BA.3 may evade immunity acquired from vaccination slightly more efficiently than BA.1 and BA.219. Therefore, the need for an additional booster vaccination may vary based on the individual\u2019s infection/vaccination histories and the circulating strains, in addition to various risk factors. In addition, our study demonstrated a strong correlation between SARS-CoV-2 neutralization titers and spike-binding antibody affinity against Omicron subvariants BA.1 and BA.2 spikes. Importantly, even at four months after the third dose the antibody binding affinities against the Omicron subvariants BA.1 and BA.2 were maintained and trended higher for the individuals with hybrid immunity. Recently, Wratil et al., reported similar findings but our data expands the antibody analyses, including delineating antibody affinity maturation and persistence of protective immunity to the Omicron subvariants up to 4 months post-third SARS-CoV-2 mRNA vaccination20. Although hybrid immunity due to prior SARS-CoV-2 infection followed by vaccination leads to broader antibody responses, it is important to understand that any infection still comes with a risk of complications. The gradual drop in antibody titers following hybrid immunity, irrespective if the infection took place before or after vaccination, suggest the immunity wanes at similar rates following vaccination and breakthrough infections with eventual loss of protection against circulating SARS-CoV-2 strains23. A possible role of immune imprinting in SARS-CoV-2 immune response due to prior SARS-CoV-2 infection/vaccination or the original antigenic sin (OAS) hypothesis, whereby adults with B-cell memory due to prior exposure to seasonal coronaviruses24 requires further investigation. Recently, we observed anti-S2 cross-reactivity in naive older children but not in the younger children (<4 years old), who share homology with HKU1, 229E and OC4325. OAS was also observed in mice immunization studies with seasonal CoV followed by SARS-CoV-2 spike26. Most of these cross-reactive antibodies do not neutralize SARS-CoV-2 and do not contribute to SARS-CoV-2 neutralization.Our study demonstrates that a third vaccination significantly boosts neutralizing antibodies against the Omicron subvariants, including BA.1 and BA.2, as recently reported32. In na\u00efve individuals, antibody affinity maturation post vaccination lags the convalescent group but may continue to evolve in the weeks following additional vaccine boosts. The breadth of variant recognition is likely to be impacted both by the antigenic distance between the infecting and vaccination strains, the rate of antibody affinity maturation through repeated GC seedings of memory B cells, and the time postinfection and last vaccination34.Our data along with previous studies suggest the presence of circulating memory B cells with expanded affinity-matured repertoires in convalescent individuals that are recalled by vaccination and likely to re-enter secondary germinal centers to undergo further somatic hypermutation (SHM) and antibody affinity maturation35. In previous studies, we had demonstrated a strong correlation between antibody affinity and protection from highly pathogenic avian influenza viruses37 and a correlation with clinical benefit in patients infected with Zika virus38, Ebola virus10, influenza virus39 and COVID-1914. Therefore, in addition to virus neutralization it is important to measure antibody affinity maturation against the SARS-CoV-2 spike not only for the vaccine strain, but also against spike proteins derived from the circulating variants of concern, that may influence the protective efficacy of vaccines against current and emerging SARS-CoV-2 variants of concern.The protective efficacy by vaccine-induced antibodies against emerging variants may be impacted by both specific amino acid mutations in the spike and the affinity of the polyclonal antibodies against the SARS-CoV-2 variants. An association was observed between high titers of low-affinity antibodies against RBD with the disease severity of COVID-19 patientsNo breakthrough infections with Omicron were reported in our cohorts. However, it will be important to demonstrate the impact of high-affinity anti-BA.1/BA.2 antibodies not only on severe outcome  but also on the clinical course and viral loads in Omicron\u00a0breakthrough infections. Based on the waning immunity, it is expected that na\u00efve adults will potentially require another vaccine booster earlier (4\u20136 months post-third vaccination) than individuals with hybrid immunity to generate protective strong neutralizing antibodies especially against the rapidly spreading Omicron subvariants BA.2.12.1, BA.4 and BA.5. In summary, our study demonstrates that hybrid immunity provides superior immune kinetics, breadth, and durable high-affinity antibodies vs. vaccine-only immunity against Omicron sublineages up to four months post-third vaccination with mRNA vaccines.Heat-inactivated de-identified samples were obtained from participants enrolled in the SPARTA  program in Athens, GA (USA) with written informed consent  or from Pfizer (BNT162b2) at 4-week or 3-week intervals between doses, respectively, between January and February 2021. All of them then received a third mostly homologous vaccine booster dose in September-October 2021. The primary series with the first two vaccine doses were homologous for each vaccinee. The boosters were either homologous or heterologous as shown in the Supplementary Table\u00a0Post-SARS-CoV-2 mRNA vaccination serum samples were collected after the second vaccination and third vaccination from 31 convalescent individuals who were exposed to SARS-CoV-2 but not hospitalized (had confirmed SARS-CoV-2 infection between March and November 2020) prior to circulation of any of the Omicron lineages as well as na\u00efve 50 healthy adults41.Sera were evaluated in a qualified SARS-CoV-2 pseudovirion neutralization assay (PsVNA) using SARS-CoV-2 WA-1 strain and the three Omicron variant lineages: BA.1 (B.1.1.529), BA.2 and BA.3 , pTrip-luc lentiviral vector and pcDNA 3.1 SARS-CoV-2-spike-deltaC19, using Lipofectamine 3000. The supernatants were harvested at 48\u2009h post transfection and filtered through 0.45\u2009\u00b5m membranes and titrated using 293T-ACE2-TMPRSS2 cells (HEK 293\u2009T cells that express ACE2 and TMPRSS2 proteins)41.Pseudovirions were produced as previously described12. For the neutralization assay, 50\u2009\u00b5L of SARS-CoV-2 S pseudovirions  were pre-incubated with an equal volume of medium containing serial dilutions  of heat-inactivated serum at room temperature for 1\u2009h. Then 50\u2009\u00b5L of virus-antibody mixtures were added to 293T-ACE2-TMPRSS2 cells (104 cells/50\u2009\u03bcL)41 in a 96-well plate. The input virus with all SARS-CoV-2 strains used in the current study were the same (2 \u00d7105 relative light units/50\u2009\u00b5L/well). After a 3\u2009h incubation, fresh medium was added to the wells. Cells were lysed 24\u2009h later, and luciferase activity was measured using One-Glo luciferase assay system . Antibody data was collected in MS Excel version 16.57. The assay of each serum was performed in duplicate, and the 50% neutralization titer was calculated using Prism 9 (GraphPad Software). Controls included cells only, virus without any antibody and positive sera. The limit of detection for the neutralization assay is 1:20. Any sample that does not neutralize SARS-CoV-2 at 20-fold dilution was given a value of 10 for representation and data analysis purposes. Two independent biological replicate experiments were performed for each sample and variation in PsVNA50 titers was <9% between replicates.Neutralization assays were performed as previously describedSteady-state equilibrium binding of post-SARS-CoV-2 vaccinated human polyclonal sample was monitored at 25\u2009\u00b0C using a ProteOn surface plasmon resonance (BioRad). The purified recombinant SARS-CoV-2 spike proteins were captured to a Ni-NTA sensor chip with 200 resonance units (RU) in the test flow channels. Serial dilutions  of freshly prepared samples in BSA-PBST buffer (PBS pH 7.4 buffer with Tween-20 and BSA) were injected at a flow rate of 50\u2009\u00b5L/min (120\u2009s contact duration) for the association, and disassociation was performed over a 600-s interval. Responses from the protein surface were corrected for the response from a mock surface and for responses from a buffer-only injection. Total antibody binding was calculated with BioRad ProteOn manager software (version 3.1). All SPR experiments were performed twice. In these optimized SPR conditions, the variation for each sample in duplicate SPR runs was <6%. The maximum resonance units (Max RU) shown in figures is for 10-fold diluted sample.14. Off-rate constants were determined from two independent SPR runs.Antibody off-rate constants, which describe the stability of the antigen-antibody complex (i.e. the fraction of complexes that decays per second in the dissociation phase) were determined directly from the interaction of human polyclonal samples with recombinant purified SARS-CoV-2 proteins using SPR in the dissociation phase only for the sensorgrams with Max RU in the range of 10\u2013150 RU and calculated using the BioRad ProteOn manager software for the heterogeneous sample model as described beforeDescriptive statistics were performed to determine the geometric mean titer values and were calculated using GraphPad. All experimental data to compare differences among groups were analyzed using lme4 and emmeans packages in R (RStudio version 1.1.463).p value was less than 0.05.The demographic characteristics of these study participants are shown in Supplementary Table\u00a0Samples were allocated randomly to each test group and tested blindly (researcher was blinded to sample identity) to minimize selection bias or detection bias. There were no exclusion criteria. All samples and data were used for analysis and presented in the study.Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileReporting Summary"}
+{"text": "Evaluating the serum cross-neutralization responses after breakthrough infection with various SARS-CoV-2 variants provides valuable insight for developing variant-proof COVID-19 booster vaccines. However, fairly comparing the impact of breakthrough infections with distinct epidemic timing on cross-neutralization responses, influenced by the exposure interval between vaccination and infection, is challenging. To compare the impact of pre-Omicron to Omicron breakthrough infection, we estimated the effects on cross-neutralizing responses by the exposure interval using Bayesian hierarchical modeling. The saturation time required to generate saturated cross-neutralization responses differed by variant, with variants more antigenically distant from the ancestral strain requiring longer intervals of 2\u20134\u00a0months. The breadths of saturated cross-neutralization responses to Omicron lineages were comparable in pre-Omicron and Omicron breakthrough infections. Our results highlight the importance of vaccine dosage intervals of 4\u00a0months or longer, regardless of the antigenicity of the exposed antigen, to maximize the breadth of serum cross-neutralization covering SARS-CoV-2 Omicron lineages.  \u2022A modeling to estimate the effects on cross-neutralization by the exposure interval\u2022The saturation time to generate a high neutralization titer differs for each variant\u2022The optimal booster vaccination interval is estimated to exceed 4\u00a0months Immune system; Immunity; Immune response; Virology Compared to the SARS-CoV-2 ancestral strain, the BA.1 virus has more than 30 amino acid mutations in the spike protein, including insertions and deletions. The BA.2 spike protein differs from the BA.1 spike protein at 27 amino acid positions, whereas the BA.5 spike protein differs from the BA.2 spike protein by four amino acids, including a L452R mutation. BA.2.75 differs the from BA.2 spike protein at nine amino acid positions, including K147E, W152R, F157L, I210V, and G257S, which are located in the N-terminal domain, and G339H, G446S, N460K, and R493Q, which are located in the receptor-binding domain (RBD). BQ.1.1 differs from BA.5 spike protein at R346T, K444T, and N460K.At the end of 2021, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron (B.1.1.529) variant emerged and rapidly spread worldwide. Since then, the Omicron variant has evolved into multiple sub-lineages, with BA.1, BA.2, and BA.5, emerging sequentially as the globally dominant variant. In August 2022, BA.5 was the most common variant circulating worldwide. In India, the proportion of BA.5 infections increased in May 2022, but the proportion of BA.2.75 (a variant of the BA.2 sub-lineage) has increased since June 2022, suggesting that the transmissibility of BA.2.75 may be higher than that of BA.5.,,Because of the numerous mutations that have accumulated in the spike protein of Omicron variants, Omicron variants are antigenically distinct from the ancestral strain and have the capacity to evade immunity introduced by a primary series of the first-generation COVID-19 vaccine, including COVID-19 mRNA vaccine containing ancestral spike antigen alone. Among Omicron sub-lineages, BQ.1.1 shows the greatest immune evasion against serum neutralization.,,,,,,,,,,,,Recently, BA.1 and BA.2 breakthrough infections in individuals vaccinated with COVID-19 mRNA vaccines were found to increase broad serum neutralizing activity against Omicron BA.1, BA.2, and prior VOCs at levels comparable to those of the ancestral strain.,One approach to account for these differences is to use Bayesian hierarchical modeling. Bayesian hierarchical modeling constructs a hierarchical structure of sub-models, and by estimating probability distributions using Bayes' theorem, it enables the extension and estimation of parameters in multiple groups based on common parameters shared among these groups.In this study, cross-neutralizing activity against SARS-CoV-2 variants, including the Omicron sub-lineages BA.1, BA.2, BA.5, BA.2.75, and BQ.1.1, was assessed using serum samples from individuals with breakthrough infections and booster vaccine recipients before or during the Omicron epidemic. Furthermore, Bayesian modeling was used to correct for the influence of different exposure intervals to enable estimation of the saturated serum cross-neutralizing responses against SARS-CoV-2 variants induced after breakthrough infection with the ideal exposure interval between vaccination and breakthrough infection.,We collected serum samples from individuals with breakthrough SARS-CoV-2 infections during the Omicron BA.1 wave and individuals who had received a booster dose of the first-generation mRNA COVID-19 vaccine containing the ancestral spike antigen alone . IndividNeutralization titers (NTs) were determined using live virus-based assays B. Serum To obtain an overall picture of the antigenicity of Omicron sub-lineages in serum samples from each exposure group, we performed antigenic cartography to locate the antigens and sera in a two-dimensional map D. Positi,,https://www.who.int/news/item/17-05-2022-interim-statement-on-the-use-of-additional-booster-doses-of-emergency-use-listed-mrna-vaccines-against-covid-19). Generally, an interval of 4\u20136\u00a0months after the second vaccination could be considered. In Japan, a vaccination interval of at least 3\u00a0months is recommended (https://www.mhlw.go.jp/stf/covid-19/booster.html). This vaccination strategy and the periods of pre-Omicron and Omicron waves resulted in distinct exposure intervals among the three exposure groups . Although a shorter vaccination interval during periods of transient surges in COVID-19 cases may benefit to the level of herd immunity, it may be insufficient to induce high levels of cross-neutralizing antibodies covering antigenically distinct variants. Previous studies have shown that in vaccine recipients, higher NTs were induced with a dose interval of 16\u00a0weeks  than with a dose interval of 4\u00a0weeks  between the first and second doses,,,,,An exposure interval of more than 128\u00a0days was required to induce broad cross-neutralizing activity against the BQ.1.1 variant, which was antigenically distant from the ancestral strain. As stated above, WHO recommends at least 4\u00a0months (approximately 120\u00a0days) after the second vaccination before booster vaccination, which is comparable to the exposure interval needed to induce broad cross-neutralizing responses. In contrast, the US Centers for Disease Control and Prevention recommends at least 2\u00a0months (approximately 60\u00a0days) between the second and third doses .Additional information and requests for resources and reagents should be directed to the lead contact, Tadaki Suzuki  under a material transfer agreement with the NIID, Tokyo, Japan.The characteristics of the participants are listed in MatchIt R functions .We used the SARS-CoV-2 ancestral strain WK-521 , Omicron BA.1 variant TY38-873 , Omicron BA.2 variant TY40-158 , Omicron BA.5 variant TY41-702 , Omicron BA.2.75 variant TY41-716 , and Omicron BQ.1.1 variant TY41-796  in this study. These variants were isolated using VeroE6/TMPRSS2 cells at NIID with ethics approval provided by the Medical Research Ethics Committee of NIID (#1178). More specifically, viruses belonging to the Omicron lineage were isolated at NIID using VeroE6/TMPRSS2 cells on respiratory specimens collected from individuals screened at airport quarantine stations in Japan and transferred to NIID for whole-genome sequencing.2.VeroE6/TMPRSS2 cells  were maintained in low-glucose Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 1\u00a0mg/mL geneticin, and 100\u00a0U/mL penicillin/streptomycin at 37\u00b0C supplied with 5% COAntibody titers for the ancestral spike (S) RBD and nucleoprotein (N)\u00a0were measured using Elecsys Anti-SARS-CoV-2\u00a0S and Elecsys Anti-SARS-CoV-2 kits according to the manufacturers\u2019 instructions.,50) SARS-CoV-2 viruses, followed by incubation at 37\u00b0C for 1 hour. Before adding of the virus-serum mixtures, 1.0 x 104 VeroE6/TMPRSS2 cells were seeded in each well of the 96-well plates to ensure a consistent cell number across all wells. The virus-serum mixtures were then placed on the cells and cultured at 37\u00b0C with 5% CO2 for 5\u00a0days. The cells were then fixed with 20% formalin and stained with crystal violet solution. NTs were defined as the geometric mean of the reciprocal of the highest sample dilution that protected at least 50% of the cells from a cytopathic effect, using two to four multiplicate series. Because of the limited volume of serum samples from individuals with breakthrough infections, this assay was performed only once. All experiments using authentic viruses were performed in a biosafety level 3 laboratory at NIID.Live virus neutralization assays were performed as described previously.Racmacs R function with 2,000 optimizations, with the minimum column basis parameter set to \u201c80\u201d.,Antigenic maps based on NTs against SARS-CoV-2 viruses were created using the https://www.r-project.org/). Measurements below the detection limit were recorded as half the detection limit. One-way analysis of variance with Dunnett\u2019s test or Tukey\u2019s test were used to compare the antibody titers. Statistical significance was set at p\u00a0<\u00a00.05.Data analysis and visualization were performed using GraphPad Prism 9.3.1  and R 4.1.2 . We inferred population means . Four independent MCMC chains were run with 5,000 steps in the warm-up and sampling iterations, with subsampling every five iterations. We confirmed that all estimated parameters showed <1.01\u00a0R-hat convergence diagnostic values and >500 effective sampling size values, indicating that the MCMC runs were convergent. The fitted model closely replicated the observed NT increases in each exposure group (https://www.r-project.org/). Information on the estimated means of saturated NTs against SARS-CoV-2 variants is summarized in Parameter estimation was performed using a Markov chain Monte Carlo (MCMC) approach implemented in rstan 2.26.1 (re group A and 3A."}
+{"text": "This study aimed to explore the influence of bone conduction hearing aid (BCHA) implantation on HRQoL and hearing disability in patients with hearing loss suffering from tinnitus. (2) Methods: Data were collected from an international hearing implant registry. Health Utilities Index Mark 3 (HUI-3), Spatial and Qualities of Hearing- 49 Questionnaire (SSQ) and self-reported tinnitus burden data for adult patients implanted with a BCHA (n = 42) who provided baseline as well as follow-up data 1-year post-implantation were extracted from the registry. Wilcoxon signed rank tests and paired samples t-tests were used to analyse outcomes data. (3) Results: Patients, with or without tinnitus, demonstrated clinically important mean improvements in HUI-3 multi-attribute utility scores, HUI-3 hearing attribute and SSQ scores. Hearing loss patients with tinnitus presented with a lower HRQoL than patients without tinnitus. (4) Conclusions: These findings demonstrate the importance of hearing rehabilitation in improving the quality of life and hearing disability of patients with or without tinnitus and in providing tinnitus relief in some patients with hearing loss and tinnitus. The term tinnitus describes a perceived auditory sensation in the absence of an associated external auditory stimulus . TinnituTinnitus is often directly related to age and hearing loss . HoweverCurrently, there is no cure for tinnitus; however, non-standardised interventions with varying efficacies exist for symptomatic relief, including auditory stimulation, education and reassurance, and cognitive behavioural therapy (CBT) . PsychotTinnitus is a severe condition comparable to other major chronic diseases . Its higAccording to the literature, some tinnitus sufferers have reported worsening symptoms in noisy areas; however, many have reported tinnitus aggravation from a lack of background noise, whereby its addition helped suppress their tinnitus ,25. TinnSubjective data on patient-reported outcomes using standardised measures on HRQoL such as the disease-specific Speech, Spatial and Qualities of Hearing Scale (SSQ) and the Health Utilities Index Mark 3 (HUI-3) are necessitated in addition to objective clinical outcome measures for optimal disease management . SSQ is Long-term follow-up based on acoustic hearing implantation influence on HRQoL in tinnitus patients are not readily available in the literature. Furthermore, these studies suffer from limited sample size and non-standardised reporting ,37. ThisThe study utilised retrospective data collected during the Cochlear Implant Recipient Observational Study (IROS). IROS was a prospective, longitudinal study that used repeated measures with intra-subject controls. The study used objective audiological measures and subjective evaluation tools such as the SSQ and HUI-3 to evaluate HRQoL and patient-related benefits of the use of implantable hearing devices, including BCHA devices. ClinicalTrials.gov with the identifier NCT02004353. The design, implementation and management of the registry have been published previously [The IROS is an international registry that also collected baseline and follow-up socio-economic information on adult recipients, including information on tinnitus burden. IROS is registered on eviously . The regThe presence of tinnitus, defined as noise or ringing in the head or ears, was assessed using a non-standardised form within the IROS patient questionnaire. Subjects were first asked to report pre-implantation if they experienced tinnitus. If yes, they were asked to complete follow-up questions based on how often it was experienced: always; sometimes; and do not know. The same questions were asked post-surgery, at the 1-year follow-up. Thereafter, a single question was asked based on how subjects would describe their tinnitus in comparison to pre-implantation: worse; same; better; and do not know. The registry contains baseline data, pre-implantation, and follow-up data for up to 3 years post-implantation, although there is significant attrition after 1-year follow-up. A total of 1164 subjects that received an implantable hearing device provided baseline data, but only 180 recipients provided data at the three-year follow-up visit . The study included all eligible adult patients receiving a BCHA device who have provided baseline as well as follow-up data after 1 year, including information on HUI-3, SSQ and self-reported tinnitus burden . EligiblThe IROS study was approved by the Ethical Review Boards of participating clinics in Colombia , Germany , Hungary , Poland , Spain , and South Africa  according to institutional and national research standards. All patients provided written informed consent prior to inclusion.Data were analysed using IBM SPSS version 29. Socio-demographic and other relevant clinical and nonclinical categorical data, including background characteristics at baseline, are presented descriptively. Categoric variables are represented as a number (n) and/or percentage. Continuous background data are presented as a mean value with standard deviation (SD).HUI-3 questionnaire data were analysed, and attribute and utility scores were calculated following standard methodology: Calculation Matrix HUI-3 Multi-attribute Utility HUI3 Multi-attribute Utility Function on Dead-healthy Scale Matrix .p-value of p < 0.05 was used to determine statistical significance.SSQ questionnaire scores from each domain were combined into mean domain-specific scores and a global mean score by taking the average score across each scale. HUI-3 and SSQ data were assessed graphically and by Kolmogorov\u2013Smirnov and Shapiro\u2013Wilk tests. These tests confirmed unequal distribution of the HUI-3 overall utility and hearing attribute scores pre- and post-implantation and normal distribution for the SSQ scores pre- and post-implantation. Therefore, the parametric Wilcoxon signed rank test for related samples was applied to determine mean utility and hearing attribute changes and paired t-tests were used for the SSQ score differences. A p-values are widely encouraged to enhance research quality [Effect size (d) values are presented as an additional measure to demonstrate the magnitude of the treatment effect, independent of sample size . Effect  quality . Point-biserial correlation tests were conducted to measure the strength of the association between tinnitus status and change in HUI-3 and SSQ scores pre- and post-implantation. Standard correlation value guidelines were applied whereby coefficient values between 0 and \u00b10.3 indicated a weak linear relationship, values between \u00b10.3 and \u00b10.7 indicate a moderate linear relationship, and values between \u00b10.7 and \u00b11 indicate a strong relationship. The positive or negative value indicates the direction of the strength of the relationship . HUI-3 mean overall HRQoL score differences of 0.03 or greater are considered clinically important, and differences of at least 0.01 may be meaningful and important in some contexts . DiffereThe study included 42 IROS participants who were implanted with a BCHA device. Forty patients received a transcutaneous device and the remaining 2 patients received percutaneous devices. The presence of tinnitus was reported by 23 participants at baseline, and 19 participants presented without tinnitus at baseline. IROS participants who had HUI-3 and SSQ data at baseline and one-year post-implantation were included. Participants who answered \u201cdo not know\u201d for tinnitus-related questions at baseline or had missing tinnitus status data were excluded from the study.The study comprised about 60% males and 40% females. The mean age of the group was 40.21 (\u00b114.58) years, and a majority of the participant group was based in Columbia (85.7%), with the remainder based in Poland (14.3%) . p = 0.218) and had a small effect size (d = 0.182). The HUI-3 mean utility score change for patients without tinnitus presented a clinically important mean improvement of 0.065 from pre-implantation (0.811) to post-implantation (0.876) (p = 0.277) and had a small effect size (d = 0.176).The HUI-3 mean scores for patients with tinnitus showed a clinically important mean improvement of 0.054 from pre-implantation (0.624) to post-implantation (0.678) . The Wil (0.876) ; howeverp = 0.139) and had a small effect size (d = 0.218). The HUI-3 hearing attribute mean score change for patients with tinnitus demonstrated a clinically relevant improvement of 0.051 from pre-implantation (0.865) to post-implantation (0.916) . Resultsp = 0.053) but displayed a medium effect size (d = 0.313).The HUI-3 hearing attribute mean score change for patients without tinnitus presented a clinically relevant improvement of 0.076 from pre-implantation (0.857) to post-implantation (0.933) . Resultsp < 0.001) and clinically relevant global SSQ mean improvement from pre (4.794) to post-implantation (6.566) for patients with tinnitus of 1.772 (p < 0.001) and clinically relevant global SSQ mean improvement from pre (5.036) to post-implantation (7.684) in patients without tinnitus of 2.648 (Results from the paired t-test in of 1.772  and dispof 2.648  and the p = 0.026) (p = 0.906). No other comparisons were statistically significant.Results show that there was a statistically significant difference in pre-operative HUI-3 score depending on tinnitus status in patients that were later implanted with a BCHA (= 0.026) , which wThe aim of this study was to explore the impact of BCHA on HRQoL and hearing disability in patients suffering from tinnitus by using standardised instruments in the form of HUI-3 and SSQ questionnaires. Subjective hearing assessments allow for a more comprehensive understanding of audiometrically assessed handicaps and provide information based on hearing ability pre- and post-intervention as well as additional hearing-related effects and its psychosocial impact ,46. The p = 0.218) and hearing attribute scores  for tinnitus patients and the overall utility change  and hearing attribute scores  for patients without tinnitus, however, yielded differences not reaching statistical significance, likely due to the small sample size. The mean score improvements between tinnitus (0.054) and non-tinnitus (0.065) patients are similar post-implantation and the correlation results show that there are no significant associations between tinnitus status and change in HUI-3 scores post-BCHA-implantation . This indicates that baseline tinnitus status is likely to have a negligible impact on change in HUI-3 multi-attribute scores between pre- and post-device implantation. Tinnitus status and pre-implant HUI-3 scores, however, are significantly negatively correlated . This indicates that patients with tinnitus are likely to present with lower HUI-3 scores before treatment than patients without tinnitus. This is also expected as individuals with untreated hearing loss without tinnitus have been shown to experience greater HRQoL than those with both untreated tinnitus and hearing loss [Important findings obtained from the HUI-3 measurements were that there were clinically relevant improvements in mean utility scores from pre- to post-implantation. The overall utility change  between tinnitus status and change in SSQ scores post-implantation. SSQ scores from a similar study were significantly lower in cochlear-implanted patients with tinnitus than without [The standardised SSQ system provides detailed information focused on patients\u2019 auditory performance in everyday situations to further enhance the data analysis . There w without . Hearing without ,51.Results from the tinnitus perception assessment pre-and post-implant demonstrated that 29% of patients felt an improvement in tinnitus symptoms post-implant. Comparable findings based on hearing healthcare professionals\u2019 survey results suggested that 60% experienced minor to major relief and 22% of patients experienced major tinnitus relief with hearing aids . Their sThe prevalence of tinnitus among BCHA patients (55%) is considerably higher than the global tinnitus prevalence rate. This can be expected for patients with hearing loss as they are more likely to experience distressing tinnitus symptoms than those able to experience a sound-filled environment ,22,28. TThe analysis of two standardised measures in the form of HUI-3 and SSQ questionnaires is a key strength of this study as they are valid instruments used to reliably measure both HRQoL as well as hearing disability ,32,57. HHearing loss patients with tinnitus present with a lower HRQoL than patients without tinnitus, however, the improvement in overall HRQoL post-implantation did not vary significantly between these patient groups. Bone conduction hearing implantation improves HRQoL and reduces hearing disability in patients with hearing loss, with or without tinnitus. Furthermore, tinnitus symptoms were improved in over a quarter of tinnitus patients 1 year post-device implantation. These findings demonstrate the importance of hearing rehabilitation in improving the quality of life and hearing disability of patients with or without tinnitus and in providing tinnitus relief in some patients with hearing loss and tinnitus."}
+{"text": "Here, we synthesized and characterized a novel two-dimensional (2D) conjugated electron donor\u2013acceptor (D-A) copolymer (PBDB-T-Ge), wherein the substituent of triethyl germanium was added to the electron donor unit of the polymer. The Turbo\u2013Grignard reaction was used to implement the group IV element into the polymer, resulting in a yield of 86%. This corresponding polymer, PBDB-T-Ge, exhibited a down-shift in the highest occupied molecular orbital (HOMO) level to \u22125.45 eV while the lowest unoccupied molecular orbital (LUMO) level was \u22123.64 eV. The peaks in UV-Vis absorption and the PL emission of PBDB-T-Ge were observed at 484 nm and 615 nm, respectively. Polymer solar cells (PSCs), comprising bulk-heterojunction (BHJ) photoactive layers of electron donors (D) and electron acceptors (A), have attracted enormous attention due to their lightweight properties, solution-processability, low-temperature printing fabrication and mechanical flexibility ,5,6,7,8.sc), and the alignment of the energy levels between electron donors and acceptors was reported to control open-circuit voltage (Voc). The crystallinity and morphology of electron donor\u2013acceptor pairs have also been reported to impact the fill factor (FF) dithiophene-4,8-dione, tin(ll) chloride dihydrate, anhydrous sodium sulfate (>99%), indium tin oxide (ITO) glass substrate, 1,3-Bis(5-bromo-2-thienyl)-5,7-bis(2-ethylhexyl)-4H,8H-benzo dithiophene-4,8-dione , tetrakis(triphenylphosphine) palladium (Pd(PPh3)4) and Y6 were purchased from Sigma-Aldrich . Triethyl germanium chloride and 2-iodothiophene were obtained from TCI . Ethanol was obtained from EMD . Isopropyl alcohol was purchased from VWR . All materials were used as received without further purification.Dichloromethane , isopropyl magnesium chloride lithium chloride complex , trimethyl tin chloride , acetone and deuterated chloroform (1) were added to the round-bottom flask under stirring condition. Then, iPrMgCl\u00b7LiCl  was added dropwise to the reaction mixture, which was left under stirring conditions for 3 h at room temperature. The mixture was quenched with cold DI water (30 mL), which was then extracted by DCM (25 mL) three times. The organic layer was collected and dried over anhydrous sodium sulfate (Na2SO4). The volatile solvent was removed under vacuum with mild temperature; then, the residue was purified by column chromatography using hexane: ethyl acetate (5:1 v/v) as an eluent with a retention factor (RF) of 0.0789 to yield the triethyl(thiophen-2-yl) germane as a colorless oil. Yield = 5.4 g (86%). 1H NMR , \u03b4 (ppm): 7.62\u20137.59, 7.25\u20137.22, 7.20\u20137.19, 1\u20131.3. Triethyl(thiophen-2-yl) germane was synthesized via a method that was adopted from Sherborne G. et al. . Under t2) in THF (45 mL) was placed in a 100 mL, two-neck, round-bottom flask. The solution was cooled to 0 \u00b0C. n-BuLi  was added dropwise into the RBF and the mixture was kept at 0 \u00b0C for 40 min before the mixture was refluxed at 65 \u00b0C under stirring for 2 h. After we cooled the mixture down, 4,8-dehydrobenzo dithiophene-4,8-dione  was added in one portion, and the mixture was refluxed with stirring for another 2 h. Tin(ll) chloride dihydrate  in 10% HCl (15 mL) was added after the mixture was cooled down to room temperature and stirred overnight. The reaction was quenched using cold DI water (40 mL), and then the crude product was extracted using diethyl ether (25 mL) three times. The organic layer was collected and dried over anhydrous sodium sulfate (Na2SO4). The volatile solvent was removed under vacuum at a mild temperature and the crude product was purified using a column chromatography with hexane: ethyl acetate (5:1 v/v) as eluents to yield 4,8-bis-(5-(triethyl germanium)thiophene-2-yl)benzodithiophene as bright yellow gel. Yield = 0.8 g (36.2%). 1H NMR , \u03b4 (ppm): 7.67\u20137.65, 7.6\u20137.58, 7.47\u20137.45, 7.3\u20137.28, 1.00\u20131.4. Under the protection of argon, a solution of triethyl(thiophen-2-yl) germane  in THF (6 mL) was placed in a 25 mL round-bottom flask. The solution was cooled to \u221278 \u00b0C using dry ice with acetone, and then n-BuLi  was added dropwise. After the addition, the mixture was kept at \u221278 \u00b0C for 1 h; trimethyl tin chloride  was added dropwise. The resulting mixture was stirred overnight at room temperature. The mixture was then poured into cold DI water (6 mL) and extracted with diethyl ether (8 mL) three times; after drying over anhydrous sodium sulfate (Na2SO4), the volatile solvent was removed under vacuum at a mild temperature. Recrystallization was used for purification using ethanol as a solvent, and then dried for two days in a vacuum at a mild temperature to yield a yellowish white crystal of -2-trimethylstannylthieno[1]benzothiol-6-yl]-trimethylstannane. Yield = 0.092 g (15.6%). 1H NMR , \u03b4 (ppm): 7.49, 7.48\u20137.46, 7.2\u2013 7.3, 1.2\u20130.9, 0.6\u20130.5. Under the protection of argon, a solution of 4,8-bis-(5-(triethyl germanium) thiophene-2-yl)benzodithiophene-4,8-dione  were dissolved in dry toluene (5 mL). The reaction container was purged with argon for 20 min, and then tetrakis(triphenylphosphine)palladium (Pd(PPh3)4)  was added. After another flushing with argon for 20 min, the reactants were heated up to reflux them at 120 \u00b0C for 24 h. The solution was cooled down to room temperature and poured into cold methanol (MeOH) (50 mL), and then washed three times using cold methanol. The product was dried under vacuum at a mild temperature for 24 h to yield a dark purple solid of PBDB-T-Ge. Yield 0.09 g. 3), respectively. The polydispersity (PDI) was observed to be 1.41. 1H NMR , \u03b4 (ppm): 7.6\u20137.75, 7.45\u20137.35, 7.2\u20137.3, 7.15\u20137.25, 6.95\u20137.05, 1.4\u20130.95, 0.9\u20130.6.In a 25 mL, double-neck, round-bottom flask, -2-trimethylstannylthieno[1]benzothiol-6-yl]-trimethylstannane  and 1,3-Bis(5-bromo-2-thienyl)-5,7-bis(2-ethylhexyl)-4d-chloroform as solvent and the internal chemical shift reference standard was selected based on the residual proton resonance of the solvent, which were acquired, processed, and analyzed using Bruker Topspin (4.1.4) software. The molecular weight of polymers was determined by gel permeation chromatography (GPC) relative to polystyrene standards with chloroform as the eluent at a concentration of 1.0 mg/ mL for at least 12 h. The optical properties were tested using a UV-Vis spectrometer (Shimadzu 3600 Plus). This characterization was performed using chloroform solution in a quartz cuvette using polymer film on a glass substrate. The polymer film samples were prepared using drop-casting on a glass substrate. The photoluminescence study was performed using Spectro-fluorophotometer (Shimadzu RF-5301PC model) using a chloroform solution. Cyclic voltammetry study was performed using a potentiostat  at room temperature to determine the redox reaction. The surface morphology characterizations on PBDB-T-Ge:Y6  composite film were performed using atomic force microscopy  using a taping mode for imaging. Further surface morphology was performed using scanning electron microscopy  with secondary electron for the imaging.The structure of PBDB-T-Ge was confirmed using an NMR spectrometer . The spectra were collected using n-butyl lithium to activate the ethylhexyl branch groups of PBDB-T-C and trialkyl silyl groups of PBDB-T-Si, as seen in Our work implemented germanium on the side branch of the PBDB-T-Ge. Previous reports utilized 1H NMR spectra in 2) is synthesized, where the three peaks between 7.1 and 7.7 ppm are assigned to the proton of the single-branched thiophene and the multiple peaks from 0.8 to 1.4 ppm are assigned to the alkyl groups. The 1H NMR spectra show that the implementation of triethyl germanium on the thiophene was successful, simplifying and enhancing product yields.onset) at 620 nm, and the maximum absorption (\u03bbmax) occurred at 477 nm. In comparison, the absorption peak of reference polymer  of 666 nm and maximum absorption (\u03bbmax) at 507 nm, while another reference polymer film  in acetonitrile as the supporting electrolyte and the ferrocene/ferrocenium as the internal reference. Cyclic voltammogram was obtained at a scan rate of 10 mV/s with a 1 mV sample interval.The HOMO and LUMO of PBDB-T-Ge were measured to evaluate the effect of the substitution of the triethyl germane sidegroup on the electronic energy levels using cyclic voltammetry. The electrochemical study was performed using conventional three electrodes configuration consisting of an Ag/AgCl electrode as a reference electrode, the glassy carbon electrode as the working electrode, and the platinum wire as the counter-electrode. We used tetraethylammonium tetrafluoroborate (Etoxonset and Eredonset), respectively, according to the Equations (1) and (2) [1/2.Fc/Fc+ is the half-potential of ferrocene/ferrocenium coupled to the electrochemical measurement system, and the energy level of Fc/Fc+ was taken as 4.8 eV below vacuum. Ferrocene/ferrocenium half-potential was measured to be 0.44 V ferrocene/ferrocenium, and Equations (3) and (4) are as follows:Solutions of PBDB-T-Ge polymer and Y6 were prepared by dissolving in chloroform and the films were deposited onto the glassy carbon button electrode via a drop-casting procedure. It was then air-dried. HOMO and LUMO energy levels were estimated from onset oxidation and reduction potentials (E and (2) .E (HOMOoxonset) and onset reduction potential (Eredonset) of PBDB-T-Ge were measured to be 1.09 and \u22120.72 V vs. Ag/AgCl, respectively. The HOMO energy level and LUMO energy level of PBDB-T-Ge were calculated to be \u22125.45 and \u22123.64 eV, respectively, as seen in sc, which is expected to enhance the photovoltaic performance. The data were collected from PBDB-T-C in 0.1 M tetrabutylammonium hexafluorophosphate (Bu4PF6) acetonitrile solution at a scan rate of 50 mV/s, while the data of PBDB-T-Si were collected using Ultraviolet Photoelectron Spectroscopy (UPS). The detailed electrochemical data for PBDB-T-Ge and its counterparts, Y6, are listed in oc, which is expected to enhance the PCE of PBDB-T-Ge-based PSC. It should be noted that the electrochemical bandgap (Egcv) of PBDB-T-Ge is 1.81 eV, which is similar to its optical bandgap of 1.86 eV.We noticed that the HOMO of PBDB-T-Ge was deepest among the three polymers and showed a smaller gap of 0.22 eV and 0.31 eV compared to PBDB-T-C and PBDB-T-Si, respectively. The small band gap is well-known to improve Jw/w) blend film was performed using atomic force microscopy (AFM) and electron-scanning microscopy (SEM), as depicted in Molecular-disorder-induced nanostructure variations in the BHJ are critical in optimizing photo-induced charge separation and transportation in OSCs. The surface morphology study and characterizations of PBDB-T-Ge:Y6  calculations were conducted to provide further insights into the fundamental aspects of the molecular architecture of germanium-based comonomers. All calculations were conducted with the Gaussian16 software package. Optimized geometries of all compounds were obtained at the B3LYP level of theory with the 6-31G(d) basis set for all atoms. Previous work ,40,41 shWe observed that the electron density distributions at the HOMO are highly localized on the electron-rich moiety of BDTT-Ge (M1) in the ground state while, in the excited state, it is distributed at the LUMO and highly localized in the electron-deficiency moiety BDD (M2). The transition of the electron density distributions at the HOMO and LUMO were observed from all cases of -C, -Si and -Ge.DFT calculations of Ge-based comonomer (triethyl-Ge) estimated the HOMO of \u22125.02 eV and LUMO of \u22122.36 eV with the band gap of 2.66 eV, while the band gaps for the counter-partner C-based comonomer (triethyl-C) and Si-based comonomer (triethyl-Si) were calculated to be 2.655 and 2.68 eV, respectively. The calculated electronic energy values of the HOMO, LUMO and band gap of the three co-monomers are summarized in A new 2D-conjugated donor\u2013acceptor (D-A) copolymer (PBDB-T-Ge) was synthesized, where two reaction methods were compared to implement triethyl Ge: the conventional n-BuLi basis and Turbo\u2013Grignard reactions. The Turbo\u2013Grignard reaction was observed to be efficient in incorporating the group IV element to the side chain with a yield of 86%. The PBDB-T-Ge exhibited a LUMO of \u22123.64 eV, which was closer to that of Y6, an electron-acceptor. The gap between the LUMO values was calculated to be ca. 0.22 eV, which is sufficient to expect efficient photo-induced charge injection between the electron-donor and -acceptor in BHJ PSCs. PBDB-T-Ge and Y6 showed a complementary absorption, indicating a wide range of light-harvesting. DFT calculations on the comonomer verified that there were no significant changes in the band gap when the Ge sidegroup of the polymer replaces carbon- and silicon-base sidegroups."}
+{"text": "Mycobacterium marinum is a prevalent nontuberculous mycobacterium (NTM)\u2010infecting teleosts. Conversely, little is known about mycobacteriosis in elasmobranchs, and M.\u00a0marinum infection has never been reported from the subclass. This study investigated the histopathological characteristics and localization of this mycobacterium through molecular analysis of two captive sharks, a scalloped hammerhead Sphyrna lewini and a Japanese bullhead shark Heterodontus japonicus, exhibited in the same aquarium tank. We detected genital mycobacteriosis caused by M.\u00a0marinum infection using molecular analyses, including polymerase chain reaction (PCR) and DNA sequencing targeting the 60\u2009kDa heat\u2010shock protein gene (hsp65), and peptide nucleic acid\u2013fluorescence in situ hybridization (PNA\u2010FISH) targeting the 16S rRNA gene. Both sharks showed granulomas in connective tissues of the gonads without central necrosis or surrounding fibrous capsules, which is unlike the typical mycobacterial granulomas seen in teleosts. This study reveals that elasmobranchs can be aquatic hosts of M.\u00a0marinum. Because M.\u00a0marinum is a representative waterborne NTM and a potential zoonotic agent, cautious and intensive research is needed to overcome a lack of data on the relationship between NTM and the aquatic environment in association with this subclass of Chondrichthyes. Peptide nucleic acid (PNA) probes have been applied to the detection of mycobacteria using clinical samples from humans  occurs circumglobally in warm\u2010temperate and tropical coastal seas .Here, we present the first reported cases of 22.1An approximately 10\u2010year\u2010old wild\u2010caught male scalloped hammerhead shark and a wild\u2010caught adult female Japanese bullhead shark of uncertain age were housed at the Shimane Aquarium in Shimane Prefecture, Japan. They were exhibited in a 1 million\u2010L tank exhibit along with other fish, including multiple teleost and elasmobranch species, and one sea turtle. Water pumped from the local sound (Sea of Japan), treated with pressure filtration, is used as the aquarium's water source. After 9\u2009years and 9\u00a0months in captivity, the hammerhead shark presented anorexia for 1\u2009month and was subsequently found dead in the tank. One month later, the bullhead shark was also found dead, but without any remarkable clinical signs. The captivity duration of this bullhead shark was unknown because Japanese bullhead sharks in the tank were not individually identified. Autopsies revealed marked hemocelom, multifocal congestions in the bilateral testes of the hammerhead shark and multifocal haemorrhages in the bilateral epigonal organ Figure\u00a0. ApparenTo assess the microbial community distribution in the rearing environment, samples of water (1\u00a0L) and filtration sand, as well as swabs of biofilms from a wall and a water intake were provided. All samples were sent to the Laboratory of Aquatic Medicine at Nippon Veterinary and Life Science University in Tokyo.2.2Samples from the brain, alimentary canal, liver, kidney, spleen, pancreas and reproductive organs were collected from both sharks, and samples from the epigonal organ, eyes and skin were collected from the hammerhead shark. The reproductive organs from the bullhead shark were frozen on site and then fixed in 10% neutral\u2010buffered formalin at our laboratory; the other remaining samples were fixed in 10% formalin on site. All samples were routinely processed to create paraffin\u2010embedded tissue blocks, sectioned at 2\u2010\u03bcm thickness, mounted on frosted glass slides, stained with haematoxylin and eosin (HE), Giemsa, Gram and Ziehl\u2013Neelsen (ZN) stains and subjected to histopathological examination.2.32.3.1Isolation of the etiological agent of mycobacteriosis was attempted from frozen organs, including the reproductive organs, kidney, spleen and liver, in both cases, and the epigonal organ from the hammerhead shark. Pretreatments were performed based on four methods, as follows: (1) non\u2010treatment; (2) decontamination by hydrochloric acid (HCl) solution, at a final concentration of 0.5\u00a0N , the beta subunit of RNA polymerase (rpoB) and superoxide dismutase (sodA), through the cycles described by Fukano et al.\u00a0. The DNA was tested for the presence of mycobacterial DNA by PCR on the C1000TM Thermal Cycler (Bio\u2010Rad) using GoTaq\u00ae DNA Polymerase (Promega) and mycobacterial universal primers targeting the 16S ribosomal RNA (16S rRNA), 65\u2009kDa heat\u2010shock protein , using the primer sets MU5\u2013MU6 and PU4F\u2013PU7Rbio for IS2404, and MU7\u2013MU8 for IS2606 . The phylogenetic tree of the hsp65 sequences was reconstructed using the neighbour\u2010joining method with Kimura's 2\u2010parameter model, implemented in MEGA X software and evaluated further in a bootstrap analysis of 1000 replicates.Purified products were sequenced by the FASMAC Corporation using the specific primers described in Table\u00a02.52.5.1M.\u00a0marinum ATCC BAA\u2010535 and M.\u00a0marinum JCM 17638T were used. To detect pathogens in the shark tissues, paraffin\u2010embedded tissue samples of the current cases, including the testes and epigonal organ of the hammerhead shark, and the ovary, uterus and shell gland of the bullhead shark, were used. As a negative tissue control, the ovary of another hammerhead shark, in which mycobacterial infection was not detected by ZN staining and by PCR assay, was also tested.To estimate the hybridizability and specificity of the designed PNA probes and to validate the PNA\u2010FISH procedure, mycobacterial isolates from the tissues of the current clinical cases and the reference strains 2.5.2M.\u00a0marinum, Mycolicibacterium llatzerense (NJB1901), M.\u00a0hodleri (NJB19061) and M.\u00a0obuense (NJB19062). Each probe was designed not to cross\u2010hybridize with the non\u2010targeted genomic sequences of the other NTM candidates, and therefore, it acted as a negative control for the other three NTM species like a sense probe. The probes were chosen with regard to purine content, avoiding hairpin formations and inverted repeats. The N\u2010terminus of all probes were labelled with cyanine 3 (Cy3) fluorescent dye. All designed probes were custom\u2010synthesized by PANAGENE Inc.With the genome and partial sequence database using BLAST, and the results of sequence analyses of DNA extracted from the present samples, we designed four PNA probes Table\u00a0, each of2.5.3The preparation of tissue sections and bacterial samples for FISH was performed as described in Lehtola et al.\u00a0 and Lefm2.5.4Microscopy was performed with a fluorescence microscope (Keyence BZ\u2010X700). TRITC OP\u201087764 and DAPI OP\u201087762 filter sets (Keyence) were used to analyse the Cy3 and DAPI signals, respectively. Digital images were captured under the fluorescence microscope, and image analysis was performed with BZ\u2010H3 software (Keyence).33.1In both testes of the hammerhead shark, foamy macrophages infiltrated into the serosal connective tissues in the resorption zone mixing with large numbers of infiltrating lymphocytes Figure\u00a0. NumerouAlthough it was somewhat difficult to distinguish inflammation cells since the sample was frozen after sampling, macrophage\u2010like cells with foamy cytoplasm infiltrated and accumulated in the ovarian connective tissue stroma of the bullhead shark Figure\u00a0. ZN staiIn the hammerhead shark, in addition to mycobacteriosis, we diagnosed panophthalmitis, corneal trephination, enteritis, dermatitis, focal lymphocytic meningitis and suspected collagenofibrotic glomerulopathy. In the bullhead shark, we additionally diagnosed focal lymphocytic gastritis, focal lymphocytic interstitial nephritis, pancreatitis and necrotic splenitis. However, in both cases, no apparent pathological features of mycobacterial suspicious lesions were detected other than in the testes or ovaries.3.23.2.1In total, three AFB strains were isolated from the testis of the hammerhead shark (NJB1901) and the ovary of the bullhead shark (NJB19061 and NJB19062). NJB1901 was cultured using Middlebrook 7H11 agar without pretreatment; on day 21 after inoculation at 30\u00b0C, it formed a smooth, white colony. NJB19061 and NJB19062 were cultured using the same agar without pretreatments; on day 14 after inoculation at 25 and 30\u00b0C, respectively, each formed a smooth, yellow colony. All purified colonies of the three isolates showed rapid growth on day 3, with Middlebrook 7H11 agar, at 25\u00b0C (NJB19061) or 30\u00b0C .3.2.2Seven isolates of mycobacteria were cultured from the environmental samples. The colony characteristics of the isolates are presented in Table\u00a03.33.3.1hsp65 gene, and from one part of the left testis (testis II) using primers to amplify the 16S rRNA, hsp65, rpoB and sodA genes. Sequence analyses indicated that M.\u00a0marinum was the most probable candidate bacterial species detected in four portions of the testes  of the hammerhead shark, and in the ovary of the bullhead shark, based on genomic evidence, whereas M. llatzerense was the most probable candidate species in testis II of the hammerhead shark, based on the high degree of molecular homology  and from the ovary, using primers to amplify the hsp65, rpoB and sodA genes. The results of sequence analyses of the isolates showed that NJB1901, NJB19061 and NJB19062 were identical with M.\u00a0llatzerense, M.\u00a0hodleri and M.\u00a0obuense, respectively , while apparent bands were observed with DNA extracted from the positive control (M.\u00a0pseudoshottsii JCM15466)  did not show positive signals indicating the presence of the targeted NTM in the tissue sections of both samples. In the negative controls, no signal was detected in the tissue sections examined Figure\u00a0. Also, n4hsp65 gene detected genomic evidence of nontuberculous mycobacteria (NTM) infection, with 100% nucleotide sequence identity to M.\u00a0marinum. The nucleotide sequence of hsp65 gene of M.\u00a0marinum shows a relatively high rate of similarity with mycolactone\u2010producing mycobacteria (MPM), such as M.\u00a0pseudoshottsii. However, in most M.\u00a0marinum strains reported previously, the insertion sequences IS2404 and IS2606 relating to mycolactone were not detected by PCR, unlike detection in MPM    (=Rhinobatos lentiginosus)  (Karsten & Rice,\u00a0Mycobacteriosis cases in this subclass have rarely been reported (Anderson et al.,\u00a0Mycobacterium marinum is a representative NTM agent in aquatic animals; it is considered an important candidate infectious agent of aquatic zoonosis because M.\u00a0marinum has been isolated from human lesions, including dermatitis (Tomas et al.,\u00a0M.\u00a0marinum infection and the potential for zoonotic transmission from an elasmobranch species have not yet been shown. Nevertheless, the present results prove that an elasmobranch species can be a host of waterborne M.\u00a0marinum and indicate that elasmobranchs, like teleosts, might act as a reservoir of M.\u00a0marinum infection in humans, especially immunocompromised individuals (Streit et al.,\u00a0The progress of granuloma pathology is usually dependent on the duration and amount of infecting bacteria (Swaim et al.,\u00a0Mycobacterium spp. in association with granulomatous inflammation (Broussard & Ennis,\u00a0M.\u00a0marinum in the reproductive organs in the current cases clarified that mycobacteria could cause gonadal lesions in elasmobranchs as in teleosts.In teleost fishes, the ovary and testis are not major organs involved in mycobacterial infection (Novotny et al.,\u00a0In conclusion, our study indicates that mycobacteriosis can be suspected in elasmobranchs even without typical caseating granulomas and it should be added to potential zoonotic diseases in aquaria housing sharks.The authors have no conflicts of interest to declare.Appendix S1Click here for additional data file."}
+{"text": "To evaluate the relationship between the fetal head\u2010circumference\u2010to\u2010maternal\u2010height (HC/MH) ratio measured shortly before delivery and the occurrence of Cesarean section (CS) for labor dystocia.This was a multicenter prospective cohort study involving four tertiary maternity hospitals. An unselected cohort of women with a singleton fetus in cephalic presentation, at a gestational age beyond 36\u2009+\u20090\u2009weeks and without any contraindication for vaginal delivery, was enrolled between September 2020 and November 2021. The MH and fetal HC were measured on admission of the patient to the labor ward. The primary outcome of the study was the performance of the HC/MH ratio in the prediction of CS for labor dystocia. Women who underwent CS for any indication other than failed labor progression, including fetal distress, were excluded from the final analysis.vs 164.5\u2009\u00b1\u20096.3\u2009cm; P\u2009<\u20090.001), larger fetal HC  and a higher HC/MH ratio  compared with vaginal delivery. Multivariate logistic regression analysis showed that the HC/MH ratio was associated independently with CS for labor dystocia ; P\u2009<\u20090.001). The HC/MH ratio had an area under the receiver\u2010operating\u2010characteristics curve of 0.77 and an optimal cut\u2010off value for discriminating between vaginal delivery and CS for labor dystocia of 2.09, which was associated with a sensitivity of 0.62 , specificity of 0.79 , positive predictive value of 0.13  and negative predictive value of 0.98 .A total of 783 women were included in the study. Vaginal delivery occurred in 744 (95.0%) women and CS for labor dystocia in 39 (5.0%). CS for labor dystocia was associated with shorter MH (mean\u2009\u00b1\u2009SD, 160.4\u2009\u00b1\u20096.6 Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.In a large cohort of unselected pregnancies, the HC/MH ratio performed better than did fetal HC and MH alone in identifying those cases that will undergo CS for labor dystocia, albeit with moderate predictive value. The HC/MH ratio could assist in the evaluation of women at risk for CS for labor dystocia. \u00a9 2022 The Authors.  abstract to view the translations.This article's abstract has been translated into Spanish and Chinese. Follow the links from the  What are the novel findings of this work?The newly described fetal head\u2010circumference\u2010to\u2010maternal\u2010height (HC/MH) ratio performs better than do fetal HC and MH alone in identifying cases that will undergo Cesarean section for labor dystocia, albeit with moderate predictive value.What are the clinical implications of this work?In an unselected population at low risk for labor dystocia enrolled within 72\u2009h before delivery, the HC/MH ratio demonstrates a fair specificity and negative predictive value for vaginal delivery. Use of the HC/MH ratio could assist in the evaluation of women at risk for Cesarean section for labor dystocia.Labor dystocia is estimated to account for half of all primary Cesarean sections (CS)Over the years, several studies have investigated the role of maternal and fetal anthropometric characteristics, such as maternal height (MH)This was a multicenter prospective observational study conducted at four tertiary maternity hospitals, in Italy  and Germany , between September 2020 and November 2021. A non\u2010consecutive series of singleton pregnancies with a cephalic\u2010presenting fetus, gestational age (GA) \u2265\u200936\u2009+\u20090\u2009weeks and no contraindications for vaginal delivery were considered eligible for the study. Patients were approached on admission due to labor induction, premature rupture of the membranes or spontaneous labor. Informed consent for study participation was obtained upon enrolment. In all included cases, the fetal HC was measured on transabdominal ultrasound in the standard transventricular plane22. In detail, a protracted active phase of labor was defined as \u2265\u20096\u2009cm of dilatation with ruptured membranes and failure to progress despite 4\u2009h of adequate uterine activity or at least 6\u2009h of oxytocin administration with inadequate uterine activity. Arrest of dilatation in the first stage requiring CS was diagnosed following two more hours of oxytocin administration with no cervical change. A diagnosis of arrest of labor in the second stage was made in the event that the duration of the active phase was at least 2\u2009h in parous women and 3\u2009h in nulliparous women on epidural analgesia, or 1\u2009h and 2\u2009h in parous and nulliparous women without epidural analgesia, respectively.A common protocol for labor management was shared across the participating units during the study period. Labor dystocia was defined based on the American College of Obstetricians and Gynecologists (ACOG)/Society for Maternal\u2013Fetal Medicine (SMFM) recommendations for the safe prevention of primary CSAccording to the local protocol of the participating units, women diagnosed with labor dystocia underwent clinical examination by the senior obstetrician responsible for patient care. Obstetric intervention for labor dystocia was performed when the criteria for arrest of dilatation or arrest of labor in the second stage were fulfilledClinical data including maternal age, ethnicity, parity, height and body mass index (BMI), as well as GA at inclusion, mode of delivery and indications for CS, and neonatal outcomes, including birth weight, umbilical artery pH and 5\u2010min Apgar score, were extracted from patient case notes. The HC/MH ratio was computed as the ratio between the measurement of the HC, expressed in mm, and that of the MH, expressed in cm. All information was collected after birth using a dedicated form. Data were recorded and stored in a Microsoft Excel secured pseudonymized database , which was accessible only to members of the research team. The primary outcome of the study was the relationship between the HC/MH ratio within 72\u2009h before birth and the occurrence of CS for labor dystocia.t\u2010test or the Mann\u2013Whitney U\u2010test was used as appropriate for continuous variables. Results are presented as n (%), mean\u2009\u00b1\u2009SD or median (range). Logistic regression analysis was used to control for potential confounding variables. The performance of MH, HC and the HC/MH ratio in predicting CS for labor dystocia was determined using receiver\u2010operating\u2010characteristics (ROC)\u2010curve analysis. The DeLong method was used for the comparison of areas under the ROC curve (AUCs). P\u2009<\u20090.05 was considered statistically significant. The study was approved by the local ethics committee of each participating unit and was reported in accordance with strengthening the reporting of observational studies in epidemiology (STROBE) guidelinesStatistical analysis was performed using Statistical Package for Social Sciences (SPSS) version 22 . The Kolmogorov\u2013Smirnov test was used to assess the normality of the distribution of the data. The chi\u2010square test was used to compare categorical variables and Student's Overall, 836 women were eligible over the study period. Of these, 15 were excluded due to failed induction of labor and 38 due to CS for suspected intrapartum fetal distress, leaving 783 cases for data analysis , higher frequency of nulliparity  and epidural use , higher GA at inclusion , larger fetal HC , higher HC/MH ratio , higher birth weight  and shorter MH , compared with vaginal delivery.The demographic and perinatal characteristics of the study population overall are shown in Table\u2009P\u2009<\u20090.001), MH ; P\u2009<\u20090.001), fetal HC ; P\u2009<\u20090.001), parity , P\u2009=\u20090.04) and epidural use ; P\u2009=\u20090.03) were associated independently with CS for labor dystocia (Table\u2009P\u2009<\u20090.001) and 0.71 , respectively, while the HC/MH ratio had the highest AUC ; P\u2009<\u20090.001). This was higher than that of the MH and of the fetal HC alone  , 2.65 ; This study demonstrates that the HC/MH ratio is associated independently with CS for labor dystocia. The HC/MH ratio had the greatest accuracy in identifying cases that would undergo CS for labor dystocia, albeit with poor predictive value in our population of women at low risk for labor dystocia leading to CS.Several studies suggest an association between MH and mode of delivery, including CS for labor dystociaThe influence of fetal size on labor outcome has also been studied extensivelyThe present study suggests that predictive parameters or models aiming to identify those laboring women at risk for labor dystocia leading to CS should ideally include maternal and fetal anthropometric characteristics.Our results demonstrate a fair specificity and NPV of the HC/MH ratio, meaning that women with a HC/MH ratio below the index threshold have a good chance of delivering vaginally. Nonetheless, our data indicate that the HC/MH ratio has a poor sensitivity and PPV for CS for labor dystocia. On one hand, this can be explained by the fact that the size of the fetus and the pelvis do not represent the only determinants of obstructed labor, as malposition, cephalic malpresentation and asynclitism are among the factors which may cause a relative mismatch between size of the fetus and that of the birth canalth percentile for the given GAMoreover, the HC/MH ratio may also support obstetricians evaluating women at risk for fetal macrosomia close to term. A randomized controlled trial demonstrated that labor induction before 39\u2009gestational weeks improved labor outcome in women at risk for fetal macrosomia based on an EFW above the 95The strengths of this study include its evaluation of a novel parameter and the prospective enrolment of a large cohort of cases. The HC/MH ratio demonstrated fair specificity and NPV for identifying cases with a good chance of vaginal delivery, which may have potential utility in the context of labor dystocia and the antenatal counseling of patients with a large\u2010for\u2010gestational\u2010age fetus. Another advantage of the parameter is that it can be quantified easily and quickly, and hence be readily available, even in emergency settings.Conversely, the non\u2010consecutive enrolment of patients could be seen as a limitation of the study findings. Furthermore, our results may not be generalizable to all centers, given that the study was conducted across four academic centers sharing a common protocol for labor management. The inclusion of the fetal scalp in the measurement of fetal HC could be also seen as a limitation, as it is not the standard recommendationIn a large cohort of women at low risk of labor dystocia enrolled within 72\u2009h before delivery, the HC/MH ratio performed better than did fetal HC and MH alone in identifying those cases that will undergo CS for labor dystocia, albeit with poor predictive value. Further studies are needed to evaluate the performance of the HC/MH ratio in high\u2010risk settings, such as in the event of labor dystocia or antenatal suspicion of fetal macrosomia."}
+{"text": "Inguinal hernia repair is one of the most commonly performed operations in general surgery. A total of 130.000 inguinal hernia repairs are performed yearly in Italy, and approximately 20 million inguinal hernias are treated worldwide annually. This report represents the trend analysis in inguinal hernia repair in Italy from a nationwide dataset for the 6-year period from 2015 to 2020.Based on regional hospital discharge records, all the inguinal hernia repairs performed in public and private hospitals in Italy between 2015 and 2020 were reviewed based on diagnosis and procedure codes. For the aim of this study, data from the AgeNas  data source were analyzed.Elective inguinal hernia repairs outnumbered urgent operations over the 6-year study period, ranging from 122,737 operations in 2015 to 65,780 in 2020 as absolute numbers, and from 87.96 to 83.3% of total procedures in 2019 and 2020 respectively, with an annual change ranging from \u2212\u200966.58%, between 2020 and 2019, to \u2212\u20092.49%, between 2019 and 2018 .This large-scale review of groin hernia data from a nationwide Italian dataset provides a unique opportunity to obtain a snapshot of open groin hernia repair activity. More specifically, there is a trend to perform more elective than urgent procedures and there is a steady decrease in the amount of open hernia repairs in favor to laparoscopy.The online version contains supplementary material available at 10.1007/s10029-023-02902-z. Inguinal hernia repair is one of the most commonly performed operations in general surgery. A total of 130,000 inguinal hernia operations are performed annually in Italy, and approximately 20 million are treated worldwide .The choice of surgical technique for inguinal hernia repair remains controversial. Quality assessments of inguinal hernia repair have previously been conducted using two different methods: either through analyzing dedicated regional/national databases (DD) \u20137 or revAnalyzing the data from the AgeNas  data source, the present study aimed to offer the first analysis of open inguinal hernia repairs in Italy covering the 6-year period from 2015 to 2020.Using the Hospital Discharge records regional Databases (HDD), all inguinal hernia repairs carried out in public and private hospitals in Italy in patients over 18\u00a0years between 2015 and 2020 were retrieved based on diagnosis and procedure codes. The HDD collected clinical and administrative informations regarding all hospital admissions for patients discharged from any hospital in Italy. The AgeNaS led the management and analysis of data. All hospital admissions for inguinal hernia repair that occurred between 2015 and 2020 were analyzed. The data source included patient demographic data  and admission and discharge data with up to six discharge diagnoses , as well as living status at discharge . It did not provide information on size of the hernia defect, individual surgical expertise, number of surgeons or number of procedures performed per surgeon. It gave information on the use of a mesh for inguinal hernia repair and allowed registration of all readmissions regardless of the primary site of surgery and type of institution (public or private). It did not provide information on hernias treated as outpatient procedures.The validity of this data system with respect to the type of surgical procedure had been already established for several surgical procedures \u20138. This Informations on consecutive admissions for inpatient inguinal hernia repair in Italy from 2015 to 2020 for patients with a diagnosis of unilateral and bilateral inguinal or femoral hernia were retrieved from the database.Sex, age and preexisting comorbidities were analyzed. Comorbidities were further divided into general comorbidities and neurological comorbidities. The presence of bowel obstruction at the time of the operation was also registered. The search coding system is summarized Table The discrimination between laparoscopic or open groin hernia repair was identified as the presence or the absence of the ICD-9-CM code 54.21 (laparoscopy). The association of inguinal hernia repair with other surgical procedures was defined by the wording \"AND cholecystectomy\" or \"AND adhesiolysis\". The presence of an association with another type of surgical procedure other than cholecystectomy and adhesiolysis was grouped as \"other\". The analysis included total hospital stay, readmission rate within 30\u00a0days from the operation, early mortality rate (during the index admission or within 30\u00a0days from the operation), late mortality rate  and 30-day morbidity.Data concerning in-hospital and 30-day mortality were obtained linking the hospital discharge records and the regional registry of mortality through unique patient codes. Complication rates were defined using pre-defined codes either as primary diagnosis or among the first five secondary diagnoses Table .Data were processed using the MedCal statistical package (version 12.5). Qualitative variables were summarized by absolute numbers and percentages, while quantitative variables were described by the median and standard deviation (SD) or range min\u2013max, for normally or non-normally distributed variables respectively.Statistical analysis was performed using Student\u2019s t test and the Cochran Armitage test for trends, as appropriate. A two-tailed p value\u2009<\u20090.05 was considered statistically significant.st of December for the period from 2015 to 2020, as reported by the Italian National Institute of Statistics (ISTAT)  per 100,000 population was calculated, assessing the changes in the considered period. The sample size was the Italian population, reported by region, according to the average yearly population on the 31), with an annual change ranging from a minimum of \u2212\u20092.26% between 2016 and 2015 to a maximum of \u2013 60.63% between 2019 and 2020 . The mean annual change was\u20142.85%  from 2015 to 2019 . Mean age ranged from 59\u2009\u00b1\u200920\u00a0years in 2015 to 61\u2009\u00b1\u200919\u00a0years in 2020. Overall comorbidity rates were stable during the study period. More details are shown in Fig.\u00a02Of this cohort, 0.02% patients (n\u2009=\u2009171 over time) presented with bowel obstruction.A total of 757,558 inpatient inguinal hernia repairs were performed in Italy from 2015 to 2020. Of these, 723,633 (95.5%) were open repairs , but, excluding 2020, the numbers of open procedures decreased steadily with a mean annual change of \u2212\u20092.75%   to a maximum of 7,146 . The mean annual change was 8.26% (95%CI 2.35%\u201414.17%) from 2015 to 2019, while a mean annual change of \u2212\u20090.98% (95%CI: \u2212\u200919.68%\u201417.72%) was observed over the whole time period  . The mean annual decrease was \u2013 1.95%  from 2015 to 2019  inguinal hernia repairs were associated with additional surgical procedures between 2015 and 2020. Of these, 2,914 (0.43%) were associated with cholecystectomy, 1,477 (0.20%) with adhesiolysis, and 9,601 (1.42%) with other types of open surgical procedures. The mean annual change of this trend was \u2212\u20091.23% (95%CI\u2009\u2212\u20094.45%\u20140.55%) if considering the total number of the associated procedures.The trend of associated cholecystectomies varied with a mean annual decrease of \u2013 11.32  over time, and of \u2212\u20093.12%  from 2015 to 2019. The association of open inguinal repair and adhesiolysis demonstrated a mean annual change of 3.55% , decreasing to 2.85%  from 2015 to 2019.A total of 3461 complications were registered, ranging from 362 in 2020 to 706 in 2015 as absolute numbers and from 0.43% to 0.52% in relative frequencies of overall procedures in 2018 and 2015, respectively. Complication rates decreased over time, with an annual change ranging from \u2212\u20096.4% between 2016 and 2015 to \u2212\u200965% between 2020 and 2019 . The mean annual change was \u2212\u20099.18%  from 2015 to 2019 . The mean annual change was \u2212\u20091.96%  between 2015 and 2019  within 30\u00a0days of the operation and 1153 after 30\u00a0days (42.7%), with a mean annual change ranging from \u2212\u200913.3% between 2016 and 2015, and 0.25% between 2020 and 2019 . The mean annual change was \u2212\u20094.60%  from 2015 to 2019.Early mortality ranged from 194 in 2016 to 254 in 2015 as absolute frequencies and from 0.48% to 0.57% in relative frequencies of overall procedures in 2018 and 2020 respectively, with an annual change ranging from \u2212\u200930.9% between 2016 and 2015 to 10.29% between 2018 and 2017 . The mean annual decrease was \u2212\u20095.92%  from 2015 to 2019.Late mortality ranged from 168 in 2020 to 211 in 2016 as absolute frequencies and from 0.14% to 0.21% in relative frequencies of overall procedures in 2017 and 2020 respectively, with a mean annual change ranging from \u2212\u200916.76% between 2018 and 2017 to 13.39% between 2018 and 2017 . The mean annual change was \u2212\u20094.26%  if considering from 2015 to 2019. Figure\u00a06Concerning the type of healthcare provided, the number of procedures performed in inpatient admission ranged from 37,093 in 2020 to 61,952 in 2015 and from 43.69% to 48.38% of overall procedures in 2019 and 2020 respectively, with a mean annual change ranging from a maximum of \u2212\u200944.4% between 2020 and 2019 to a minimum of \u2212\u20092.56% between 2018 and 2017  . The mean annual change was \u2212\u20092.05% (95%CI \u2212\u20094.43% \u2013 0.33%) between 2015 and 2019. Figure\u00a07.Mean hospital stay was 5\u00a0days between 2015 and 2019 and 4 in 2020Regional data are provided as supplemental material.This study provides a picture of the epidemiological trends over a six-year period for open inguinal hernia repair surgery in Italy. Although there have been previous studies involving \u2018\u2018hernia audits\u2019\u2019 in regional populations , 9, 10, Currently, however, the treatment of inguinal hernias is not standardized across Europe and the choice of the surgical approach remains debatable and subject to bias, including individual surgeon experience and preference. The open approach remains prevalent in both elective and emergency settings worldwide \u201319, consThe results reported in both elective and emergency inguinal hernia repairs performed between 2015 and 2020 are consistent with the literature , 20, 21.Primarily, there was a gradual and significant decline in the number of open hernia repairs both in elective (p\u2009<\u20090.0001) and emergent setting (p\u2009<\u20090.0001). This decrease coincided, in part, with the increase in use of laparoscopy, and, additionally, with the gradual decline of inpatient hospital stay for groin repair (p\u2009<\u20090.001). Similarly, a reduction in the number of admissions for inpatient surgery was noted (p\u2009<\u20090.0001). Other international studies have noted a similar reduction in the use of inpatient procedures due to several factors, including broader use of local anesthesia for elective inguinal hernia repair , which hThe reduction in inpatient directed care, probably coincided with an increase in the ambulatory service. The choice of ambulatory service could be further investigated; however, other authors have reported the steady implementation of ambulatory services into their routine practice , 22.The otherwise well-established ambulatory set-up for elective groin hernia repairs , 26\u201328 hComplications and readmission rates did not significantly change in the considered period, when the decrease observed reflects the overall decrease of the overall procedures. A large study conducted in Denmark identified a hospital readmission rate for hernia of 1.8% . In our Our data also demonstrated that the number of days spent in hospital for elective admissions did not change over the decade considered. A likely explanation for this is that, despite gradual improvements in healthcare provision, the greater selection of patients referred for elective hospital admissions over the years has altered the case mix.In-hospital mortality remained relatively constant. A significant reduction in both early and late mortality rates was observed across the whole period but more significantly decreased in the period between 2015 and 2019. This trend reflected the drastic drop of hernia cases in 2020.The global necessity to adapt to the unprecedented challenges associated with the rapid spread of Sars-Cov-2 in early 2020 resulted in a significant disruption in all elective and benign surgery. Over a few short weeks, surgeons witnessed a dramatic change in their practice, with a rapid decrease in the number of surgical interventions reported worldwide .General surgeons were particularly affected due to the wide variety of procedures they perform, many of which are carried out routinely, many in outpatient settings .A nationwide survey  highlighThe limit of this paper is intrinsic in the search methodology and the data source. The system involves AD that collect data from all public and private hospitals of a given region or country, obtaining such information from hospital admissions and discharge charts. The main advantage of the AD system is the ability to retrospectively record almost 100% of the surgical procedures performed using the coding system based on the ICD-9-CM. Additionally, AD are readily accessible at low cost. However, they often result in inaccurate data registration as well as inexact follow-up and recurrence/complication information; additionally, they often lack case mix adjustment.Inguinal hernia repair could be used as an indicator of the surgical quality offered in different health institutions and countries, thereby establishing a scientific basis from which to make decisions, given the rate with which day surgery and outpatient surgery are developing. Such solutions could help improve elective surgery efficiency, better sustainability across healthcare services, and offer elective patients shorter waiting times. Finally, this dataset can be used as a tool to help present validated recommendations for the management of patients with groin hernias, with a potential to implement a registry of ambulatory hernia repairs, following the example of other European countries.This large-scale review of inguinal hernia data from a nationwide Italian dataset provides a unique opportunity to obtain a snapshot of inpatient open hernia repair activity. Generally, a steady substantial decrease of open procedures could be observed over the six-year period taken into consideration and a simultaneous increase of laparoscopic adoption, in the setting of a global reduction observed in 2020 due to the COVID pandemic. The trend of day care surgeries remained stable throughout the whole period.Supplementary file1 (DOCX 19 KB)Supplementary file2 (DOCX 25 KB)Supplementary file3 (DOCX 28 KB)Supplementary file4 (DOCX 944 KB)Supplementary file5 (DOCX 630 KB)Supplementary file6 (DOCX 942 KB)Supplementary file7 (DOCX 941 KB)Supplementary file8 (DOCX 19 KB)Below is the link to the electronic supplementary material."}