We have studied whether growth factors, cytokines, hormones, neurotransmitters, and local hormones (autacoids) promote the proliferation of hepatic parenchymal cells  using in vitro primary cultured hepatocytes. The indicators used for this purpose include changes in DNA synthesis activity, nuclear number, cell number, cell cycle, and gene expression. In addition, the intracellular signaling pathways from the plasma membrane receptors to the nucleus have been examined in detail for representative growth-promoting factors that have been found to promote DNA synthesis and cell proliferation of hepatocytes. In examining intracellular signaling pathways, the effects of specific inhibitors of presumed signaling factors involved have been pharmacologically confirmed, and the phosphorylation activities of the signaling factors  have been evaluated. As a result, it has been found that there are many factors that promote the proliferation of hepatocytes , prostaglandin (PG)), and serotonin (5-HT)), while there are very few factors  that inhibit the effects of growth-promoting factors. We have also found that 5-HT and GH promote the proliferation of hepatocytes via different autocrine factors . Using primary cultured hepatocytes, it will be possible to further study the molecular and cellular aspects of liver regeneration. The liver is the central organ of metabolism and is responsible for the homeostasis of the body by metabolizing carbohydrates, proteins, lipids, and other substances. Furthermore, detoxification and regeneration are characteristic functions of the liver. These abilities are thought to have been acquired during the long evolutionary process to protect animals from catastrophic damage to liver tissues caused by food toxins  .The mechanisms of liver regeneration have been studied for many years, and numerous reports have been published. There are also excellent review articles integrating those results ,3,4,5,6.Therefore, we used a simpler in vitro primary cultured hepatocyte system to examine the actions of individual hepatocyte growth factors (and candidate substances) and their intracellular signaling pathways. Primary cultured hepatocytes retain metabolic activity comparable to that in vivo and express many kinds of receptors, each of which is known to be responsive to agonists ,7. It isIn this review, an in vivo model of liver regeneration is briefly described. In addition, based on our results, the factors that promote or inhibit the proliferation of hepatocytes are classified into their bioactive characteristics, and their respective intracellular signaling pathways are briefly summarized. Then, we will discuss the physiological significance of how each of these factors may play a role in liver regeneration in vivo.0 → G1 → S → G2 → M → G0 phase), which stops when the organ returns to its original size. During the post-PHx, the blood levels of the intracellular enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increase rapidly due to the destruction of the liver tissue , tumor necrosis factor-α (TNF-α), interleukin (IL)-6, serotonin (5-HT), and bile acid levels in the blood also increase after PHx . TherefoAlthough the aforementioned in vivo study of liver regeneration using live animal models has its advantages, there are limitations when studying the intracellular signaling mechanisms in detail. The main reason is that the liver tissue is composed of several different cell groups , and the mediators released by each cell affect each other in liver regeneration. Therefore, in the early 1970s, a method was developed to isolate, purify, and culture hepatocytes by using collagenase to perfuse the liver. This technique made it possible to study the intracellular signaling pathways for the proliferation of hepatocytes. As a result, it is now believed that liver regeneration does not initiate and progress by a single humoral factor, but rather by the interaction of a number of growth-promoting factors that exert a vigorous regenerative capacity.The hepatocytes are isolated from normal livers by the two-step in situ collagenase perfusion technique devised by Seglen to facilitate disaggregation of the adult rat liver . The hepAfter 3 h of incubation, the culture medium is replaced with a serum-free, defined medium, and the incubation is continued for an arbitrary period of time (0–21 h). Under the serum-free, defined culture conditions, the various effects of growth regulators on hepatocyte proliferation can be observed when different types of growth factors (and candidate substances) and signal-transducing factor inhibitors are added alone or in combination .0) and have stopped proliferation. However, in our culture system, the cell density at the time of cell seeding is relatively low, and the concentration of dexamethasone is reduced without adding insulin or EGF, which are considered essential for cell survival. By doing so, we have found that the effects of individual growth factors on hepatocyte proliferation can be observed much earlier than before.Under normal physiological conditions, hepatocytes are in the quiescent phase of the cell cycle  increases significantly after about 3–4 h A,B.We also found that there are many factors that promote hepatocyte proliferation, but there are few factors that can strongly inhibit the effects of these hepatocyte growth-promoting factors . Recentl2, 5-HT, and GH, which indirectly promote hepatocyte proliferation by secreting autocrine factors. In contrast, some factors, such as transforming growth factor-β1 (TGF-β1) and glucocorticoids, strongly suppress mitogen-induced hepatocyte proliferation . The physical properties, receptors, intracellular signaling pathways, and bioactive characteristics of representative hepatocyte growth-promoting and growth-inhibitory factors are briefly summarized below , and IGF-I. Co-mitogens, by themselves, do not promote hepatocyte proliferation, but, when used in combination with mitogens, the co-mitogens enhance the activity of the mitogens, and these include adrenergic α- and β-agonists and glucagon. In addition, there are many indirect mitogens, such as TNF-α, IL-1β, prostaglandin (PG) Eed below .EGF is a protein with a molecular weight of approximately 6 kDa, and it has three disulfide bonds. EGF binds to the EGF receptor (EGFR) and activates intracellular signaling pathways by phosphorylating the receptor tyrosine kinase (RTK); the EGF receptor is phosphorylated in vivo 30–60 min after PHx . IL. IL24]. TNF-α is a protein consisting of 157 amino acids and is a potent proinflammatory cytokine similar to IL-1 and IL-6. TNF-α promotes the proliferation of hepatocytes via TNF-α type 1 receptors . The majWe investigated the proliferative effects of TNF-α on hepatocytes in culture. The results showed that TNF-α alone promotes DNA synthesis and the proliferation of hepatocytes and that its effects are mediated via its autocrine secretion of TGF-α from the hepatocytes  25]..25].2. The physiological process involves constitutive COX-1 expression, while the inflammatory process involves inducible COX-2 expression. Biosynthesized PG products exhibit a variety of physiological effects, but inactivation is relatively rapid. PGs act on cells of the immune system to enhance inflammatory responses  by promoting the release of IL-1 and TNF-α. PGI2 is produced by vascular endothelial cells, regulates blood flow, and inhibits platelet aggregation. It has been reported that PGs stimulate hepatocyte DNA synthesis in culture [Prostaglandins (PGs) are low-molecular-weight local hormones. The receptors for PGs include EP, IP, and TP types, all of which are G protein-coupled. PGs are synthesized under the influence of cyclooxygenase (COX) from arachidonic acid and excised from the plasma membrane by phospholipase A culture .2 and PGI2, which promote DNA synthesis and the proliferation of hepatocytes. As a result, we found that both PGE2 and PGI2 indirectly promote DNA synthesis and the proliferation of hepatocytes via the secretion of the autocrine factor TGF-α from the hepatocytes  cells in the small intestinal epithelium and acts as a local hormone. 5-HT is stored in platelets and released upon stimulation and is involved in blood coagulation by promoting platelet aggregate formation (hemostatic thrombus). 5-HT has been reported to be involved in the promotion of liver regeneration [5-HT is a low-molecular-weight substance with receptor subtypes designated 5-HTneration . That is2B/Gq/phosphoinositide-specific phospholipase C (PLC)/Ca2+ pathway. We then found that the secreted TGF-α directly promotes DNA synthesis and hepatocyte proliferation  priming, 2) progression, and (3) termination. It has been recognized that liver regeneration begins early in each of these phases due to the interplay of numerous regulatory factors [ priming, progress2 receptors enhance the proliferative effects of EGF and HGF separately. Then, it is conceivable that transported endocrine mediators, either locally or somewhat distantly via the bloodstream, serve as signals to enhance the proliferation of hepatocytes at an early stage in vivo  to phagocytose and remove necrotic tissues. In addition, an early hemostatic response occurs at the site of the vascular injury. In other words, platelets adhere and release stored mediators  to promote the aggregation reaction. The platelets also release their own growth factors, such as HGF and PDGF. PGs and 5-HT, which are involved in inflammation and hemostasis, have been shown to act as indirect growth-promoting factors mediated by autocrine factors in vitro  17,26,2,226,27. Moreover, we also found that the branched-chain amino acid leucine and vitamin C stimulate hepatocyte proliferation via an autocrine mechanism ,30. HoweTGF-β1 strongly inhibits the hepatocyte proliferation-promoting effects of EGF, HGF, and TGF-α in culture ,32. In vRecently, the molecular mechanisms of action of various factors that regulate hepatocyte proliferation in vitro have been gradually elucidated. Based on these results, the in vivo mechanisms of liver regeneration are also becoming better understood. By integrating the results of these basic biological studies, we hope to extract useful insights that can be appropriately applied to several clinical issues  .


To study the protective effect and mechanism of icaritin (ICT) in a SH-SY5Y cells with virus-loaded TAR DNA-binding domain protein 43(TDP-43) by examining the effect of ICT on the expression of autophagy-related proteins in TDP-43-infected SH-SY5Y cells.A TDP-43-induced neuronal cell injury model was established by transfecting well-growing SH-SY5Y cells with virus loaded with the TDP-43 gene. The changes in cell viability were detected by the CCK-8 method. After successful transfection, the establishment of the model was verified by real-time quantitative PCR (qPCR) and Western blot methods. After the cells were subjected to drug intervention with ICT, the changes in the expression levels of TDP-43, cleaved Caspase-3, LC3 II/I, Beclin-1 and p62 were detected by Western blotting.After ICT intervention, it was found that compared with that of the TDP-43 group, the cell viability of the TDP-43+ICT group increased, the expression level of TDP-43 decreased, and the expression levels of the apoptotic protein cleaved Caspase-3, autophagy protein Beclin-1, and LC3-II/I decreased, while the expression level of the autophagy protein p62 increased.ICT has a protective effect on the SH-SY5Y cell injury model transfected with TDP-43. This protective effect may be related to reducing the protein expression of TDP-43 and inhibiting autophagy. TAR DNA-binding domain protein 43 (TDP-43) is a highly conserved rib nucleoprotein body consisting of 414 amino acids located on chromosome 1p36, which contains six exons. TDP-43 plays multiple roles in regulating gene expression at the transcriptional and translational levels . When exIcaritin (ICT) is a flavonoid compound and is a metabolite of icariin (ICA), both of which are the active ingredients in the extract of epimedium . The molTherefore, combined with the previous results of this research group, TDP-43-transfected SH-SY5Y cells were the research focus of this study and we used ICT for drug intervention in SH-SY5Y cells carrying the viral TDP-43 protein. Then, we initially explored whether ICT has a protective effect on nerve cell damage caused by TDP-43 transfection. At the same time, it was clear that it exerts a protective effect through the autophagy pathway, which provides a certain basis for the treatment of TDP-43 proteinopathy and drug development.2 culture flasks and cultured in a 5% CO2 incubator at 37 °C. The medium was replaced every 24–48 h and was composed of DMEM-F12, 15% fetal bovine serum, and 1% penicillin. When it grew to occupy 80–90% of the bottom of the bottle, the cells were passaged. The packaged lentivirus was transfected into cells, 200 µL of virus-containing medium was added to each well of a 96-well plate, and 1 ×104 cells were inoculated on the plate at an MOI of 0–60. The cell morphology was observed after 12 h of culture and the best MOI was selected.SH-SY5Y human neuroblastoma cells donated by the Collection Center of Wuhan University were inoculated into 25 cm4/mL with culture medium, and the suspension was seeded in a 96-well plate with 200 µL per well. After culturing for 1 day, the medium was changed, 10 µL of CCK-8 solution was added to each well, and the cells were incubated in a 37 °C incubator for 2 h. Then, a Synergy HTX microplate reader was used to measure the absorbance at 450 nm and calculate the cell viability.The CCK-8 assay was used to detect cell viability. The cells were grown under good conditions, the cell concentration was diluted to 5 ×10First, RNA was extracted, 200 µL of TransZol Up was added to the successfully transfected cells, 40 µL of chloroform was added and shaken for 30 s after the precipitate disappeared, and the mixture was allowed to stand. Then, 500 µL of CB9 and 500 µL of WB9 were added to the tube and discarded by centrifugation. The supernatant was finally added to 50 µL of RNase-free water, placed in an RNase-free tube and centrifuged to extract RNA. At a constant temperature of 65 °C, the mixing solution consisting of the primers shown in The cells were placed on ice, RIPA lysis buffer was added, and the cells were lysed on ice for 30 min. After centrifugation, the supernatant was taken as the protein sample. Then, the BCA method was used to determine the protein concentration. A standard curve was drawn at 562 nm on the microplate reader, and the blank of the standard curve was used as a control to determine the protein content of the sample. The protein was placed in boiling water at 100 °C and heated for 5 min to denature the protein. The separation gel and concentrated gel were selected according to the molecular weight of the target protein. Each 12% SDS–PAGE gel was added to 25 µg of total protein for separation. The concentrated gel electrophoresis voltage was set to 90 V, and electrophoresis was performed for approximately 30 min. After the same level, the separation gel electrophoresis voltage was set to 120 V, electrophoresis was performed for approximately 90 min, the electrophoresis was stopped when the sample band reached the bottom layer of the separation gel, and then transmembrane the protein. The nitrocellulose (NC) membrane was sealed with 5% skimmed milk powder for 1 h and washed with TBST three times after the sealing was completed, each time for 10 min. The NC membrane was incubated with the primary antibody overnight. After washing the membrane, the NC membrane was incubated with HRP-anti-rabbit antibody for 2 h. A color developing agent was added to the film and exposed in a fully automatic chemiluminescence gel imager, and then protein detection was performed after exposure.P < 0.05 was considered statistically significant.All statistical analyses were performed with IBM SPSS Statistics 22.0. If the data met normality, the mean ± SEM was used. The difference between the means of more than two groups was analyzed by one-way analysis of variance (ANOVA). If there were two variables in the statistical plot, two-way ANOVA was performed. When ANOVA showed significant differences, pairwise comparisons of means were made by Bonferroni’s post-hoc t test with correction. A value of Transfection was carried out with different concentrations of virus MOI. When the MOI was 30, the cell dendrites were clear, the growth state was good, and the transfection efficiency was high. When the MOI was 10 and 20, the cell state was good, but the transfection efficiency was low; when the MOI was 40, the transfection in the staining efficiency was good, but the cell status was less than when the MOI was 30, and the amount of cell death was greater than when the MOI was 30; the cell status was poor, the amount of cell death was greater and the transfection efficiency was low when the MOI was 50 and 60. Therefore, in this experiment MOI 30 was selected for transfection, as shown in After the cell model was established, ICT was added at different concentrations, including 0.0, 0.1, 1.0, and 10.0 µmol/L, to the TDP-43 group, and ICT was added for 12 h, 24 h, and 48 h to detect the cell viability of each group. The results showed that the cell viability of each group was the highest when the ICT concentration was 1.0 µmol/L and the incubation time was 48 h, as shown in The expression levels of LC3-II/I, cleaved Caspase-3, Beclin 1 and TDP-43 in the TDP-43 group were significantly higher than those in the Con group, and the expression levels of LC3-II/I, cleaved Caspase-3, Beclin 1 and TDP-43 in the TDP-43+ICT group after 48 h of ICT treatment were clearly decreased than those in the TDP-43 group. At the same time, the expression of p62 in the TDP-43 group was lower than that in the Con group, and the expression of TDP-43+ICT after 48 h of ICT was significantly higher than that in the TDP-43 group, as shown in TDP-43 is a member of the nuclear heterogeneous ribonucleoprotein family and includes two domains of RNA recognition motifs located on both sides of the N-terminal and C-terminal regions. The C-terminus affects the binding of heterogeneous and homologous proteins, while the N-terminus is mainly involved in the formation of homodimers . TDP-43 Autophagy is a regulated self-degradation process in cells that is highly conserved in many species, such as mammals and yeast. Autophagy avoids the accumulation of misfolded or aggregated proteins, which is essential to maintain the normal function of the central nervous system. The autophagy process includes the process of initiating autophagy after receiving the signal, forming autophagosomes, autophagolysosomes formed by the combination of lysosomes and autophagosomes, degrading autophagolysosomes into small molecules and reusing decomposition products . The ini1−42 contents (Previous research by our group showed that ICT can inhibit β-amyloid precursor protein cleavage enzyme 1 (BACE 1) to block the production of amyloid precursor protein (APP) to reduce the secretion of Aβ . We founcontents . Our grocontents .Moreover, studies have shown that ICA can act on diseases through autophagy and other pathways . TherefoTherefore, in this study, after transfecting SH-SY5Y cells with a virus carrying TDP-43, the cell morphology shrank, axons and dendrites were shortened or even disappeared, and cell death occurred. At the same time, the mRNA and protein expression of TDP-43 was measured. A significant increase in cell viability measured by CCK-8 decreased, and the cells in this state could better simulate the damage caused by TDP-43 in SH-SY5Y cells. Therefore, this experiment selected a virus carrying TDP-43 to transfect SH-SY5Y cells. Cell-based methods were used to establish cell models for research. Based on related literature and preliminary experiments, it was determined that the duration of virus transfection has different effects on the cells. Therefore, in this experiment, the cell viability of SH-SY5Y cell models after different transfection times was measured, and it was found that the cell viability was 12 h after transfection. The cell morphology changes were viewed under the microscope. At the same time, the cell viability was restored after ICT was added, and the cell morphology was partially restored to normal. Considering that TDP-43 has a greater impact on cytotoxicity, in this experiment the virus that carried TDP-43 was selected. SH-SY5Y cells were stained for 12 h as the experimental conditions for establishing cell models.After the model was successfully established, according to the experimental results of this research group and previous experiments, the cell viability was the best when the SH-SY5Y cell model transfected with TDP-43 was treated with ICT at a concentration of 1 µmol/L for 48 h. After ICT drug intervention for 48 h, the cell morphology and growth status were significantly improved compared to before the drug was added. CCK8 assays showed that SH-SY5Y cell viability increased significantly after TDP-43 transfection compared with before the drug was added. Western blot results showed that after drug intervention, the expression of TDP-43 in the TDP-43+ICT group was lower than that in the TDP-43 group. At the same time, the expression of the apoptosis protein cleaved Caspase-3, autophagy protein Beclin-1, and LC3-II/I decreased after adding ICT, and the expression of the autophagy protein p62 increased, suggesting that ICT may affect the protein expression of cleaved Caspase-3, Beclin-1, LC3-II/I and p62, and it also has a protective effect on the autophagy pathway in the injury model of TDP-43-transfected SH-SY5Y cells.in vivo level and explore its protective mechanism to provide new ideas for the development of novel drugs for the treatment of TDP-43 proteinopathy.In summary, this study used TDP-43-carrying virus to transfect SH-SY5Y cells and establish a TDP-43 transfection cell injury model. After ICT intervention, it was found that ICT reduced the autophagy pathway Beclin-1 and LC3-II/I protein expression by reducing cleaved Caspase-3 and increasing the expression level of p62 protein, thereby protecting the TDP-43-induced cell injury model. However, the current research on ICT is still in the preliminary stage, and the specific mechanism of its protective effect on the TDP-43-induced cell injury model is still unclear. This study explores the protective effect of ICT at the cellular level. In a follow-up study, our group will further verify its protective effect at the ICT has a protective impact on the SH-SY5Y cell injury model transfected with TDP-43. This protective effect may be relevant to regulating TDP-43, inhibiting the expression of cleaved Caspase-3, Beclin-1 and LC3-II/I and promoting the expression of p62 protein.10.7717/peerj.13703/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13703/supp-2Supplemental Information 2Click here for additional data file.


Team development and idea generation are key intertwined steps in translational science that need a framework to accommodate unstructured, participatory interactions. To this end, we introduced Un-Meetings to the Clinical and Translational Science Awards (CTSA) Program, innovative events that facilitate cross-disciplinary idea generation and informal discussions between translational scientists, policy makers, community members, advocates, and public health professionals. Here we describe a mixed methods study to characterize the conceptual diversity and clusterization of ideas generated through an Opioid Crisis Un-Meeting.An Un-Meeting targeting translation science approaches to the opioid crisis were hosted at the University of Rochester Center for Leading Innovation and Collaboration (CLIC). We used semantic analysis and conceptual mapping of keywords to analyze how attendee-led idea generation sessions identified topics for breakout discussions.One hundred and two individuals from 40 institutions proposed 150 unique ideas that were grouped into 23 breakout sessions. Network analysis showed that diverse pools of experts were bridged by topics addressing the complexities of the opioid crisis. Two clusters emerged: (1) systems, contexts, and community engagement, and (2) technologies, innovations, and treatment advancements.The cross-disciplinary nature of topic areas that bridge across thematic communities provide opportunities for CTSA programs to engage and support development of diverse translational teams. Potential opportunities for team building include technological advancements of opioid prevention, treatment, surveillance, systems approaches, and studies focusing on special populations and health disparities. The analysis method here may be useful in identifying naturally emerging teams of experts and community gaps when addressing large problems. Translational research involves bridging between different stages of the translational spectrum  to find, test, and implement solutions that address complex biomedical and health problems . InnovatUn-conference is an innovative meeting that shifts from traditional didactic lectures and presentations to a more participatory format that engages attendees from multiple disciplines to co-develop ideas and form collaboration networks [The networks . Un-confnetworks ), which networks ,9.The University of Rochester Center for Leading Innovation and Collaboration (CLIC), the National coordinating center for the National Institutes of Health Clinical and Translational Science Awards (CTSA) Program, adapted the methodology of Un-conferences  to develThe unstructured format of the Un-Meeting allows researchers to form social clusters that may lead to early transdisciplinary team formation. In addition to formal organizational affiliations (e.g. institutions and departments), invisible communities of research and collaboration can form based on shared interests, values, and scientific worldviews that cross traditional organizational structures. These “invisible colleges” , “invisiThe data collected through an Un-Meeting provide a unique opportunity to study the emergence of translational ideas for future research from the perspective of investigators interested in developing emerging collaborations, and to identify potential invisible research communities. Previous efforts identifying such ideas and communities have been largely limited to semantic analysis of translational research topics, heavily focused on bibliometric analysis of research outputs to identify hot topics and emerging fields  or commIn contrast, analytic clustering of dynamic Un-Meeting topic keyword data allows a multifaceted analysis of attendee interests, dominant fields, and common frameworks. Such keyword analysis may identify bridging topics and cross-disciplinary approaches for future scientific team formation to address urgent research problems. Network analysis can identify topics that connect attendees from disparate research areas, similar to identifying bridging keywords in research publications that cross scientific fields –24.In this exploratory study, we describe content analysis of topics generated by participants in an Un-Meeting on the “opioid crisis” at University of Rochester. We investigated the clustering patterns and bridging action words, as indicators of dominant fields and bridging opportunities.The Center for Leading Innovation and Collaboration (CLIC), housed at the University of Rochester, hosted the first CTSA-wide “Un-Meeting” on June 2, 2018 addressing the opioid crisis. The Un-Meeting is a participatory event focused on engaging translational scientists, policy makers, community members, advocates, and public health professionals in a dynamic process of idea generation and translational conversation . The Un-After the talks, attendees used sticky notes to write topics of interest, questions, or ideas for breakout sessions and placed them on an idea generation board. Assisted by the organizing team, conference participants were encouraged to group the ideas into common themes that would determine the eventual topics for the subsequent concurrent small group breakout sessions. Facilitators and participants re-arrange and aggregate these notes into small group session topics in specific rooms and time slots. Attendees then self-select which sessions they would like to join. The resulting breakout sessions were 45 minutes long, unmoderated, and included between 8 and 25 participants. A second round of 4×4 talks, topic generation, and breakout sessions occurred in an afternoon session.Photographs of the notes posted on the grid were used to collect the final clustering, and were transcribed into machine-readable format by hand. Each note was treated as a separate unit of text and was pre-processed by removing stop words  and common terms using the Automap program . The resWe used two analytical techniques to explore the patterns of topic clusterization:Keyword cluster analysis: We clustered keywords based on their co-occurrence in notes and linked them with their corresponding breakout sessions using a density-based spatial clustering (DBSCAN) algorithm [lgorithm  with a cKeyword-to-session network mapping: To identify keywords that bridged across fields and disciplines, we performed a separate analysis on keywords that were assigned to more than one breakout session. We expected that this analysis would provide clues to translational opportunities to address the opioid crisis and opportunities for cross-disciplinary team formation. We developed a keyword-to-session two-mode matrix and map, with nodes representing keywords and breakout sessions, and edges representing the relation between keywords and sessions. The nodes (keywords and sessions) were arranged in a two-dimensional graph using multi-dimensional scaling (MDS) of geodesic distances between nodes [en nodes  and ploten nodes .In total, 102 individuals from 40 institutions  participated in the Un-Meeting. A third of participants came from within a 100-mile radius of the Un-Meeting location, the remainder represented 16 states and the District of Columbia.The goal of this analysis was to visualize the conceptual distance between and within breakout sessions. Such a mapping can show the conceptual diversity of the conference, grouping of words within the breakout sessions, provide an unsupervised clustering window into the breadth or focus of a particular breakout, and cross-connections between breakouts.1). Unsupervised clustering of the 248 keywords in 150 sticky note ideas resulted in 18 clusters , Criminal Justice (Session 2), Clinical trials (Session 3), Community Engagement (Session 8), Special Populations (Session 11), and Health Insurance/Policy (Session 23). Most sessions addressed systemic and cross-disciplinary issues, involving community, governmental, and policy sectors.We next examined the linkage between breakout sessions and keyword clusters. Breakout sessions also varied in terms of the breadth of the distribution of their keywords across clusters. Six sessions with highest between-cluster breadth shared keywords with five or six clusters. They included Most interestingly, we found that the content of breakout sessions rarely corresponded with keyword clusters, as the content of clusters usually referred to multiple breakout sessions. Thus, clusters related to general medical aspects of addiction and systemic and organizational themes distributed across breakout sessions. Themes related to special populations and disparities did not appear in many sessions and were more specific to the sessions corresponding to these topics.We next analyzed the relationship between keywords and sessions, based on their distribution in a network map, aiming to identify meaningful regions and distinct communities. Figure ‘community engagement’ , ‘technologies and innovations’ , ‘treatments and medications’ , ‘clinical and best practice’ , ‘prevention and education’ , ‘special populations and disparities’ , and ‘system/context/complexity’ .After reviewing, the common features of keyword clustered in different regions of the map and sessions that they bridged. We labeled seven main regions, denoted by the large blue labels in Fig. In summary, we identified thematic regions in the map that can indicate to potential/invisible communities among researchers with common interests and approaches in opioid research. The main clusters included clinical research, translational research using technologies, population health, community engagement, special populations and disparities, and systemic and structural approaches to opioid disorders.In this report, we describe the use of unsupervised clustering based on semantic analysis of concepts and keywords to identify bridging concepts across expert domains and hidden communities of interest within an Un-Meeting involving investigators and research users interested addressing the opioid crisis. We used two complementary approaches to identify clusters of keywords and illustrated the overlap between sessions and keyword clusters, and areas that bridged across potential communities of research. We found that keywords related to treatment, pain, medication, access, and communities span across many breakout sessions that represented focused areas of investigation, while important concepts such as disparity, discrimination/racism, and policy were less frequent and were limited to few focused communities. The network analysis showed that focused communities were bridged by topics pertaining to the complexities of the opioid crisis, including systems, contexts, and community engagement, on one hand, and technologies, innovations, and treatment advancements on the other hand. These bridging concepts represent translational opportunities in opioid research that connect disciplines and research groups.Results of the thematic analysis confirmed the conceptual diversity, with a majority of breakout sessions related to more than one topic area/disciplinary field, and spanning socio-political, technological, and clinical disciplines. This supports the translational nature of opioid research, similar to many other complex fields, in which, the expertise and research focus could not be easily classified by traditional departmental and disciplinary boundaries . This unpreclinical, clinical research, community engagement, computational science and data informatics, and workforce development and implementation science [The opioid crisis affects several aspects of healthcare systems, spanning from prevention and public health to emergency care, and from primary care to specialties. The opioid crisis is even more complicated by a gradual change in the demographics of those most affected, and emergence of new behavioral trends ,32. Thes science . While iCollaboration between clinical and basic sciences researchers to develop new medications and treatments. This is a dominant area in the center of Fig. Community engagement region in Fig. Collaboration between health researchers and community members as well as community-based institutions, to engage stakeholders in research and to address disparities. The System/Context/Complexity in Fig. Special populations/Disparities, which underscores the role of systemic approaches to improve health equity in this area.Collaboration between clinical researchers, system scientists, dissemination & implementation scientists, and sociologists to study the complexities of social systems and organization of care. The region labeled Inspection of the bridging concepts in the keyword-to-session map provides clues for translational opportunities in opioid research to be fostered by CTSA programs. These opportunities include:While the results of our study at a minimum provide a potential roadmap of opportunities for translational advancement in opioid research among this core subgroup of scientists, there are several limitations to be acknowledged. The keywords used in the mapping are based on limited content provided on sticky notes and might miss the complexities and breadth of subject matters to which they correspond. This may affect the richness of conceptual clusters and resulting interpretations. The assignment of topics to breakout sessions was moderated by a facilitator and hence does not reflect a completely unsupervised process of clustering. The topics also reflect the areas of expertise of this particular group of attendees and may not be generalizable. Nevertheless, this method of semantic analysis with thematic network analysis and keyword clustering may be useful for analyzing Un-Meetings with the aim of identifying hidden communities, areas of potential collaboration, and potential gaps in a research network’s ability to impact a chosen translational research problem. The value of these clustering techniques in identifying emerging communities and future collaboration potentials could be assessed using mixed methods studies to incorporate the perspectives and reflections of investigators involved in idea generation activities into the analysis.Our case study of mapping the generated ideas in an Un-Meeting on the opioid crisis, an inherently translational issue, provided insights on the thematic clusters and topic areas that bridge across them. Our findings add to the scientific body of literature on using thematic mapping to reveal the complexities of inter-disciplinary research  and provThe cross-disciplinary nature of topic areas that bridge across thematic communities provide opportunities for CTSA programs to engage and support diverse communities of researchers and users to develop translational teams. Potential opportunities to support translational collaborations include technological advancements of opioid prevention, treatment, and surveillance; studying systems, contexts, and complexities of the opioid crisis, and studies focusing on special populations and health disparities.


In vitro-in vivo discordance in the activity of β-lactams against MBL-producing Enterobacterales has been described. This discordance is likely attributed to the supra-physiologic zinc level in the in vitro testing media, which facilitates the bicyclic β-lactam ring hydrolysis. In this study, we compared the outcome of empirical non-MBL-active β-lactam therapy (carbapenems and ceftazidime/avibactam) and MBL-active β-lactam therapy (ceftazidime/avibactam plus aztreonam) among patients with bloodstream infections due to NDM-producing Klebsiella pneumoniae. Validation of the efficacy of carbapenem in a murine septicemia model was conducted. In vitro susceptibility testing conditions were altered to better predict the in vivo outcome.A retrospective observational study of patients admitted to hospitals in Italy. The primary outcome was 14-day all-cause mortality. Cox regression analysis was performed to evaluate primary outcome. Kaplan Meier survival and log-rank test were used to compare 14-day mortality between patient’s cohorts. Septicemia was induced in mice via intraperitoneal inoculation with the isolates retrieved from the patients then clinical exposure of meropenem  was given for 2 days. Survival was recorded for 4 days and compared with sham controls. Unbound zinc levels were measured in human and infected mice plasma. MEM MICs were determined in Mueller Hinton Broth (MHB) and MHB adjusted to the physiologic zinc levels.Of the patients identified, 29 received empirical non-MBL-active β-lactams for median duration 4 days while 29 received MBL-active β-lactams. The 14-day mortality rate was 21% in the non-MBL-active group vs 14% in the MBL-active group (P = 0.73) and survival patterns were not significantly different . Cox regression showed that use of non-MBL-active therapy was not associated with significantly increased 14-day mortality . MEM treatment resulted in protection from mortality in mice . MEM MICs in zinc-adjusted MHB were 1- to > 16-fold lowered relative to MICs in MHB ( ≥ 64 mg/L).Our data provide foundational support to help establish PK/PD relationships using MICs derived in physiologic zinc levels which may better predict β-lactam therapy outcome.Kamilia Abdelraouf, PhD, Evopoint Biosciences Co., Ltd: Grant/Research Support|Venatorx Pharmaceuticals, Inc.: Grant/Research Support Christian M. Gill, PharmD, Everest Medicines, Shionogi, Cepheid: Grant/Research Support Matthew Gethers, PhD, Thermo Fisher: Employee Marco Falcone, MD, PhD, GILEAD: Grant/Research Support|GSK: Honoraria|MENARINI: Advisor/Consultant|MENARINI: Grant/Research Support|MSD: Honoraria|PFIZER: Honoraria|SHIONOGI: Grant/Research Support Francesco Menichetti, n/a, Aneglini: Advisor/Consultant|Aneglini: Board Member|Aneglini: Grant/Research Support|Aneglini: Honoraria|Astellas: Advisor/Consultant|Astellas: Honoraria|Becton: Advisor/Consultant|Becton: Honoraria|bioMérieux: Advisor/Consultant|bioMérieux: Honoraria|Biotest: Advisor/Consultant|Biotest: Board Member|Biotest: Honoraria|Bristol-Myers Squibb: Advisor/Consultant|Bristol-Myers Squibb: Honoraria|Correvio: Advisor/Consultant|Correvio: Speaker honoraria|Dickinson: Advisor/Consultant|Dickinson: Honoraria|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Janssen: Advisor/Consultant|Janssen: Honoraria|MSD: Advisor/Consultant|MSD: Speaker honoraria|Nordic pharma: Board Member|Nordic pharma: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Honoraria|ViiV: Advisor/Consultant|ViiV: Honoraria David P. Nicolau, PharmD, Shionogi: Grant/Research Support.


To face crises like the COVID-19 pandemic, resources such as personal protection equipment (PPE) are needed to reduce the infection rate and protect those in close contact with patients. The increasing demand for those products can, together with pandemic-related disruptions in the global supply chain, induce major local resource scarcities. During the first phase of the COVID-19 pandemic, we witnessed a reflex of ‘our people first’ in many regions. In this paper, however, we show that a cooperative sharing mechanism can substantially improve the ability to face epidemics. We present a stylized model in which communities share their resources such that each can receive them whenever a local epidemic flares up. Our main finding is that cooperative sharing can prevent local resource exhaustion and reduce the total number of infected cases. Crucially, beneficial effects of sharing are found for a large range of possible community sizes and cooperation combinations, not only for small communities being helped by large communities. Furthermore, we show that the success of sharing resources heavily depends on having a sufficiently long delay between the onsets of epidemics in different communities. These results thus urge for the pairing of a global sharing mechanism with measures to slow down the spread of infections from one community to the other. Our work uses a stylized model to convey an important and clear message to a broad public, advocating that cooperative sharing strategies in international resource crises are the most beneficial strategy for all. It stresses essential underlying principles of and contributes to designing a resilient global supply chain mechanism able to deal with future pandemics by design, rather than being subjected to the coincidental and unequal distribution of opportunities per community that we see at present. To keep a pandemic like the one caused by COVID-19 . UnfortuDue to an exponential increase in demand during epidemic waves, local production increases are rarely fast enough to cover the high need . MoreoveThe primary production of medical products comes from global supply chains (GSC). GSCs have been optimized for cost-efficiency and may therefore lack resilience, such as redundant supply lines or large buffers along the chains. For this reason, a major topic of debate is if and how these GSCs should be restructured or replaced ,10,11. AConsequently, due to the resource scarcity threat in the first wave of the COVID-19 pandemic, multiple countries started to stock, confiscate, and prohibit the exportation of PPE ,15 and dInstead, this paper investigates the question of whether implementing a resource-sharing policy among different regions or countries can enhance essential-resource stock resilience. A PPE-sharing policy differs from increasing the overall resilience of GSCs: instead of ensuring the transport lines from producers to each individual consumer region, a sharing policy entails creating flexible transport lines between the consumer regions. For this reason, it may provide a more feasible approach that is effective at suppressing future pandemics. The current research field into epidemic spreading and COVID-19 lacks studies on sharing strategies for sharing this type of resource. However, the effects of vaccine sharing between two populations have already been studied using a classical multi-compartmental susceptible–infected–recovered (SIR)-based model , concludIn this article we will first present the stylized model we created to investigate cooperative resource sharing, followed by a simulation of sharing scenarios between different GDP-sized community combinations. In brief, the model concerns two communities that can be interpreted as countries, regions, or even cities. We modeled their local epidemics and the spread of infections across the communities, in combination with their (unspecified) PPE resources. PPE availability lowers the infection rate by offering protection and therefore enables suppressing the infection spread to some degree. Of interest are the effects of simulating changes in resource-sharing behavior on the capacity of going through the entire epidemic without fully depleting the PPE stock.The specific contribution of this study is thus not the novelty of our modeling techniques, but presenting an important message to a broad public by using a stylized model. Motivated by the non-cooperative strategies observed at present, it has the purpose to understand at a high level the impact of sharing essential resources and which factors should be taken into account to reach a better local situation for all communities involved.Here follows a functional description of the model needed to understand the results. Readers interested in more technical details are referred to the Extended Methods in S will be used to refer to the PPE ‘stock’ from the DSP model, as susceptibles (from the SIR model) are never referred to outside of the appendix.We developed a simplified demand–stock–production (DSP) model to emulate the usage and production of PPE. PPE can be interpreted here as any physical equipment that may help limit disease transmission. To cover the disease dynamics, including the transmission-mitigating effects of PPE, the DSP model is coupled to a classical susceptible–infected–recovered (SIR) model ,22 withoFor the inter-model coupling, the following two assumptions are used: (1) more protective equipment is needed when the amount of infected increases ,9; and  reaches stock depletion. Although this is already a successful sharing example for A, the time difference is still too short for the combined stocks to be restored sufficiently for the second epidemic in B. That being said, we can see that when the time difference between A and B epidemic onsets is increased further, both communities can achieve a low infected ratio (blue state). In the latter case, there was enough time to replenish the stock levels between the consecutive epidemics to save both communities.The size of the time difference (We defined the medium stock category with this maximum stock of B (the community that gets infected secondly) still reaches high infected numbers even diminishes for the lower maximum stock communities when The observation that sufficient time difference increases the chance of having one or both sharing communities reach a low infected ratio also holds for more intermediate maximum stock communities. This becomes apparent from In summary, the most important result for equal maximum stock community sharing is that communities with insufficient stock to weather the epidemic by themselves can, when agreeing on sufficiently low sharing thresholds, pass the epidemic without reaching the maximal infected ratio. Crucially, for these communities, a large-scale epidemic can be averted only for either or both of them when the time difference between epidemic onsets is sufficiently long. This requires a tailored strategy depending on the dynamics of the disease at hand, but always with a strong focus on mitigating disease spread between different regions by, for example, imposing travel restrictions early in epidemic outbreaks.Upon reading the equal maximum stock results, one could start fearing that sharing poses a risk to the second community. However, as we will further show by presenting the unequal maximum stock sharing results, this fear is unjustified for two reasons: (1) for a low- or medium-stock community, sharing never leads to worse results than non-sharing for both equal and unequal maximum stock combinations; and (2) for higher-stock communities there is hardly any risk in unequal sharing-even when the total shared stock turns out to be insufficiently high for both communities, a higher-stock second community can still weather the epidemic with a low infected ratio .B in the figure). This should again stress the importance of implementing measures to sufficiently slow down infection spread between communities, rather than discouraging higher-stock communities from sharing. Furthermore, in Only for very specific low time differences between epidemic onsets, a secondly affected higher-stock community could possibly be worse off, i.e., experience so-called ‘second community disadvantage’  the time differences between these onsets and agreeing on a low sharing threshold.In other aspects however, the epidemic onset ordering does impact sharing outcomes, which is indicated by some asymmetries that can be observed in the phase plots around An interesting illustration of the effects of increasing the time difference between onset in a lower-stock and higher-stock community is shown in By increasing the threshold, the system passes from a state of depleted stock for the first community, via the maximal total infected ratio and a state of second community disadvantage, to both communities reaching low infected ratios. This effect is indeed asymmetric around The results discussed here solely focus on the three stock categories, as we defined before, to ease result interpretation. For more intermediate stock values, we refer to Summarizing, the unequal maximum stock sharing results presented here again stress the positive combined effect of a low sharing threshold and a sufficient time difference between epidemic onsets. Additionally they show (1) that there is no downside from sharing for lower-stock communities and (2) how high-stock communities may substantially improve the overall epidemic resilience for both communities combined without much increased risk for their own population, as long as sufficient measures are implemented to reduce epidemic spread between the communities.We have introduced a stylized PPE production model with a sharing mechanism for communities facing an epidemic. The model convincingly shows that involved PPE stock sharing can greatly improve total and local epidemic resilience for all combinations of maximum stock communities. Crucially, these sharing strategies should be accompanied by disease spread mitigation measures to increase the time between epidemic peaks. This ensures that both communities have access to large quantities of stock when they need it most. First, we saw that completely blocking the circulation of essential supplies can lead to local stock shortages and subsequently epidemic proliferation. Then, we showed that for all realistically possible scenarios , sharing leads to better overall and local epidemic outcomes than non-sharing. Even if a high-stock community would exist, sharing can never lead to a more negative outcome for any cooperating community as long as the epidemic onset time differences are long enough. It is good to additionally realize that investing in the global reduction of infection rates helps to restrict the total viral circulation and the chance of reinfection in each region. Therefore, these investments are beneficial to all and crucial for final pandemic mitigation. The results thus strongly advocate for a willingness to share big stock proportions, with a special focus on accompanying measures to slow down epidemic spread between communities. In this way, pandemics can be fought way more efficiently and successfully for all parties involved with their respective PPE production limitations.We emphasize that the numbers used in the examples are not intended to be realistic and are merely for illustrational purposes. In particular, what time difference would be ‘sufficient’ will depend on the properties of the virus, the modes in which people have contacts with other people, the infrastructure and production capabilities of the PPE, the preventive measures in place, and so on.As mentioned before: increasing the delay between epidemic onset should evidently be a major focus when implementing this sharing mechanism. To achieve this, participating countries must directly focus on, e.g., limiting international travel when a community gets infected. Interestingly, the process of resource sharing itself could then start posing a threat, by increasing the chance of importing infection cases . This con, and PPE production rate P could differ substantially between communities. At the moment, the only characteristic we varied between communities was their maximum stock There are some simplifications in our model, which could possibly be accounted for in further research. For example, the effective reproduction number n communities and r resource types. Lastly, in all its simplicity, our model could serve as a basis for models of higher complexity that more closely resemble real-world scenarios. After fitting the data of manufacturers, resource usage, and epidemic dynamics, optimal sharing strategies could potentially be estimated with some precision for combinations of collaborating communities, depending on the uncertainties involved. A similar work of fitting is done to predict SARS-CoV-2 evolution [Further interesting extensions could be to include more communities and to tailor the DSP model to specific and possibly multiple resource types. When multiple communities are involved, the necessary individual sharing amount to sufficiently help a community in need reduces as the load can be divided over contributing groups. In our model we have seen that two low-stock communities had too little combined stock to save either of them, even when sharing major proportions of stock. But a bigger group of solely low-stock communities could potentially have enough combined stock to successfully avoid high infected ratios. However, one should note that in this more complex scenario, a more complex sharing strategy decision structure also arises. To model the politics of this, game-theoretic approaches could be used ,25. For volution .Overall, we can see that increasing global resilience to future pandemics is a complex problem that requires an interdisciplinary effort. Although myriad extensions could be made to this model, these additions would have unnecessarily distracted the reader from our main aim of communicating to policymakers how cooperation can improve epidemic resilience. The strengths of this model are its simplicity and comprehensibility, conveying a clear message and recommendations.In summary, this work demonstrates how trade barriers for essential healthcare resources can worsen pandemic outcomes, while cooperation between different communities can successfully avoid resource scarcity and prevent high-infected incidences for all parties involved. We used a stylized model encompassing disease spread and a resource supply chain in two communities. If enough measures were taken to slow down infection spread between them, both communities were able to avoid resource exhaustion at any point during their epidemics by cooperative sharing. This shows that collaboration is a viable solution to avoid resource scarcity. This message is similarly true for vaccines, as we have learned during the current COVID-19 pandemic [The model serves as a proof-of-concept that illustrates how sharing protective equipment resources between communities is a crucial step in epidemic mitigation. Although the model is stylized, we believe that its general insights are easy to understand and essential to disseminate to all stakeholders involved. It advocates for involved sharing strategies between all types of communities, showing that with a high willingness to share and a strong focus on slowing international infection spread, total epidemic mitigation outcomes can be improved greatly. It also successfully demonstrates that high GDP countries should not be afraid to offer help to countries that need it, as potential negative effects for their own population can be abolished if the right disease spread mitigation measures are in place. Furthermore, reducing viral circulation globally reduces each region’s chance of reinfection and is thus in the interest of all. By taking these insights into account, we believe that global initiatives can help prevent essential-resource exhaustion during pandemics. Our study thus shows that mutual resource sharing can potentially support all participating communities during their local epidemics and ultimately lead to the collaborative mitigation of a pandemic.


Azadirachta indica). SuperGrainbags® were more effective in maintaining seed moisture at acceptable levels close to pre-storage conditions than LDPE bags. Both seed germination and seedling coleoptile length were significantly greater in hermetic than LDPE bags. Neem had no effect on seed moisture, germination, or coleoptile length. SuperGrainbags® were also more effective in abating seed damage during storage, although inclusion of neem within LDPE bags also had significant damage. Quantification of seed predating insects and diseases suggested that SuperGrainbags® also suppressed Coleopteran pests and blackspot, the latter indicative of Fusarium graminearum. Conversely, where farmers used LDPE bags, neem also had an additional though limited pest suppressive effect. Post-storage treatment scoring by farmers revealed a strong preference for SuperGrainbags® and no preference differences for or against neem. This study demonstrates a process by which farmers can be involved in the participatory co-design and testing of alternative wheat storage options, and stresses the need to develop SuperGrainbag® supply chains so hermetic storage can be made widely available.Smallholder farmers in Bangladesh often use low-density polyethylene (LDPE) bags contained within woven polypropylene bags to store wheat seed during the summer monsoon that precedes winter season planting. High humidity and temperature during this period can encourage increased seed moisture and pests, thereby lowering seed quality. Following a farm household survey conducted to inform trial design, eighty farmers were engaged in an action research process in which they participated in designing and conducting trials comparing traditional and alternative seed storage methods over 30 weeks. Factorial treatments included comparison of hermetic SuperGrainbags® (Premium RZ) against LDPE bags, both with and without the addition of dried neem tree leaves ( •Surveys identified seed storage methods for participatory trial co-design in Bangladesh.•Storage trials were then conducted with 80 wheat farming households.•Hermetically stored seed had greater germination and coleoptile length than LDPE bags.•Neem slightly reduced damage rates for traditionally but not hermetically stored seed.•Both men and women farmers expressed strong preferences for hermetic storage. These are in addition to Lepidopteran pests such as the Angoumois grain moth ) and some ant (Hymenoptera: Formicidae) species  to stored grains (Sitophilus zeamais Motschulsky) and bean weevils  in Bangladesh and Ethiopia, respectively  and the Department of Agricultural Extension (DAE), studies were conducted in Faridpur, Rajbari, and Gopalgonj districts in central Bangladesh. Both BARI and DAE proposed these districts as locations in which wheat is a crop of importance to food security for smallholder farmers, but in which farm households had challenges with wheat seed storage and in which no prior experimental work evaluating wheat seed storage methods had been conducted .Fig. 1Lo2.2n = 28, 22, and 23 in Faridpur, Gopalgonj, and Rajbari districts, respectively) across six villages . Villages were selected by DAE officials as locations in which field-level extension staff had an interest in research and where farmers had requested guidance on improved seed storage management recommendations. Survey respondents were first randomly drawn from a list of farm households supplied by DAE as having had at least a five-year history of growing wheat in these villages. From this initial list, DAE next identified households for survey based on criteria including (a) their willingness to participate in household trials testing alternative seed storage methods, (b) willingness to include their own saved seed in research trials, and (c) households in which male and female household heads both agreed to be surveyed. When the pre-trial surveys were administered, 72 had been the maximum number of farm households (including husbands and wives) in the selected villages that were willing to be surveyed and met the above criteria. Survey respondents thus included both male and female household heads (husbands and wives) who were interviewed separately by trained male and female enumerators, respectively, using a pretested questionnaire in February of 2012.To assist in the design of participatory household seed storage trials, we conducted a survey of 72 farm households  with paired male and female discussion facilitators in each village during March of 2012. In these meetings, a descriptive summary of survey results was presented in the form of pie-charts on posters, after which a facilitated discussion led by researchers aimed to further elucidate men's and women's perceived wheat seed storage problems and interest in potential solutions. During FGDs, farmers were also introduced to the concept of hermetic storage; combined with DAE staff's partial familiarity with hermetic storage, this spurred farmers' and DAE's interest in testing hermetic SuperGrainbags® (Premium RZ), which had recently begun to be promoted for seed storage in Bangladesh. Further interactions with farmers and subsequent presentation of an initial trial protocol draft in each village in April 2012 aided in the finalization and farmers' endorsement of trial design.2.4Following completion of the participatory trial design process, storage trials were initiated using the protocol co-designed with farmers in each of the villages in which farmers had been surveyed . In addiThe first experimental factor ‘neem’ had two levels - with and without neem addition - based on survey data and FGDs that highlighted how farmers sometimes mix dried neem tree leaves with stored seed to reduce pest losses. The second factor ‘storage device’ (two types: low-density polyethylene ‘LDPE’ bag and SuperGrainbags®) represented traditional and alternative storage devices, respectively. Trials were conducted under household conditions, and with the objective of assessing the performance of storage devices and methods under conditions that could be implemented by resource-poor farmers. As farmers were however largely unfamiliar with hermetic storage principles and SuperGrainbags®, all husbands and wives in each household were given a standard and brief training on how to use the experimental storage devices and maintain experimental treatments.https://www.grainpro.com/grainpro-bag-zipper). The size of both the SuperGrainbags® and the LDPE bags was 70 by 40 cm, the former and later with a thickness of 78 ± 10 and 60 ± 10 μm, respectively. Each household participating in the trials therefore had a total of four bags to store seed in their houses.We supplied each farmer with a new LDPE bag, which they contained within a woven polypropylene bag, as a control treatment. Fertilizer bags in Bangladesh are typically prepared using high strength but low density woven polyethylene strips and are used to contain LDPE bags in grain storage; as such, this treatment mimicked this common farmers' practices. The second storage device was the hermetic storage Premium RZ SuperGrainbag®, hereafter reffered to as a SuperGrainbag®, provided by GrainPro Inc.  were subsequently mixed with seed before storage. The quantity of neem applied was chosen by households during FGDs as an average of what they considered to be the requisite ratio for neem leaves to seed stored  both immediately before and after storage. Following recommendations from BARI for on-farm experiments with a large number of replicates and treatments, we retrieved five randomly selected sub-samples of 50 seeds both before and after storage. Seed germination testing was conducted immediately thereafter in BARI's Seed Testing Laboratory in Gazipur. The five sub-samples of 50 seeds from each treatment combination  from each household were first visually examined for signs of fungal presence and damage signified by cracking or evidence of predation. They were then placed evenly on a moistened and a distilled water damped germination paper towel at 8 a.m. and placed in an incubator at 20 °C for eight days. Moisture was maintained by misting with distilled water at 8 a.m., 2 p.m. and 8 p.m. daily. After the eighth day, normally germinated seeds that showed emerged sprouts were distinguished from dead and abnormally germinated seeds and subsequently counted from each of the five sub-sample groups. Sub-sampled data were then averaged to provide a single germination percentage for each treatment from each household replicate. In addition to germination, seed vigor can be a useful parameter for identifying the potential of seed to perform well after storage . Hence, 2.5.2Prior to removing seed from storage devices after storage, four composite samples of seed equal to 500 g were randomly retrieved from storage devices and placed in bowls. Samples were then thoroughly examined following 2.5.3At the conclusion of storage, male and female household heads together extracted seed from storage devices. Following a visual inspection, they were each independently requested to provide a score between 1 and 4 representing their perception of the effectiveness of each treatment option to maintain seed quality, with 1 being poor, and 4 being high, performance.2.6Farm household survey data across villages and districts were analyzed descriptively and presented as percent total in pie-chart form to farmers during FGDs, and in tabular form in this manuscript. Data for different parameters observed in the household storage trials were analyzed using three models. In the first statistical model, before and after storage measurements of seed moisture content, germination, coleoptile length and damaged seed were analyzed using a repeated measures two-factor factorial ANOVA, with the addition or absence of neem as the first within-subjects factor, and storage device type as the second within-subjects factor. The timing of sampling (before or after storage) was considered as the between-subjects factor. Both within- and between-subjects factors were treated as fixed effects, with replicate considered as a random effect. This model permits the examination of two- and three-way interactions that compare the second (with or without neem) and third (SuperGrainbag® or LDPE bag) factor treatment performance to each other, while accounting for any variation observed in the first factor (before and after storage). In the second statistical model, post-storage data for insects and indicators of disease were analyzed using a factorial ANOVA, with neem (with or without), and storage device (LDPE or hermetic), as the first and second factors, respectively. These factors were also considered as fixed, while farmer replicate was again treated as a random effect. Finally, in the third statistical model, farmer's post-storage preference scoring for storage methods was analyzed using a three-factor design/ANOVA, with storage device, neem, and sex of the survey respondents considered as fixed factors, with household replicates as a random factor.X + 1]), where X is the original observation) before they were subjected to ANOVA. If significance was detected at α = 0.05, treatment means were separated by using the Student's-t or Tukey's Honestly Significantly Different (HSD) test for non-interacting and interacting sources of variation, respectively.All analyses were conducted using the restricted maximum likelihood (REML) procedure in JMP  tablets per storage bag, although most indicated that naphthalin was expensive and regularly not available in the market. The remaining households used various types of insecticides  as protection measures. Only 1% of farmers surveyed used rodenticides.During FGDs, farmers expressed that neither they could neither reliably access nor afford synthetic insecticides as an additive to stored wheat seed. For this reason, they expressed an interest in experimenting with neem leaves, which are occasionally used in the study area  as a pot3.2As described in Section 3.2.1P < 0.001) mean increase (2.8%) in moisture across treatments. The inclusion of neem had no effect on seed moisture, though a significant (P < 0.001) three-way interaction was observed, with SuperGrainbags® preventing seed moisture from rising above 10.4%, while seed moisture in LDPE bags exceeded 12.1%. Seed moisture content after storage demonstrated that of the 160 first factor treatment comparisons, 103 of the observations of LDPE storage had moisture contents >12% before storage, while SuperGrainbags® had only 28  decreased across treatments following storage. The percentage of damaged seed conversely significantly (P < 0.001) increased by 1.5% across treatments (P < 0.001) interaction between sampling time and storage device was observed, with SuperGrainbags® maintaining a germination percentage when compared to pre-storage levels. LDPE bags however were associated with significantly reduced germination by 3.7–4.1% lower than pre-storage levels. A nearly significant (P = 0.08) three-way interaction was also observed, with SuperGrainbags® maintaining germination percentage on-par with pre-storage conditions, yet with neem inclusion to LDPE bags tending towards a slight increase. As with germination percentage, significant (P < 0.001) differences between storage devices were found for coleoptile length when comparing pre- and post-storage conditions. Coleoptile lengths measured following SuperGrainbag® storage were not different than pre-storage lengths. Conversely, coleoptile lengths were 5.0–9.5 mm shorter, on average, following storage in LDPE bags, and were significantly different from pre- or post-storage SuperGrainbag® measurements.Both germination percentage and coleoptile length significantly  between sampling time and inclusion of neem. Each two- and three-way interaction that considered sampling time (before or after storage) as a factor affecting seed damage was significant at either P < 0.05 or P < 0.001. While no differences between treatments were found, and damage levels were low (0.3–0.5%) before storage, the addition of neem to SuperGrainbags® maintained these low levels, while SuperGrainbags® without neem had a significantly higher damage rate of 1.4% after storage. Considering the LDPE bag-based treatments, use of neem lowered damage rates from 3.6 to 2.2% compared to LDPE bags alone, though both LDPE treatments (with or without neem) had higher damage than the SuperGrainbag® treatments.There was a significant interaction  ranged from 0 to 6 for the former family and from 0 to 9 for the groupings of Chrysomelid, Tenebrionid and Bostrichid beetles, with a mean density of 0.5 for Curculionidae and the grouping of other beetles  and no differences among treatments. Our household surveys also suggested that insect pest presence was dominated by Coleoptera. The primary family identified was Curculionidae, with species including  beetles .Table 3IP < 0.05) were found for populations comparing with and without neem addition (−1 seed stored) than the former . Similarly, hermetic storage without neem had a highly significant effect (P < 0.001) on reducing weevil density, with 0.15 individuals kg−1 seed stored compared to when LDPE storage without neem . A significant (P < 0.01) interaction was also found when comparing both factors. In a statistical sense, hermetic storage with or without neem were both equally suppressive of Curculionidae populations, although for LDPE storage, neem addition lowered weevil density by 0.41 individuals kg−1 seed stored , Tenebrionid beetles such as red flour beetle (Tribolium castaneum) and darkling beetle (Coleoptera tenebrionidae), and Bostrichid beetles such as auger beetles (Heterobostrychus sp.) and borers beetles (Rhyzopertha dominica) were found. A single lined flat bark beetle Laemophloeus minutus of the superfamily Cucujoidea was also observed. As each of the population density of these species was relatively low on an individual basis, we bulked all non-Curculionidae beetles into a general category for analysis. Neem addition had similar effects as found with Curculionidae with significant (P < 0.01) differences, and population density 0.35 individuals kg−1 seed stored lower than without neem. Hermetic storage had a similar suppressive effect on population density of Coleoptera, with highly (P < 0.001) significant differences between LDPE bags and SuperGrainbags®, the latter nearly eliminating non-weevil beetle populations relative to the former. A mildly significant (P < 0.05) interaction was also found when these factors were compared, mirroring the same pattern as found for Curculionidae.Considering non-Curculionidae species, Chrysomelid beetles such as bean weevil  different when neem was added, though at low levels of one percentage point less when neem was added to storage devices. SuperGrainbags® had a slightly stronger and significant (P < 0.001) effect, with an incidence of blackspot 1.6% less than LDPE bags. Mildly significant (P < 0.05) interactions were observed, with the highest percentage of blackspot (5.1%) in LDPE bags without neem and dropping to 3.5% in LDPE bags with neem, the latter statistically similar to the degree of disease suppression observed with SuperGrainbags® without neem. Neem addition to SuperGrainbags® however had the greatest (2.9%) blackspot suppressive effect.Following storage, we observed blackspots on seeds indicative of attack by fungus of Order Hypocreales family Nectriaceae 3.2.4P < 00.01) preference for SuperGrainbags® compared to LDPE bags for wheat seed storage (data not shown). No differences in preferences were found between men or women, nor for inclusion or exclusion of neem, under any storage device option.After storing seeds in the LDPE bags and SuperGrainbags® for 30 weeks with or without neem, both male and female (husband and wife) household members indicated significantly greater  of replicate samples. The The characteristics of high germination rate and rapid coleoptile expansion is desirable where farmers sow their wheat seed deeply into the soil, or in zero or reduced tillage systems where shoot emergence may be hampered . Our obs4.2.3The results of the current study suggest that both neem addition and/or hermetic storage could be used to reduce densities of Coleopteran pests when storing wheat over the monsoon in the sub-tropics. During storage, insect and disease-causing organisms’ activities release moisture and energy in the form of heat into the spaces between seeds. Moisture therefore builds up faster and to higher levels from insects and other organisms than from grain respiration alone . Our obsFusarium graminearum can cause blackspots and molds can cause mycotoxins and aflatoxins in stored grain (P < 0.001) effect, with lower incidence of blackspot than LDPE bags. Mildly significant (P < 0.05) interactions were observed, with the higher percentage of blackspotted seed found for LDPE bags without neem than for LDPE bags with neem, the latter statistically similar to the degree of disease suppression observed with SuperGrainbags® without neem. Neem addition to SuperGrainbags® however had the greatest blackspot suppressive effect.Insects can also enhance fungal development because they increase moisture and temperature, open the testa for infection, and can supply inoculum. The fungal pathogen ed grain . Our resThe presence of mycotoxins and aflatoxins as reflected by the presence of molds and blackspots in stored grain suggests the need for the development of control measures to avoid potential losses due to molds and fungal and insect attacks . An impo4.2.4P < 00.01) preference for SuperGrainbags® compared to LDPE bags for wheat seed storage. There were however no differences in preferences between men or women farmers, nor for inclusion or exclusion of neem, under any storage device option. These data suggest that farmers consider SuperGrainbags® to be an effective device for wheat seed storage and that the co-design and participatory implementation of trials by farming households can help inform opinions with experience and evidence. Farmers however also expressed concerns about the SuperGrainbag® as they are not produced domestically and are still not widely or commercially available in Bangladesh. The current cost for an imported SuperGrainbag® is around USD 1.9 compared to USD 0.4 for a locally made LDPE bag. Although these costs appear to be miniscule, farmers who have not participated in trials may have an aversion to investing in hermetic storage out of a perception that LDPE bags are sufficient, especially when used in combination with neem and/or insecticides. Such costs are also higher than those reported by At the conclusion of the storage period, when participating farmers were asked about the relative preference of different storage treatments both male and female (husband and wife) household members indicated significantly greater (We suggest that further action research be conducted by extension services to familiarize farmers with the viability of different storage options. Research to identify Bangladeshi farmers’ willingness to pay for SuperGrainbags® could also help to inform commercialization efforts and/or assist in the development of supportive market policy and incentives for farmers to purchase and access hermetic storage options. Finally, our study suggests that in conducting participatory on-farm research trials, it is important to include novel treatments suggested by farmers – such as the inclusion of neem – as a benchmark from which they can compare among their own traditional and alternative options, as also suggested by 5This study aimed to elucidate farmers’ wheat seed storage practices obtained from a household survey, and to use the data collected during surveys to co-design and implement participatory farm household trials with farming households comparing hermetic SuperGrainbags® and LDPE bags storage, both with and without neem. Our results suggest that SuperGrainbags® are more effective in maintaining seed moisture levels close to pre-storage conditions and in increasing germination and coleoptile length than LDPE bags, with no effect of neem. Hence, SuperGrainbags® could potentially lend additional agronomic advantages due to rapid coleoptile expansion that could aid in wheat seedling emergence and establishment, though this hypothesis requires confirmation under field trial conditions.Fusarium graminearum. For these reasons, where farmers are unable to access or afford hermetic storage bags, the indigenous technique of applying dried neem leaves appears to confer some, albeit limited benefits for pest control. Conversely, the comparatively better and more consistent performance of SuperGrainbags® for the range of parameters and higher preference rankings by participating farmers, suggests that agricultural development programs advising wheat farmers on storage techniques in Bangladesh may wish to focus more on making hermetic storage options widely available and affordable. Lastly, this study demonstrated the value of participatory approaches that actively consult and involve farmers in the selection and design of experimental treatments, and the implementation of long-duration storage trials within their own homes. Such participatory approaches can help validate the performance of promising storage options under real-world, and hence highly relevant conditions, while also serving as a pedagogic tool for extension services and farmers alike.SuperGrainbags® were also more effective in reducing insect damage to seed during storage. Inclusion of neem with LDPE bags also had statistically significant damage abatement effects, although at levels lower than those associated with SuperGrainbags® with or without neem. Hermetic storage bags suppressed the primary seed-predating arthropod pest of Coleoptera order with Curculionidae as the most abundant family, and weevils and other families of beetles. Where farmers used LDPE bags, neem inclusion had some additional pest suppressive effect, with similar patterns for damaged seed and the blackspot Conceptualization: TJK, MMU. Methodology: TJK, MEB, MKG. Formal Analysis: TJK, KH. Investigation: TJK, MMU, MEB, MZK. Data curation: KH, MZH, Writing-Original Draft: TJK, JT. Writing – Review & Editing: TJK, KH, MZH, JT. Project Administration: TJK, MMU. Supervision: TJK, MKG, MMU. Funding acquisition: TJK.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Staphylococcus aureus (MSSA) infections are an important cause of morbidity and mortality in neonatal intensive care unit (NICU) infants. MSSA colonization  is a risk factor for subsequent invasive infection . Active surveillance programs coupled with decolonization measures for MSSA-colonized infants have been associated with decreased invasive MSSA infection rates in the NICU setting.Invasive methicillin-susceptible In June 2020, an active surveillance program was implemented to detect MSSA nasal colonization for infants admitted to the St. Louis Children’s Hospital NICU, a 140-bed quaternary NICU. MSSA-colonized infants were decolonized with a 7 day course of intranasal mupirocin twice daily plus topical chlorhexidine baths on days 1, 4, and 7 . MSSA bloodstream infection (BSI) rates were followed quarterly pre- (September 2018-May 2020) and post- (June 2020-March 2022) implementation of MSSA surveillance and decolonization. Mean MSSA BSI rates pre- and post-implementation were compared via paired T-test. The number of infants treated with mupirocin was calculated pre- and post-implementation.Figure): 0.22 per 1000 patient-days pre-implementation, 0.16 per 1000 patient-days post-implementation (p=0.37). Since initiating MSSA decolonization in June 2020, the number of NICU infants exposed to mupirocin increased nearly fourfold: mean 10 infants treated per month pre-implementation, mean 38 infants treated per month post-implementation.After implementing MSSA surveillance and decolonization there was no significant change in the mean MSSA BSI rate .All Authors: No reported disclosures.