Antimicrobial resistance is a global public health concern, with extensive associated health and economic implications. Actions to slow and contain the development of resistance are imperative. Despite the fact that overuse and misuse of antibiotics are highlighted as major contributing factors to this resistance, no sufficiently validated measures aiming to investigate the drivers behind consumer behaviour amongst the general population are available. The objective of this study was to develop and investigate the psychometric properties of an original, novel and multiple-item questionnaire, informed by the Theory of Planned Behaviour, to measure factors contributing to self-reported antibiotic use within the community.A three-phase process was employed, including literature review and item generation; expert panel review; and pre-test. Investigation of the questionnaire was subsequently conducted through a cross-sectional, anonymous survey. Orthogonal principal analysis with varimax rotation, cronbach alpha and linear mixed-effects modelling analyses were conducted. A 60 item questionnaire was produced encompassing demographics, social desirability, three constructs of the Theory of Planned Behaviour including: attitudes and beliefs; subjective norm; perceived behavioural control; behaviour; and a covariate – knowledge.Three hundred seventy-three participants completed the survey. Eighty participants (21%) were excluded due to social desirability concerns, with data from the remaining 293 participants analysed. Results showed modest but acceptable levels of internal reliability, with high inter-item correlations within each construct. All four variables and the outcome variable of antibiotic use behaviour comprised four items with the exception of social norms, for which there were two items, producing a final 18 item questionnaire. Perceived behavioural control, social norms, the interaction between attitudes and beliefs and knowledge, and the presence of a healthcare worker in the family were all significant predictors of antibiotic use behaviour. All other predictors tested produced a nonsignificant relationship with the outcome variable of self-reported antibiotic use.This study successfully developed and validated a novel tool which assesses factors influencing community antibiotic use and misuse. The questionnaire can be used to guide appropriate intervention strategies to reduce antibiotic misuse in the general population. Future research is required to assess the extent to which this tool can guide community-based intervention strategies. Antibiotics are an antimicrobial agent defined as “a chemical substance produced by a microorganism that kills or inhibits the growth of another microorganism” . Since tappropriate antimicrobial use as the “cost effective use of antimicrobials which maximises clinical therapeutic effect whilst minimising drug-related toxicity and development of antimicrobial resistance” [From an evolutionary standpoint, AMR is unavoidable  due to bistance” .Existing literature highlights consumer or patient demand and behaviour, as a driving force behind antibiotic misuse –20. UndeA subsequent survey conducted by the WHO 2015) questioned 9772 individuals across two member states in the six WHO regions. In this survey it was found that 65% of respondents had used antibiotics in the previous 6 months, with 81% (range 56–93%) indicating that they had obtained them from a medical professional. The WHO survey reported that 25% of respondents believed it acceptable to use antibiotics given to them by a friend or family member, 43% thought it acceptable to buy antibiotics or seek them from a doctor if they were sick with symptoms that they believed were effectively treated by antibiotics in the past, and 64% incorrectly believed viruses such as colds and influenza could be treated by antibiotics [ questionAccording to Wise et al., (1998), 20% of human antibiotic use occurs within the hospital sector, whilst 80% is within the community sector. Within this community portion, 20–50% may be questionable and unnecessary . Within Both the TNS Opinion & Social (2010) and WHO (2015) surveys had numerous limitations, including various sampling techniques, bias toward more educated responders, and an absence of checks upon socially desirable responding. Furthermore, neither survey was theory informed in order to enable prediction of consumer antibiotic use, other than the potential impact of poor knowledge about antibiotics and AMR, and neither reported detailed psychometric properties of the questionnaires. They do, however, confirm previous research which has identified a range of key factors contributing to patient behaviour with respect to antibiotic use, including attitudes and beliefs, subjective norms, self-efficacy and knowledge , 24.There are few measures which currently exist in this area. Many are specific to population sub-groups, including physicians, parents , 25–27, The current study sought to develop a questionnaire that predicts the factors influencing a consumer’s intentions to indiscriminately obtain and use antibiotics. Given that attitudes and beliefs , the opiOne of the most extensive TpB reviews, focusing on prospective behaviours across 237 studies, was conducted by McEachan, Conner, Taylor and Lawton, , who fouLimitations surrounding the TpB include its sole reliance upon self-reported behaviour, potentially inspiring socially desirable and less accurate predictions of objective behaviour . ArmitagThus, the aim of the current study is to develop and investigate the psychometric properties of an original, novel and multiple-item quantitative questionnaire, aiming to identify factors contributing to antibiotic use within the community, informed by the TpB. Considering the adaptive nature of the TpB , knowledA three-phase process was employed to develop the Antibiotic Use Questionnaire (AUQ) utilised within this study. Phases included: a literature review and item generation; expert panel review; and pre-test. Investigation of the AUQ was then subsequently conducted through a cross-sectional, anonymous and voluntary survey.An opening list of 43 items were drawn from a literature review of previous studies investigating consumer characteristics and self-reported antibiotic use, using search terms such as ‘antibiotic use’, ‘AMR’, and ‘antibiotic use influences’. Questions were then grouped under discrete categories, including demographics, knowledge, TpB constructs , and an outcome factor - self-reported antibiotic use behaviour. Items were adapted where required to suit the cultural and sociodemographic context of the target population. For example, ‘Aboriginal or Torres Strait Islander’ was added as an option for ancestry to reflect the Australian population.The original 43 item questionnaire was examined for content validity by a panel of eight experts, organised to represent a range of fields including psychology, business and health. Questions were evaluated with respect to the extent to which, on face value, they aligned with the TpB variables (including knowledge), their repetition, clarity and cultural relevance. Additional questions were generated in areas under-represented, such as social norms, and redundant questions removed. A subset of six items from the Marlowe-Crowne Social Desirability Scale  were ranBefore administration of the questionnaire to participants, a group of 10 participants pre-tested the survey to examine face validity. Feedback was gathered on time to completion, question clarity, perceived relevance, and face validity. Minor adjustments were made based on feedback received.The finalised questionnaire was distributed via an anonymous cross-sectional survey conducted between July – August 2018. Tacit consent was obtained, inferred through anonymous completion and return of the questionnaire. Survey Monkey was used to create a soft copy version of the questionnaire, with the link being distributed via non-moderated e-mail services and social media, predominantly incorporating snowballing techniques. Hard copy questionnaires were also distributed, mainly to participants who were unable to be reached via e-mail or social media, and for the purposes of purposive sampling after a mid-data collection review identified disparities in demographic representation. All hard copy questionnaires were completed in the presence of a researcher. Purposive sampling took place in popular public spaces, including a local shopping mall and retirement club, with a desire to achieve balance from older age groups, males, and those of lower socioeconomic status and education level. Hardcopies were returned directly to the researcher after completion, and manually entered into a Microsoft Office Excel, Version 10, spread sheet. The current study was approved by the Health and Medical Human Research Ethics Committee .n = 338), with the remaining participants recruited in person . Eighty participants (21%) were excluded from the analysis, due to concerns regarding the accuracy and reliability of their responses, after scoring equal to or higher than five in social desirability. Data from the remaining 293 (79%) participants was analysed.All recipients of the questionnaire, aged 18 years and over, were invited to partake in the research. Completed questionnaires were received from 373 participants. The majority of participants were recruited via online platforms . All completed questionnaires were screened for missing data, outliers and coding errors. Participants answered at least 83% of the questions  =3.23). Descriptive statistics for patient demographics were reported, expressed as raw numbers and percentages. An orthogonal principal component analysis with varimax rotation (factoran Matlab function) was utilised to assess the factor loadings of the questionnaire items for the four dimensions of the TpB and the covariate – knowledge. Cronbach alpha was used to determine the internal reliability of items relating to each of the five factors. Furthermore, linear mixed-effects modelling was employed to study the influence of the TpB factors on intended antibiotic use behaviour. The moderation from knowledge on the link between attitude/belief and intended behaviour was modelled as an interaction term between knowledge and attitude/belief. The fixed effects of the model included the interaction between attitudes and beliefs, and knowledge, PBC, social norms, age, gender, education, whether the participants had children, health trained, health worker in the family, frequency of antibiotic consumption, financial security, and most recent antibiotic consumption. The random effect was the participants.n = 52) were female with 55% (n = 44) of respondents having a bachelor degree qualification or higher. 59% (n = 47) of respondents were aged between 18 and 44 years, with the 18–24 year category the highest (n = 25). Whilst majority of respondents were not personally trained in a health-related field , 58% (n = 46) had a family member or friend with a health-related occupation. 55% (n = 44) of respondents excluded had not taken an antibiotic within the past year.Participant demographics are reported in Table n = 217) identified themselves as infrequent antibiotic users, consuming antibiotics once a year or less, with less than a third of respondents  consuming antibiotics within the past 6 months. Consistent with previous research, although 83% (n = 242) of participants correctly identified that antibiotics should be used for the treatment of bacterial infections, 25% (n = 74) of these respondents also incorrectly identified that they work for viral infections and/or fungal infections.Of the remaining 293 participants whose data was included, majority , and covariate, knowledge, are reported in Table Results showed modest but acceptable levels of internal reliability  within each variable: attitudes and beliefs = 0.60; social norms = 0.59; PBC = 0.64; knowledge = 0.61; behaviour = 0.76 . Inter-ip = 0.001), Social Norms , interaction between attitudes and beliefs and knowledge , and the presence of a healthcare worker in the family , were all significant predictors of antibiotic use behaviour. All other predictors tested did not produce a significant relationship with the outcome variable.A Linear-mixed model was run with the following fixed effects: the interaction between attitudes and beliefs and knowledge; PBC; social norms; age; gender; education; whether the participants had children, were health trained, had a health worker in the family; frequency of antibiotic consumption; financial security; and most recent antibiotic consumption. The random effect was the participants. Fixed effects coefficients can be found in Table An alternative model was tested with the same variables except for the moderator knowledge. AIC and BIC were slightly higher in this case (1040.9 and 1096.1 respectively) and the model explained a smaller proportion of variance (ordinary and adjusted R-squared: 0.6950 and 0.6820).Scores for rational antibiotic use were calculated using the factor loading coefficients from the questionnaire items which loaded to the construct of behaviour. Calculations were made as follows: 0.561*Behaviour Item 5 + 0.83*Behaviour Item 6–0.544*Behaviour Item 13 + 0.602*Behaviour Item 14. Scores were subsequently normalised in order to have 0 as the minimal score, and 10 as the maximum score. High scores are reflective of rational antibiotic use, whilst low scores are reflective of less rational behaviour. Figure The aim of the present study was to develop and investigate a novel quantitative measure, modelled on the TpB. The study sought to assess the factors influencing community antibiotic use and misuse, including: TpB variables ; knowledge; and key demographic characteristics .The confirmatory factor analysis identified items corresponding to the three variables of the TpB, the outcome variable (behaviour), and the covariate knowledge. The selected items demonstrated good psychometric properties in terms of internal reliability and convergent and discriminant validity. The internal reliability values are particularly encouraging considering the small number of items and the fact that when utilising Cronbach’s Alpha as a measure of internal consistency, the greater the number of items in the pool, the better the chance of obtaining a positive value, indicating greater internal consistency .A linear-mixed effects analysis revealed that intent of antibiotic use behaviour can significantly be explained by each of the TpB variables  and that the TpB construct predicted 70% of the variance in antibiotic use and misuse. This amount of predicted variance exceeds that of past literature using the TpB model to predict health related behaviours –41, 48, To our knowledge, this is the first sufficiently validated measure which assesses factors influencing antibiotic use and misuse within a general population and the first application of the TpB to the prediction of antibiotic use behaviour. The measure provides an opportunity for targeted intervention programs to reduce antibiotic misuse in the general community, and may inform public policy decisions. For example, while O’Neill has observed that greater accessibility to antibiotics is associated with an increase in indiscriminate use, there are limited empirical studies exploring this relationship . Our finOur study also contributes to an understanding of the role of knowledge, and hence the value of public educational programs, on antibiotic use intentions. Previous research into this relationship is largely contradictory; whilst some indicate a relationship between lesser antibiotic knowledge and more indiscriminate antibiotic use , 25, othIn our study we observed an interaction effect between knowledge and attitude/beliefs. This finding may provide evidence as to why information-intensive or educational-driven interventions alone are not entirely efficacious or maintain long-lasting results , 58, andConsistent with the TpB, subjective norm contributed to the prediction of antibiotic use behaviours, suggesting that antibiotic use behaviours are influenced by peers, family and community/cultural factors. This is a complex relationship given that in our study we found that the presence of family or friends working in a health-related field is associated with indiscriminate antibiotic use. Scaioli et al., indicated a similar finding, whereby those with a family member working in a health-related field were more likely to use non-prescribed antibiotics and keep left-over antibiotics . It is lThe remaining demographic variables did not significantly influence self-reported antibiotic-use behaviour. These results are not entirely surprising, given that the current literature is contradictory when examining the relationship between demographic variables and antibiotic use. Conflicting findings in the context of antibiotic-use behaviour are apparent for education level , 59, 60;Secondly, the sample size and representation is modest. This might explain why there was not a greater relationship between demographic variables and predictors of antibiotic use behaviours. A replication of this study with a larger and more representative sample would add to the value of the AUQ. Related to this, the questionnaire was distributed to an Australian sample and may not generalise to other nations or cultures. Further research is needed to determine whether the relationships identified in this study are replicated in other international samples.intent, and actual antibiotic use behaviours. Application of the AUQ to a population cohort and an informed intervention based on the identified drivers of antibiotic use behaviours is required.Finally, the utility of the study in informing intervention programs needs to be tested. This relates in particular to the verification of antibiotic use This study successfully developed and validated an original, theory driven tool which assesses factors influencing community antibiotic use and misuse. Notwithstanding the above mentioned limitations, the research highlights the pervasive influence that people-driven factors have upon antibiotic-use behaviours, likely contributing to the growth of AMR on a widespread scale. Furthermore, these findings have implications for the development of sustainable, multi-dimensional interventions that reflect the multitude of factors influencing antibiotic misuse. While AMR is a multifactorial problem requiring intervention at many levels of policy, drug discovery, and molecular biology, the role of end-point users, consumers, is a vital component of the worldwide effort to address AMR.


Escherichia coli O157:H7 formation. We have previously found that expression of dicC is reduced by 20.08-fold in VBNC E. coli O157:H7 compared to non-VBNC cells. Little is known about DicC except that it, along with DicA, appears to act as a regulator of cell division by regulating expression of the cell division inhibitor DicB. First, our results showed that the VBNC cell number increased in the ΔdicC mutant as well as the DicA-overexpressing strain but decreased in the DicC-overexpressing strain induced by high-pressure carbon dioxide, acid, and H2O2. Furthermore, the growth rates of both the DicA-overexpressing strain and the ΔdicC mutant were higher than that of the control strain, while DicC-overexpressing strain grew significantly more slowly than the vector strain. The level of the dicB gene, regulated by dicA and dicC and inhibiting cell division, was increased in the DicC-overexpressing strain and decreased in the ΔdicC mutant and DicA-overexpressing strain, which was consistent with the growth phenotypes. In addition, the dwarfing cell morphology of the ΔdicC mutant and DicA-overexpressing strain were observed by SEM and TEM. Taken together, our study demonstrates that DicC negatively regulates the formation of the VBNC state, and DicA enhances the ability of cells to enter the VBNC state. Besides, the cell growth rate and dwarfing cell morphology may be correlated with the formation of the VBNC state.The viable but non-culturable (VBNC) state, in which bacteria fail to grow on routine culture media but are actually alive, has been widely recognized as a strategy adopted by bacteria to cope with stressful environments. However, little is known regarding the molecular mechanism of VBNC formation. Here, we aimed to elucidate the specific roles of cell division regulatory proteins and the cell growth rate during VBNC  Escherichia coli and Vibrio cholerae from seawater could not be cultured on their routine medium but possessed metabolic activity. Based on this finding, they proposed the concept of the VBNC state, in which bacteria cannot grow on the routine culture medium but are actually alive. Subsequently, they further proved that these bacteria can recover from the VBNC state and become culturable again when the environment is suitable, which is termed resuscitation (The viable but non-culturable (VBNC) state, a unique biological state, is a positive response strategy used by a number of bacteria to cope with different adverse environments . Xu et acitation . A growicitation . To datecitation . Thus, VA range of stressful environmental conditions during food processing and preservation can also induce the entry of many foodborne pathogens into VBNC state, for instance, high-temperature sterilization of milk, chlorination of wastewater, extreme temperatures, ultraviolet (UV) radiation, ultrasound, irradiation, drying, pulsed electric field (PEF), high-pressure carbon dioxide (HPCD) and high-pressure stress, as well as the addition of preservatives and disinfectants . TherefoEscherichia coli is one of the most widely studied bacteria in VBNC research, and there are many studies on the conditions associated with induction and recovery of the VBNC state of this species as well as on the formation mechanisms  and thrombotic thrombocytopenic purpura, and produce mortality . E. coli O157:H7 was cultured overnight in Luria-Bertani (LB) broth at 37°C . The overnight culture was transferred to fresh LB broth at a dilution of 1:100 and inoculated with shaking (200 rpm) at 37°C to the exponential phase (OD600 = 0.8). LB agar was obtained by adding 1.5% (w/v) agar. When culturing the resistant strains or inducing protein expression, antibiotics or inducers were added into the culture as follows: ampicillin (100 μg/mL), kanamycin (25 μg/mL), arabinose (0.2% [w/v]), IPTG (1 mM).The bacterial strains and plasmids used in this study are summarized in dicC deletion mutant, the ORF of DicC was replaced with a kanamycin resistance (Kmr) gene cassette. The cassette was amplified from the pKD4 plasmid  by PCR using primer pairs containing an E. coli O157:H7 dicC-specific sequence  strains was purified and cloned into pTrc-His A with the appropriate restriction sites for BamHI and HindIII  to form the pTHA-DicC plasmid, which was then transformed into the ΔdicC strain.For the construction of the dicC and dicA genes were amplified by PCR with E. coli O157:H7 genomic DNA as the template using primer pairs  with restriction enzyme linkers (BamHI and HindIII)  . The ampE. coli O157:H7 of the WT strain, mutant strains and recombinant strains were induced by HPCD at 5 MPa at 25°C for 40 min. These strains were also induced into the VBNC state under acid or oxidation treatment. The condition of acid induction was treatment with pH 2.5 sodium chloride (0.85% w/v) for 3 h, and the condition of oxidation induction was 50 mM H2O2 treatment for 6 h. The number of VBNC cells was determined by staining with the LIVE/DEAD BacLight Bacterial Viability Kit  in accordance with the manufacturer’s instructions and a previously published reference . Briefly, cells were lysed, and RNA samples were extracted with the RNAprep Pure Cell/Bacteria Kit  according to the manufacturer’s instructions. RNA samples were reverse transcribed using the iScriptTM gDNA Clear cDNA Synthesis Kit  according to the manufacturer’s instructions. Primers  with ssoAdvancedTM SYBR® Green Supermix . The relative expression of the genes was calculated using the 2–ΔΔCt method , 4% SDS, 20% glycerol, 0.2% bromophenol blue, 5% β-mercaptoethanol] and boiling for 10 min. The proteins were separated on 15% SDS-PAGE gels and then transferred onto nitrocellulose membranes. The membranes were blocked in TBST  with 5% defatted milk, followed by incubation with primary anti-His antibody diluted 1:5,000  in TBST for 1 h at 37°C. The membranes were washed with TBST three times after incubation. Then, incubation with a horseradish peroxidase (HRP)-linked goat anti-rabbit secondary antibody  diluted 1:3,000 in TBST was conducted at 37°C for 45 min. The bands of the target proteins in the membranes were detected by using an Amersham Imager 600 .E. coli O157:H7 cells, ΔdicC mutants and recombinant strains was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in accordance with the procedures described previously . Analysis of variance (ANOVA) was performed by IBM SPSS Statistics 20.0 , and dicC gene was downregulated by 20.08-fold in VBNC E. coli O157:H7 cells induced by HPCD  were constructed. The DicC protein accumulation induced by IPTG in the DicC strain was verified by western blot analysis  as well as the vector, DicC and DicA strains induced by HPCD were detected using the LIVE/DEAD BacLight Bacterial Viability Kit followed by flow cytometry analysis. The results showed that compared with the WT strain, a larger number of ΔdicC mutant strain cells entered the VBNC state, while fewer VBNC cells were found in the ΔdicC/DicC strain  and 50 mM H2O2. The results showed that compared with the WT strain, the proportion of ΔdicC mutant strain cells entering the VBNC state was higher under acid and H2O2 stress, while the proportion was significantly lower for the ΔdicC/DicC complementary strain  phage, one of nine cryptic prophages in E. coli, contains three genes encoding the DicA, DicB and DicC proteins and one sRNA transcribed by DicF , could induce a burst of dicB expression and inhibit cell division (dicA gene resulted in an increase in the cell growth rate, implying that the expression levels of dicB decreased and cell division was suppressed, which is consistent with previous studies.In nditions . The Dic by DicF . FtsZ, a by DicF . Neverth by DicF . Under ny ceases . A previicB gene . In addidivision . In thisdicC gene are relatively rare. Previous studies have shown that the DicC protein can complement the function of the DicA protein, thereby inhibiting the expression of the dicB gene, but the evidence is unclear (dicC was overexpressed, the cell growth rate of the DicC strain decreased. In the ΔdicC mutants, the expression level of the dicB gene decreased, and the cell growth rate became higher than that in the WT strain. To further explain this phenomenon, the regulatory relationship between the dicA and dicC genes was demonstrated. It was demonstrated for the first time that the expression level of the dicB gene increased and that dicA was downregulated in the DicC-overexpressing strain, which suggested that the increased expression of dicB was due to the inhibition of dicA. This finding also indirectly indicated that dicA plays a major role in inhibiting the expression of dicB. In addition, the results also implied that DicC might inhibit the expression of the dicA gene to some extent, possibly because these two genes share a common promoter region, but the direction of transcription is the opposite. dicA gene could significantly inhibit the expression of dicC, which was consistent with the results of this study. Here, we confirmed that overexpression of dicC can also inhibit the expression of DicA, thereby initiating dicB transcription and finally inhibiting bacterial growth. The construction of the dicA deletion mutant was not successful in this study, which may be due to the deletion of dicA leading to significantly increased expression of dicB and bacterial growth was suppressed, so positive bacterial colonies could not be screened.Relevant studies on the function of  unclear . In thisftsZ, ftsA, ftsQ, and ftsH, was downregulated in VBNC V. cholerae, while minC and slmA were upregulated, leading to inhibition of cell division. E. coli. In addition to these omics studies, a recent study found that the cpdA gene, encoding cAMP phosphodiesterase, delayed the formation of the E. coli VBNC state by reducing intracellular cAMP levels, which negatively regulated cell division and growth under stress conditions. That is, the lack of cAMP-CRP effectively led to retention of high colony forming activity and promoted cell division, which was not conducive to the formation of the VBNC state (E. coli cells was negatively correlated with VBNC formation. Contrary to the results in this study, our results indicated that the bacterial growth rate was positively correlated with the formation of the VBNC state. A previous report proposed that VBNC state may be part of a dormant state in which active cells exist stochastically (Vibrio vulnificus, suggesting that VBNC cells existed stochastically during logarithmic-phase growth (dicC mutant strain, the number of VBNC cells randomly generated during logarithmic growth may also increase, eventually leading to an increase in the number of VBNC cells induced by stress; however, this hypothesis needs to be further verified.To date, there have been some reports regarding the relationship between cell division and VBNC formation; however, these studies were conducted at the omics level, while few studies regarding specific mechanisms have been reported. NC state . These rstically . Interesstically . It was e growth . TherefoSalmonella typhi cells exhibited gradual rounding after entering the VBNC state. The change from a bacillary to a coccoid form was also observed in VBNC Vibrio parahaemolyticus, with the occurrence of wrinkles and herpes-like structures on the cell surface (E. coli cells, accompanied by enriched intracellular DNA (Campylobacter jejuni and Edwardsiella tarda were found to transform from the spiral to coccoid form and from short rods to the coccoid form, respectively (dicC gene and overexpression of the dicA gene, an increase in bacterial growth rate was accompanied by the formation of a short and round shape compared with the WT strain, which was similar to the morphology of VBNC bacteria. Moreover, an increased number of cells of the two strains entered the VBNC state after induction by different stress factors. Hence, these results indirectly indicated that changes in morphology could affect bacterial entry into the VBNC state. It was presented for the first time that a short and round shape might be favorable for bacterial entry into the VBNC state.Changes in cell morphology and cell size are distinct characteristics of VBNC cells . Compare surface . Similarular DNA . In addiectively . CurrentdicC and dicA. Our investigation demonstrated that the cell growth rate regulated by DicC and DicA with changes in cell morphology may be positively correlated with the formation of the VBNC state. Elucidation of the molecular mechanism of the VBNC state will provide insights into bacterial stress responses and the physiological characteristics of the VBNC state, which may provide inspiration for the development of novel detection methods for VBNC bacteria or direct prevention of the entry of cells into this hard-to-detect state.This study proved for the first time that the formation of the VBNC state induced by HPCD was associated with the cell division-related genes The datasets generated for this study are available on request to the corresponding author.HP and KD carried out the experiments and wrote the manuscript. LR gave advice and assistance during the experiments. YW and LZ designed the experiments and revised the manuscript. XL revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acute respiratory tract infections (ARIs) are common in the outpatient setting. Although they are predominantly viral, antibiotics are often prescribed for the treatment of ARIs.Using the U.S. Medical Expenditure Panel Survey , we estimated the national prevalence and predictors of outpatient antibiotic prescribing for ARIs by provider type. We categorized the trends of antibiotic prescriptions  for ARIs by provider type . The outcome variable was defined as receipt of an antibiotic prescription during a consultation with a provider for an ARI (including outpatient clinic visit or doctor’s office visit).p < 0.0001). Associations of patient- and provider-level variables with antibiotics prescription were examined using binary logistic regression. Blacks were more likely to receive antibiotics than whites , and antibiotic prescription was more likely if the patient-provider race was concordant . Although the majority of patients with ARI were cared for by physicians, APPs were seeing an increasing number of ARI patients.There were 64,081,892 ARI antibiotic prescriptions written, with a decrease from 10.9 (2010) to 9.7 million (2015) during the study interval  contains supplementary material, which is available to authorized users. Antibiotic resistance is a major public health concern, and its development is primarily due to the overuse, or inappropriate use, of antibiotics , 2. CompAnywhere between 20 and 50% of the antibiotic prescribing in the United States outpatient setting is thought to be unnecessary; however, stewardship programs are not as prevalent in the outpatient setting as they are in the inpatient setting –11. AcutThe prevalence of antibiotic prescriptions written by physician assistants and nurse practitioners have increased . AccordiThe Medical Expenditure Panel Survey (MEPS) took its current form in 1996 and is administered by the Agency for Healthcare Research and Quality (AHRQ) . MEPS paThe current study utilized full-year consolidated household component files which contain expenditure and utilization data for the calendar year from several rounds of data collection. The household component includes the prescribed medicines file, which contains both medicine names and National Drug Codes (NDC) for all prescriptions. Prescription information, including NDC, is verified with the patient’s pharmacy. The prescribed medicines file also includes conditions associated with the medication, the start date of the medication, total expenditure, and sources of payment. Conditions are defined by truncated International Classification of Disease, 9th Revision, Clinical Modification (ICD-9-CM) codes, the truncated codes protect the privacy of survey participants. Trained professional coders complete the MEPS coding and determine the appropriate diagnosis code based on verbatim text from the participant. One respondent provides information for an entire household, and the consolidated full-year dataset, associated files, documentation, and codebooks are available through the AHRQ website .We analyzed outpatient ARI antibiotic prescribing categorized by provider from 2010 to 2015. Conditions of interest were those considered viral ARIs and were identified by ICD-9-CM and Clinical Classification System (CCS) codes. When a CCS code included a bacterial diagnosis, the more specific ICD-9-CM code was used. Events for inclusion were those with diagnosis codes for acute nasopharyngitis, ARI, acute bronchitis and bronchiolitis, laryngitis and tracheitis, influenza, and viral pneumonia.Antibiotics of Concern list and included quinolones, macrolides (azithromycin and clarithromycin), amoxicillin/clavulanate, ketolides , cephalosporins (2nd and 3rd generations), and clindamycin [ARI events were examined for antibiotic use. Antibiotic classes were identified using the NDC directory and generic names. Broad-spectrum antibiotics were defined based on the National Committee for Quality Assurance’s (NCQA) ndamycin , 18–20. Variables of interest were determined through literature review. Demographic variables included age, gender, race, geographic location, and income. Income was categorized as above and below the median for the sample , and insurance coverage was categorized as private, public, or none. Race was described as a categorical variable including: White, Black, Asian, and Other. Geographic location was included as it can help bring to light regional variations in provider practice and its categorization is based on the US Census geographic regions: Northeast, South, Midwest, and West. Provider types were categorized as physician and APP as above. A race dyad was included to indicate whether the race of the provider was the same as the patient, with race determined based on respondents’ answers to survey questions. Number of comorbidities was included as a proxy of general health status. MEPS includes codes for fifteen distinct comorbidities . A further patient characteristic included was an SF-12 indicator, the SF-12 being a population health measure and a suitable measure of self-reported health status in epidemiological studies (SF-12 is a 12-question short form) –23.Antibiotic prescription prevalence was defined as the percentage of ARI events wherein an antibiotic was prescribed; the prevalence was further expressed as the percent of ARI events wherein a broad-spectrum antibiotic was prescribed. A sensitivity analysis was conducted by comparing prevalence of antibiotic prescribing for ARI among the overall group and a new group wherein comorbidities that were considered likely to warrant antibiotic use including mastoiditis, otitis media, soft tissue infection, urinary tract infection, chronic obstructive pulmonary disease, human immunodeficiency virus/acquired immunodeficiency syndrome, cystic fibrosis, and diabetes mellitus were removed. As the two analyses showed no meaningful difference, those with the aforementioned comorbidities were retained for all analyses. . Trend analyses year to year were performed utilizing a chi-square statistic. All statistical analyses were carried out utilizing the weighting as provided by AHRQ and using the survey procedures available in SAS version 9.4 .(Table ). Females were more frequently seen by a provider for a ARI than males, and therefore females were more frequently prescribed an antibiotic for ARI (62.42% vs. 37.58%). Those aged under 10 years accounted for the majority of visits (21.53%), and the percentage of ARI visits receiving an antibiotic were highest in the Southern region of the US (42.24%). As the number of comorbidities increased , the number of antibiotics prescribed for a ARI decreased. Likewise, a higher SF-12 which indicates the patient believes they are in good health, was associated with an increased percentage of antibiotic receipt . Physicians wrote 94.19% of antibiotic prescriptions for ARI visits compared to 5.81% written by advanced practice providers (nurse practitioners and physician assistants). Among whites seen for ARI, 48.9% saw a white provider whereas among Asian ARI visits, 39.7% saw an Asian provider and among blacks seen for ARI, 12.3% saw a black provider.During the study period 2010–2015) there were 461,647,174 visits to providers associated with a ARI diagnosis 0–2015 th(Table 2). The number of ARI visits increased during the study period from 70.6 million in 2010 to 82.4 million in 2015. Although a majority of ARI visits are managed by a physician that number is slowly decreasing as more ARI visits are managed by both nurse practitioners and physician assistants


Drosophila testis, dedifferentiated germline stem cells (GSCs) derived from spermatogonia increase during lifespan, leading to the model that dedifferentiation counteracts the decline of GSCs in aged males. To test this, we blocked dedifferentiation by mis-expressing the differentiation factor bag of marbles (bam) in spermatogonia while lineage-labeling these cells. Strikingly, blocking bam-lineage dedifferentiation under normal conditions in virgin males has no impact on the GSC pool. However, in mated males or challenging conditions, inhibiting bam-lineage dedifferentiation markedly reduces the number of GSCs and their ability to proliferate and differentiate. We find that bam-lineage derived GSCs have significantly higher proliferation rates than sibling GSCs in the same testis. We determined that Jun N-terminal kinase (JNK) activity is autonomously required for bam-lineage dedifferentiation. Overall, we show that dedifferentiation provides a mechanism to maintain the germline and ensure fertility under chronically stressful conditions.Exhaustion of stem cells is a hallmark of aging. In the  From the heart to the brain, our bodies are made of a collection of cells that are specialized to perform precise roles. Yet, certain organs host ‘stem cells’, which can become any kind of tissue. For example, the testicles of the fruit fly contain germline stem cells; when one of these cells divides, a daughter remains unspecialized, while the other specializes – or differentiates – to become sperm. Despite previous beliefs, a cell that is undergoing specialization can dedifferentiate to become a stem cell again.As the organism gets older, stem cells become ‘exhausted’: they divide less, and lose their ability to remain unspecialized. Scientists therefore proposed that dedifferentiation could be a way to replenish a dwindling pool of stem cells, and ward off the effects of age. However, this line of thought has not been tested in the laboratory.Here, Herrera and Bach tried to test this assumption by creating two populations of male fruit flies. One was genetically intact and the other was modified so that the cells that would become sperm cells could not dedifferentiate to become germline stem cells again. The insects were then raised in either a standard environment  or in stressful conditions (periods of starvation with or without mating).The experiments showed that dedifferentiation was important to maintain a robust germline stem cell pool, both in the short and long term. This, however, was only the case in the difficult environment; the ability to dedifferentiate made no difference in the easier living conditions. In addition, Herrera and Bach observed that, in the flies’ testicles, stem cells obtained through dedifferentiation divided much more often than the ‘original’ stem cells. Finally, further analyses highlighted a series of genes that are required for dedifferentiation.That stem cells coming from dedifferentiated cells divide at a higher rate could be relevant to scientists across various fields. For example, this knowledge may help those who study how tissues regenerate after injury, a process that involves dedifferentiation. It also may be used as a cautionary tale for researchers who work on induced pluripotent stem cells – which are created in the laboratory by dedifferentiating specialized cells. This may be especially important because these cells could one day be put in patients to treat diseases such as Parkinson’s or Alzheimer’s. A robust stem cell pool is critical to the maintenance of highly proliferative tissues during an organism’s lifetime. Adult tissue stem cells typically reside in niches, anatomically defined as microenvironments that support their ‘stemness’ and promote their proliferation, resulting in some daughter cells that retain stem cell characteristics and some that begin differentiation . DepletiDrosophila intestine, complete starvation induces the loss of all intestinal stem cells, and polyploid enterocyte cells undergo a reduction in ploidity  and transform into intestinal stem cells , differentiating spermatogonia revert to the stem cell state and become functional GSCs , which is necessary and sufficient for their differentiation  cause all GSCs to differentiate. However, upon silencing of these triggers, spermatogonia break apart, migrate back to the niche, outcompete the resident CySCs and become functional GSCs by transducing JAK/STAT signals and repressing bam expression  signaling is activated in germ cells during recovery from stress. By inhibiting JNK activity in bam-expressing spermatogonia, we demonstrate that this pathway is essential for dedifferentiation of these cells.Here, we have developed a methodology that enabled us to inhibit specifically dedifferentiation of bam-Gal4 line, expressed specifically in 4- and 8-cell spermatogonial cysts, to drive UAS-Flippase (Flp) expression, and this Flp in turn excises an FRT-stop-FRT from the ubiP63E-FRT-stop-FRT-GFP cassette  should prevent them from undergoing dedifferentiation . For reasons unknown to us but also observed by another group  . We note, however, that due to the fact that some of the bam-expressing cells might not undergo recombination (due to incomplete Flp efficiency), we cannot rule out the possibility that unlabeled cells may dedifferentiate and help to maintain the GSC pool under normal laboratory conditions. Additionally, prior work has shown that symmetric renewal, whereby a gonialblast swivels to gain direct access to the niche, occurs at low levels in testes from wild type males  . Furtherpe males . It is pbam > LacZ flies to 15 days of protein starvation, followed by a 9-day time course of refeeding  causes a 25% reduction in total GSC number . Furtherefeeding . We asseefeeding . In accoefeeding . We notebam > LacZ controls, we observed variable numbers of bam-lineage dedifferentiated GSCs per testis. Most testes contained both GFP-negative wild type and GFP-positive dedifferentiated GSCs at the niche, however, some testes did not contain any dedifferentiated GSCs and some testes contained all dedifferentiated GSCs. We hypothesized that testes with more dedifferentiated GSCs had a faster recovery of the GSC pool after starvation and refeeding. Comparing testes with at least one dedifferentiated GSC , in contrast to the bam > LacZ controls (bam > bam) retards the recovery of the GSC pool in a similar manner to control bam > LacZ testes devoid of bam-lineage dedifferentiation  females could force GSCs in the testis to cope with a higher demand for sperm production and thus accelerate the age-related reduction of the GSC pool. We note that while mating is a normal physiological event, it is stressful as mating significantly decreases both male and female lifespans . Howevered males . We nexted males . These dbam lineage. We aged bam > LacZ and bam > bam flies for 45 days in the constant presence of wild type ROregon females. The rate of dedifferentiation in control bam > LacZ testes significantly increased from 7.0% at 0 days to 30.8% at 45 days when males are housed with females  smaller in mated bam > bam flies  to establish clone induction frequency and at 15 dpci to monitor clone retention. As expected, clones were generated at equal frequency in both unmated and mated conditions -LacZ . Two puc-Gal4 lines with independent origins were used for these experiments , showing similar results.We were unable to detect real-time JNK activity reporters uc)-LacZ  or TRE-G had germline labeling . Of these 17 testes, most (n = 15) had at least 1 GSC labeled as well as various gonia; only two testes had gonia but no GSCs labeled. We speculate that high number of cases where an entire germline lineage is labeled results from the considerable time (>24 hr) required to activate puc, recombine the lineage-tracing cassette and induce GFP expression. Our observations are consistent with JNK-activated gonia breaking apart and liberating gonial cells to dedifferentiate into GSCs. However, we cannot rule out the possibility that during refeeding JNK signaling is directly activated in resident GSCs that have not dedifferentiated. In support of our model that spermatogonia fragment and revert to the GSC state after autonomous JNK activation, we observed one testis at 3 days of refeeding where a 4-cell gonia labeled with puc > GFP appeared to fragment into a 3-cell gonia and a GSC at the niche . Concomitantly, we lineage traced bam-Gal4 cells. If JNK activity is necessary for dedifferentiation, we predict that blocking its activity would reduce dedifferentiation upon refeeding. Indeed, after one cycle of starvation and refeeding, the increase in bam-lineage GSCs was no longer detectable in bam > puc or DNbam > bsk testes compared with bam > LacZ controls . Instead, we find that under normal but stressful conditions such as mating or under challenging conditions such as starvation and refeeding, dedifferentiation is important for both the quick recovery of the GSC pool in the short term and the preservation of the stem cell number in the long term. These results lead us to propose that bam-lineage dedifferentiation is akin to a regenerative response aimed to preserve the number of gonadal stem cells under adverse situations but is dispensable under optimal life conditions. This model is consistent with previous results demonstrating an increase in dedifferentiation after damage induced by irradiation , the biadiation . FurtherDrosophila whether it is to reconstruct an appendage or recover a pool of stem cells.Our results reveal a critical role of the JNK pathway in spermatogonial dedifferentiation. We have detected its activation during the refeeding phase after starvation and have proven its requirement in promoting this phenomenon . As mentbam-lineage-traced, dedifferentiated GSC in the testis during protein starvation  we failed to detect real time expression of bam-Gal4 in GSCs (bam > bam) blocked the increase in labeled GSCs in every scenario we tested; and (3) under challenging conditions, the GFP-positive GSCs were demonstratively more proliferative than their unlabeled siblings, a result that is inconsistent with spurious labeling.One caveat of our results is the possibility that spurious activity of  in GSCs ; (2) prebam-Gal4) is restricted to 4- and 8-cell spermatogonia and does not label gonialblasts  and 2-cell spermatogonia. To the best of our knowledge, there are no Gal4 lines specific to the lattermost cell types and at the same time excluded from GSCs. This limitation makes it impossible for us to track dedifferentiation of gonialblasts and 2-cell spermatogonia and likely causes undersampling of all dedifferentiation events. We note that examples of dedifferentiation from 2-cell gonia have been documented by live imaging  and trithorax-group (trx-g) genes, as has been shown in several regenerative contexts : ROregon-R (Ore), Ubi-p63E(FRT.STOP)Stinger, UAS-RedStinger, UAS-LacZ, K53RUAS-bsk, E69-Gal4, puc-IT.Gal4puc. Additionally, we used the following fly stocks: UAS-puc , mouse anti-FasIII , 1:50), mouse anti-α-Spectrin , mouse anti-γTubulin , rabbit anti-phospho-Histone3-Ser10 . 5‐ethynyl‐2′‐deoxyuridine  labeling was carried out as previously described .In all the experiments, flies were raised at 25°C. For standard aging conditions, virgin males were collected and kept isolated from females. Flies were kept in vials with food at a density of 20 males per vial (1-inch width) and transferred into fresh new vials every 2 days.Flies were maintained on standard fly food. For starvation (protein deprivation) periods, males were transferred into vials with 10% sucrose/1% agar, replaced by fresh ones every 2 days.Oregon-R) virgin females. Flies were transferred to new vials every 2 days, and the females were replaced by new young virgins every 2 weeks.For aging in the presence of females , a maximum of 20 males were placed in the same vial with 40 young (no older than 1 week) wild type  it is Vasa-positive (Vasa is expressed only in germline cells in the testis); (2) it is a single cell that is not part of a spermatogonial cyst; (3) it makes direct contact with the niche (FasIII-positive cells). In some experiments, the second criterion was evaluated by assessing the presence of dot fusomes when stained with α-Spectrin, as this is a hallmark of GSCs.In the course of our experiments, we realized that distinct genotypes have a different number of GSCs at 0 days (see bam-lineage-positive) GSCs divided by the total number of GSCs in each individual testis and (2) each testis in a particular genotype was averaged.The percentage of dedifferentiation is calculated as (1) the proportion of GFP-positive .Thank you for submitting your article "The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife. However, there are several issues that need to be addressed prior to acceptance. Since reviewers' individual comments are not conflicting and in agreement, we decided to provide full review comments as is such that the authors can see the full spectrum of the basis of our discussion.As is provided below in reviewers' individual comments, all felt that this study provides significant insights into biological significance of dedifferentiation, warranting publication in The outline of essential revisions we ask is the following:1) There are strong concluding statements throughout the manuscript that are not necessarily backed up by the experimental evidence. all of dedifferentiated cells, because 2-cell SGs do not express bam-gal4. However, 2-cell SGs are considered to dedifferentiate more than 4- and 8-cell SGs due to their proximity to the niche. Thus, one would have to assume that there are many more dedifferentiated GSCs that are negative for lineage-marker than those positive for lineage-marker. Throughout the Results section, the authors do not sufficiently acknowledge this (highly likely) possibility, and draw strong conclusions, essentially assuming that all of linage-negative cells have never undergone dedifferentiation. This includes their conclusions of 1) centrosome misorientation does not correlate with dedifferentiation. 2) dedifferentiated GSCs proliferate faster. These overstatements/ over-interpretations have to be corrected.- There are several concerns regarding Bam-gal4-mediated lineage tracing of dedifferentiated GSCs. Although Bam-gal4-mediated lineage tracing indeed marks dedifferentiation, it does not mark 2) Dedifferentiation and re-differentiation are likely a dynamic process, and simple lineage tracing may not be sufficient to draw some conclusions described in this manuscript. Especially, the reviewer felt that it is not yet convincing to rely on lineage tracing (and counting clone number) to measure the activity of GSCs. It is equally conceivable that dedifferentiation causes fragmentation of the cysts, creating multiple 'clones' by fragmentation . The authors would need additional evidence to make a point that dedifferentiated GSCs are more proliferative – ideally live imaging, or additional cell cycle markers such as mitotic index. If such experiments cannot be conducted to obtain conclusive results, the authors should not make such a statement.puc>GFP lineage tracing, the authors should present a time course to show whether labeled cells always originate from 4-cell and 8-cell cysts, as opposed to GSCs, GBs, and 2-cell cysts.3) In regards to the Reviewer #1:Drosophila testis. They used an elegant genetic technique to block dedifferentiation  and found that dedifferentiation is required to maintain the GSC pool under 'challenging' conditions such as protein starvation. They further show that JNK pathway regulates dedifferentiation. The experiments are conducted to a high standard, supporting an exciting conclusion. I have several comments to be addressed prior to publication .In this manuscript, Herrera and Bach reports an exciting study that examines the biological significance of dedifferentiation in bam-gal4 mediated experiments can only manipulate 4-cell spermatogonia and later, excluding the contribution of dedifferentiation from gonialblasts and 2-cell spermatogonia from testing (and those earlier SGs are considered to be more prone to dedifferentiation). Therefore, I suggest the authors to be cautious in stating 'dedifferentiation is not required under unchallenged conditions'. Their results demonstrate that the dedifferentiation from 4/8 SGs do not contribute to GSC maintenance during aging under 'normal' conditions. This clarification does not decrease the impact of this work, but I think it is important to mention throughout the text.- bam-gal4 lineage tracing) and we speculated in this paper that this may reflect dedifferentiation from GB or 2 cell SGs . I think it is important to acknowledge the likely contribution of GB and 2-SG in dedifferentiation .- Subsection “Dedifferentiation accelerates the recovery of the GSC pool after starvation”, last paragraph: the authors state that both native and dedifferentiated GSCs have equally high frequency of centrosome misorientation. Again this comes back to the point of 'GSCs dedifferentiated from gonialblast or 2-cell SGs'. Indeed, our prior work  observed that centrosome misorientation increase during aging even in 'unmarked' GSCs (not marked by bam>bam flies decrease GSC pool). This is exciting . I wonder if constantly supplying virgin females might further reveal the importance of dedifferentiation. They add virgin females in 'challenging' conditions , but 'encountering virgin females periodically' should not be considered a challenge , and examining the impact of such a condition might reveal 'physiological role' of dedifferentiation.- In Figure 3 they show that presence of female impact dedifferentiation, and reveals the need of dedifferentiation .- Discussion starts by stating 'dedifferentiation plays no role under standard aging conditions', which is an overstatement in my opinion because their experimental scheme cannot test the contribution of dedifferentiation from GB and 2 cell SG. They only discuss this possibility in the middle of Discussion, whereas they have never discussed this important possibility until this point. The authors should acknowledge this much earlier and fully integrate this possibility in the interpretation of their results starting in the Results section.-Figure 4 title: typo, “comprised” should be compromised.Reviewer #2:Drosophila testes during chronically stressful conditions. Using a clever technique for genetically manipulating partially differentiated germ cells, the authors find that dedifferentiation of germ cells that have reached the bam(+) stage are important for replenishing the GSC pool following chronic starvation or continuous mating. They also show that dedifferentiated GSCs have increased rates of proliferation relative to GSCs that have not been derived from a bam(+) germ cell, and that JNK signaling is upregulated during chronic stress and required for efficient dedifferentiation. In general, I think their observations are novel and will be of broad interest. I also think that the data provide strong support for the central claims of the paper.This paper describes an investigation into the role of dedifferentiation in maintaining the GSC pool in Reviewer #3:Drosophila testis. The authors use a FLP-out lineage tracing system and over-express bam to block normal differentiation. They find dedifferentiation contributes little to the GSC pool in well-fed virgin males. By contrast, dedifferentiation does impact GSC numbers under "challenging" conditions, including mating and starvation. The authors provide evidence that GSCs arising from dedifferentiated cells are more active than their wild-type GSC siblings and that the JNK pathway is necessary for germ cell dedifferentiation in specific contexts.In this manuscript, Herrera and Bach examine how the dedifferentiation of developing germ cells helps to maintain the germline stem cell pool in the bam-Gal4 lineage in maintaining the GSC pool during aging under normal laboratory conditions." I think the authors should be much more explicit in the description of the conditions they are testing in both cases. They should clearly state that this observation is made in testes from unmated/virgin males, since I would consider mating a "normal" activity of male Drosophila. In regards to this experiment, the authors should also investigate or cite the relevant literature that describes how GSCs behave in unmated males. Are they as proliferative as those from mated males? Are they lost at the same rate ? If GSCs in unmated males are simply less active and are maintained for longer periods of time, the observed result is exactly what one would expect. Although the authors do mention the possibility in their discussion, making this statement in the Abstract ignores the possibility that two and four cell cysts (pre-bam expressing cells) dedifferentiate in unmated males. Examining centrosome orientation (which they use in later experiments) and live cell imaging could help clarify whether no dedifferentiation is occurring in these samples.The authors make several strong conclusions which are not necessarily supported by the data. In the Abstract, the authors write "Strikingly, blocking dedifferentiation under normal laboratory conditions has no impact on the GSC pool." Again, in the Results they state "Although unexpected and contrary to our predictions, these results preclude an important role for dedifferentiation of the bam-GAL4 marked GSC "fully recovered" the pool of GSCs in 3 days, as opposed to those that have 0 dedifferentiated GSCs, which recovered the GSC pool in 7 days. This is a correlation, and the authors should characterize this phenotype in greater depth before making any firm conclusions. For example, the rate and levels of dedifferentiation may be exactly the same in both samples, but different testes may exhibit differences in cell death or GSC loss, in addition to any number of other variables, such as GSC proliferation and symmetric renewal. Again, live cell imaging may provide a more complete picture of how these cells are behaving in different circumstances.The authors draw the conclusion that dedifferentiation accelerates the recovery of the GSC pool after starvation. This conclusion is based on the observation that testes which have at least 1 The authors observe that dedifferentiated GSCs and their wild-type siblings display the same level of centrosome mis-orientation. This appears to be directly at odds with Cheng et al. Do the authors make this observation in all cases of dedifferentiation, or only in those testes from males subjected to starvation and refeeding? Further discussion of differences between this study and previous work is warranted.The authors observe that dedifferentiated GSCs are more proliferative than their wild-type siblings based on the numbers of GFP positive cells within the testes. However, one cannot be certain that a particular labeled cell/cyst originated from a dedifferentiated GSC as opposed to a cyst that is in the process of breaking down. Dedifferentiation and redifferentiation may be a constant dynamic process. Do the authors have evidence that dedifferentiating cysts have to take on GSC identity before producing differentiating progeny? Again, the observation that dedifferentiated GSCs proliferate at a higher rate than wild-type cells contradicts previous studies. I would like to see live cell imaging that shows higher rates of cell division in the dedifferentiated GSCs and/or additional cell cycle/mitotic markers.puc>GFP lineage tracing, the authors should present a time course to show whether labeled cells always originate from 4-cell and 8-cell cysts, as opposed to GSCs, GBs, and 2-cell cysts?In regards to the JNK triggers dedifferentiation during chronic stress to maintain the germline stem cell pool in the Drosophila testis" for further consideration at eLife. Your revised article has been favorably evaluated by Marianne Bronner (Senior Editor), a Reviewing Editor, and two reviewers.Thank you for resubmitting your work entitled "The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The reviewers appreciate that the authors addressed most of concerns raised during the first round of the review. Reviewer #3 raised a few points that other reviewers agreed to be important points. These points can be simply addressed by textual changes, and we would like the authors to edit the text to reflect on those points. After that, the reviewing editor should be able to editorially accept the manuscript.Reviewer #2:The authors have addressed my concerns and I think the paper is now ready for publication.Reviewer #3:The authors have addressed many of my previous concerns. I have just a couple of points that need further clarification.bam mis-expression by bam-Gal4.". They then present data that shows GSC numbers do not drop further when dedifferentiation is blocked, opposite from their expectations.The authors state: "Prior work has shown that the number of GSCs decreases slightly during aging under normal laboratory conditions, and this has led to the model that dedifferentiation provides a means to offset normal GSC loss during lifespan . If this hypothesis is correct, we would expect a further reduction in GSC number after Previous results from the Matunis lab have shown that GSCs can undergo symmetric renewal. Perhaps blocking dedifferentiation forces the testis to use this alternative mechanism for maintaining GSC numbers. This does not necessarily mean that dedifferentiation does not play a role in maintaining GSC numbers in older flies. By blocking dedifferentiation, one may be shifting the equilibrium towards this and other potential mechanisms to maintain GSC numbers.bam-Gal4 lineage in maintaining the GSC pool during aging under normal laboratory conditions." This conclusion seems at odds with the data that precedes it. Indeed, in the abstract the authors state "blocking bam-lineage dedifferentiation under normal conditions in virgin males has no impact on the GSC pool." Perhaps the authors could rephrase their concluding sentence in the Results section and mention the aforementioned alternative mechanisms.The authors go on to write "Although unexpected and contrary to our predictions, these results strongly suggest an important role for dedifferentiation of the JNK triggers dedifferentiation during chronic stress to maintain the germline stem cell pool in the Drosophila testis" The authors state in the final paragraph of the Results section- "To functionally test the relevance of JNK activity in spermatogonial dedifferentiation, we blocked its activity in the bam-Gal4 lineage by mis-expressing the JNK inhibitor puc or a dominant negative form of the JNK basket (bsk). Concomitantly, we lineage traced bam-Gal4 cells. If JNK activity is necessary for dedifferentiation, we predict that blocking its activity would reduce dedifferentiation upon refeeding. Indeed, after one cycle of starvation and refeeding, the increase in bam-lineage GSCs was no longer detectable in bam>puc or DNbam>bsk testes compared with bam>LacZ controls . Taken together, these results indicate that JNK pathway activity is autonomously necessary for germline cells to undergo dedifferentiation."The paper is entitled "puc or basketDN survive and continue to differentiate? I do see bam-lineage positive cyst in Figure 6J, which partially speaks to this point. The worry is that over-expression of puc or DNbsk reduces germ cell viability, which could interfere with the interpretation of the data.Can the authors clarify whether cells expressing  […] The outline of essential revisions we ask is the following:1) There are strong concluding statements throughout the manuscript that are not necessarily backed up by the experimental evidence. - There are several concerns regarding Bam-gal4-mediated lineage tracing of dedifferentiated GSCs. Although Bam-gal4-mediated lineage tracing indeed marks dedifferentiation, it does not mark all of dedifferentiated cells, because 2-cell SGs do not express bam-gal4. However, 2-cell SGs are considered to dedifferentiate more than 4- and 8-cell SGs due to their proximity to the niche. Thus, one would have to assume that there are many more dedifferentiated GSCs that are negative for lineage-marker than those positive for lineage-marker. Throughout the Results section, the authors do not sufficiently acknowledge this (highly likely) possibility, and draw strong conclusions, essentially assuming that all of linage-negative cells have never undergone dedifferentiation. This includes their conclusions of 1) centrosome misorientation does not correlate with dedifferentiation. 2) dedifferentiated GSCs proliferate faster. These overstatements/ over-interpretations have to be corrected.bam-Gal4. We do not know the degree to which these events occur, but we now acknowledge that we are not monitoring all dedifferentiated cells. Furthermore, from Results onwards and in figures and legends, we specifically mention that we will refer to dedifferentiated cells as bam-lineage derived GSCs and the unlabeled GSCs as bam-lineage negative cells. We now discuss all conclusions in the context of bam-lineage cells.We thank the reviewers for pointing this out. In the revised manuscript, we added several sentences/paragraphs to the Results and the Discussion that we are underlabeling dedifferentiated cells because it is believed that gonialblasts and 2-cell spermatogonia dedifferentiate but they are not labeled by bam-lineage GSCs have significantly more mis-oriented centrosomes than lineage-negative GSCs . This is in accordance with Cheng et al., 2008 . At 45 days in fed, unmated, normal laboratory conditions, we find similar rates of mis-oriented centrosomes (~40-45%) in both bam-lineage positive and lineage-negative GSCs. This is quite similar to the 50 day time point reported by Cheng et al.  where there was no significant difference between LacZ -positive and LacZ-negative GSCs. Additionally, we monitored centrosome mis-orientation after 4 cycles of chronic challenging conditions and found no significant difference in rates between bam-lineage positive and negative GSCs . These new data have been added to the revised manuscript  and to the sixth paragraph of the Discussion.  We have made two points regarding these results in the revised manuscript. First, we acknowledge that the lineage-negative GSCs likely include cells derived from gonialblasts and 2-cell gonia that are not label by bam-Gal4. We write: “It is possible that the lineage-negative GSCs are in fact dedifferentiated from gonialblasts and 2-cell gonia. While we cannot experimentally test this hypothesis, if both types of GSCs were in fact derived from dedifferentiated gonia, they would account the roughly equal rates of mis-oriented centrosomes in both pools.” Second, as there is no difference in centrosome mis-orientation between bam-lineage positive and negative GSCs in challenging conditions, we conclude that there is likely another explanation for the increased rate of proliferation of lineage-positive GSCs after 4 cycles. We write: “These data suggest that in some challenging conditions, factors independent of centrosome orientation may regulate GSC proliferation rates”.We have also performed additional experiments to address the proportion of mis-oriented centrosomes in various conditions. At day 0, we find that bam-lineage positive GSCs compared to lineage-negative ones. Therefore, even with the considerations described in the previous paragraph, lineage-positive GSCs do have a higher proliferation rate. This suggests that factors in addition to centrosome orientation regulate GSC proliferation. In the revised manuscript we speculate that lineage-positive GSCs have experienced autonomous JNK activity and this may “epigenetically reprogram” these cells, which may impact proliferation rates. In the revised manuscript, we carefully and precisely frame our results in the context of lineage (bam vs not bam) and still observe a significant increase in the rate of proliferation of lineage-positive GSCs.As addressed in the next point, we found a higher rate of S- and M-phase in 2) Dedifferentiation and re-differentiation are likely a dynamic process, and simple lineage tracing may not be sufficient to draw some conclusions described in this manuscript. Especially, the reviewer felt that it is not yet convincing to rely on lineage tracing (and counting clone number) to measure the activity of GSCs. It is equally conceivable that dedifferentiation causes fragmentation of the cysts, creating multiple 'clones' by fragmentation . The authors would need additional evidence to make a point that dedifferentiated GSCs are more proliferative – ideally live imaging, or additional cell cycle markers such as mitotic index. If such experiments cannot be conducted to obtain conclusive results, the authors should not make such a statement.bam-lineage GSCs have a higher proliferation rate. To address this point, we subjected males to 4 cycles of challenging conditions and stained testes with anti-phospho Histone H3-Ser10 (pH3) (Millipore) that was recommended to us by experts in the field . Using this antibody, we found that there were significantly more pH3-positive bam-lineage GSCs compared to lineage-negative GSCs . These data have been added to the revised manuscript in Figure 5C and are discussed in the Results subsection “bam-lineage dedifferentiated GSCs are more proliferative than their siblings”. Taken together with the significant increase in S-phase in bam-lineage positive GSCs as well as the number of offspring , we conclude that bam-lineage GSCs have a higher proliferation rate than lineage-negative GSCs. Given the ~2 month time frame for submitting a revision and the fact that we have not optimized technically-challenging, live imaging methodologies in our lab, we have not been able to perform live imaging experiments for this study.We agree with the reviewers’ concern about the need for additional evidence to support our conclusion that 3) In regards to the puc>GFP lineage tracing, the authors should present a time course to show whether labeled cells always originate from 4-cell and 8-cell cysts, as opposed to GSCs, GBs, and 2-cell cysts.puc>GFP lineage labeling at two additional time points during refeeding (at 2 and 3 days). Based on prior work from the Matunis and Yamashita labs, we predicted that spermatogonia during the refeeding would experience transient JNK activity, fragment and migrate back to the niche. If this were the case, then we would expect to see only spermatogonia but not GSCs labeled at earliest time point. However, of the 97 testes examined, 17 (17.5%) had any germline labeling. Of these 17 testes, most (n=15) had at least 1 GSC labeled as well as various gonia. In fact, only 2 testes had gonia but no GSCs labeled. We speculate that the high number of cases where an entire germline lineage is labeled results from the considerable time (>24 hours) needed to induce puc, recombine the lineage-tracing cassette and then induce GFP. Nevertheless, our observations are consistent with JNK-activated gonia fragmenting and some of these gonial cells returning to the niche to revert to GSCs. We acknowledge that we cannot exclude the possibility that JNK signaling is directly activated in resident GSCs that have not dedifferentiated during the refeeding period. However, in support of our model that spermatogonia fragment and revert to the GSC state after autonomous JNK activation, we observed one testis at 3 days of refeeding where a 4-cell gonia labeled with puc>GFP appeared to fragment into a 3-cell gonia and a GSC at the niche . Within the 3-cell gonial cyst, each germ cell had a dot spectrosome, characteristic of fragmenting germ cells during dedifferentiation and very early germ cells in wild type testes. These data are discussed in the fourth paragraph of the subsection “The JNK pathway is necessary for bam-lineage spermatogonial dedifferentiation”.We thank the reviewers for pointing this out. To address this concern, we surveyed Reviewer #1:[…] I have several comments to be addressed prior to publication .- bam-gal4 mediated experiments can only manipulate 4-cell spermatogonia and later, excluding the contribution of dedifferentiation from gonialblasts and 2-cell spermatogonia from testing (and those earlier SGs are considered to be more prone to dedifferentiation). Therefore, I suggest the authors to be cautious in stating 'dedifferentiation is not required under unchallenged conditions'. Their results demonstrate that the dedifferentiation from 4/8 SGs do not contribute to GSC maintenance during aging under 'normal' conditions. This clarification does not decrease the impact of this work, but I think it is important to mention throughout the text.bam-lineage derived and the other GSCs  as lineage-negative.We thank the reviewer for pointing this out. We acknowledge this and as discussed above in the response to reviewers, in the revised article we refer to dedifferentiated cells as - Subsection “Dedifferentiation accelerates the recovery of the GSC pool after starvation”, last paragraph: the authors state that both native and dedifferentiated GSCs have equally high frequency of centrosome misorientation. Again this comes back to the point of 'GSCs dedifferentiated from gonialblast or 2-cell SGs'. Indeed, our prior work  observed that centrosome misorientation increase during aging even in 'unmarked' GSCs  and we speculated in this paper that this may reflect dedifferentiation from GB or 2 cell SGs . I think it is important to acknowledge the likely contribution of GB and 2-SG in dedifferentiation .We agree that dedifferentiation of gonialblasts and 2-cell spermatogonia likely contribute to the pool of GSCs in testes from aged and challenged males. This is now acknowledged in numerous places in the text (see above).- In Figure 3 they show that presence of female impact dedifferentiation, and reveals the need of dedifferentiation (bam>bam flies decrease GSC pool). This is exciting . I wonder if constantly supplying virgin females might further reveal the importance of dedifferentiation. They add virgin females in 'challenging' conditions , but 'encountering virgin females periodically' should not be considered a challenge , and examining the impact of such a condition might reveal 'physiological role' of dedifferentiation.We agreed with this reviewer and with reviewer #3 that mating is a normal physiological response of males to the presence of females. However, mating is also an energy-intensive activity that can negatively impact males. For example, mated males have significantly shorter lifespans than unmated males . Hence, we conclude that mating, while normal, is stressful to males. In the text of the revised manuscript, we acknowledge both of these facts. Additionally, in response to reviewer #3 (see below), we have found that mating provokes an overall increase in GSC proliferation compared to aged-matched, unmated males . This suggests that mating induces a systemic response that increases GSC division rates. Furthermore, again as described below, we find that neutral GSC clones are lost significantly from the testis niche upon mating, presumably due to increased proliferation rates of GSCs as well as outcompetition of resident GSCs by dedifferentiating germ cells.- Figure 5: EdU incorporation cannot be used as a measure of cell cycle on its own, unless combined with mitotic index, because high EdU can simply mean that many cells are spending more time in S phase (instead of cycling faster).bam>LacZ males after 4 cycles of challenging conditions with the anti-pH3 described above. We found significantly more bam-lineage positive GSCs in M-phase compared to lineage-negative GSCs (p<0.053). These raw data are in the new Supplementary file 2 and also presented in Figure 5C in the revised manuscript. Taken together with our prior EdU incorporation results (which was significantly higher in bam-lineage GSCs), we conclude that bam-lineage GSCs cycle faster than lineage-negative GSCs.We agree with the reviewer. To address this point, we stained testes from control - Discussion starts by stating 'dedifferentiation plays no role under standard aging conditions', which is an overstatement in my opinion because their experimental scheme cannot test the contribution of dedifferentiation from GB and 2 cell SG. They only discuss this possibility in the middle of Discussion, whereas they have never discussed this important possibility until this point. The authors should acknowledge this much earlier and fully integrate this possibility in the interpretation of their results starting in the Results section.We thank the reviewer for point this out. As discussed in detail above, we have now incorporated this possibility into the first paragraph of the Results where we describe the lineage tracing. We now treat in greater detail this possibility in the Discussion.-Figure 4 title: typo, “comprised” should be compromised.Thank you for pointing out this typo. We have fixed this in the revised manuscript.Reviewer #3:[…] The authors make several strong conclusions which are not necessarily supported by the data. In the Abstract, the authors write "Strikingly, blocking dedifferentiation under normal laboratory conditions has no impact on the GSC pool." Again, in the Results they state "Although unexpected and contrary to our predictions, these results preclude an important role for dedifferentiation of the bam-Gal4 lineage in maintaining the GSC pool during aging under normal laboratory conditions." I think the authors should be much more explicit in the description of the conditions they are testing in both cases. They should clearly state that this observation is made in testes from unmated/virgin males, since I would consider mating a "normal" activity of male Drosophila.bam-lineage dedifferentiation does not impact the GSC pool in unmated males but does play an important role in maintaining this pool of stem cells in mated males and/or stressful conditions (like starvation).We thank the reviewer for pointing this out. As described above, we now clearly indicate that these initial experiments were performed on unmated males. We have also updated the Abstract to reflect the fact that In regards to this experiment, the authors should also investigate or cite the relevant literature that describes how GSCs behave in unmated males. Are they as proliferative as those from mated males? Are they lost at the same rate ? If GSCs in unmated males are simply less active and are maintained for longer periods of time, the observed result is exactly what one would expect. Although the authors do mention the possibility in their discussion, making this statement in the Abstract ignores the possibility that two and four cell cysts (pre-bam expressing cells) dedifferentiate in unmated males. Examining centrosome orientation (which they use in later experiments) and live cell imaging could help clarify whether no dedifferentiation is occurring in these samples.bam-lineage-positive GSCs vs. lineage-negative GSCs in both conditions. We found that both types of GSCs have significantly higher mitotic rates compared to those in unmated controls (6.44% vs. 9.56% in lineage-negative cells (p<0.068) and 6.15% vs 13.61% in lineage-positive cells (p<0.034)). These data suggest that there is a systemic response to mating that increases proliferation of all GSCs.The reviewer has raised some important concerns. First, we were unable to find any literature on the effects of mating on GSC proliferation. As such, we performed experiments to directly address this. We show in Figure 3—figure supplement 1 of the revised manuscript that the rate of mitosis (pH3-positive GSCs) is significantly higher in testes from mated males compared to age-matched, unmated males. Moreover, we analyzed the mitotic rate of 40AFRT MARCM clones with which we have considerable experience  in unmated vs. mated males at 2 and 15 days post clone induction (dpci). Both types of males had similar clone induction at 2 dpci, as expected. However, there were significantly fewer GSC clones at 15 dpci in the testes of mated males compared to unmated males. The GSC clone loss in mated flies is compatible with the increased rate of GSC proliferation discussed in the previous paragraph. Furthermore, it is also consistent with the presumed competition of dedifferentiating germ cells with resident GSCs for niche occupancy. These data are included in new Figure 3—figure supplement 1 and are discussed in the Results subsection "bam-lineage dedifferentiation preserves both the GSC pool and spermatogenesis during chronic challenging conditions”.We also performed the clonal analysis suggested by the reviewer. We made neutral The authors draw the conclusion that dedifferentiation accelerates the recovery of the GSC pool after starvation. This conclusion is based on the observation that testes which have at least 1 bam-GAL4 marked GSC "fully recovered" the pool of GSCs in 3 days, as opposed to those that have 0 dedifferentiated GSCs, which recovered the GSC pool in 7 days. This is a correlation, and the authors should characterize this phenotype in greater depth before making any firm conclusions. For example, the rate and levels of dedifferentiation may be exactly the same in both samples, but different testes may exhibit differences in cell death or GSC loss, in addition to any number of other variables, such as GSC proliferation and symmetric renewal. Again, live cell imaging may provide a more complete picture of how these cells are behaving in different circumstances.bam-lineage-positive GSCs and the recovery of the GSC pool. We now write: “Although we cannot exclude other variables such as germ cell death, GSC loss, and GSC gain through symmetric renewal, this correlation strongly suggests that bam-lineage dedifferentiation accelerates the recovery of the GSC pool under challenging conditions.” As noted above, given the time frame for revision and not having optimized live imaging methodology in our lab, which is technically challenging, we have not performed live imaging experiments for this study.We concur with the reviewer and have now moderated our conclusions about the correlation between the number of The authors observe that dedifferentiated GSCs and their wild-type siblings display the same level of centrosome mis-orientation. This appears to be directly at odds with Cheng et al. Do the authors make this observation in all cases of dedifferentiation, or only in those testes from males subjected to starvation and refeeding? Further discussion of differences between this study and previous work is warranted.In the original submission, we had examined centrosome orientation in GSCs after 15 days of starvation and 5 days of refeeding. Based on this reviewer’s concern, we examined centrosome orientation in several other conditions, specifically, 0 days, after 45 days in fed but unmated males, and after 4 cycles of starvation, refeeding and mating. We already provided a detailed response to this concern above in our answer to the first general point (see above).The authors observe that dedifferentiated GSCs are more proliferative than their wild-type siblings based on the numbers of GFP positive cells within the testes. However, one cannot be certain that a particular labeled cell/cyst originated from a dedifferentiated GSC as opposed to a cyst that is in the process of breaking down. Dedifferentiation and redifferentiation may be a constant dynamic process. Do the authors have evidence that dedifferentiating cysts have to take on GSC identity before producing differentiating progeny? Again, the observation that dedifferentiated GSCs proliferate at a higher rate than wild-type cells contradicts previous studies. I would like to see live cell imaging that shows higher rates of cell division in the dedifferentiated GSCs and/or additional cell cycle/mitotic markers.bam-lineage-positive GSCs labeled with pH3 compared to lineage-negative GSCs (see above). Regarding dedifferentiation and redifferentiation, we have not been able to perform live imaging studies that may reveal such characteristics. However, in response to reviewer 3’s next comment , we have now analyzed a time course of puc-lineage activation during the refeeding period. Please see above for a detailed response, but suffice it to say here, we find some evidence for fragmentation of 4-cell gonia and migration of one or more germ cells from this cyst back to the niche to re-acquire GSC status.We agree with the reviewer and as described above we measured mitotic rate with pH3. This experiment revealed that there were significantly more In regards to the puc>GFP lineage tracing, the authors should present a time course to show whether labeled cells always originate from 4-cell and 8-cell cysts, as opposed to GSCs, GBs, and 2-cell cysts?This is an excellent suggestion. Please see above answer to general question 3.Literature cited:Drosophila Testis. PLoS Genet. 2016;12(1):e1005815. 10.1371/journal.pgen.10058151) Amoyel M, Anderson J, Suisse A, Glasner J, Bach EA. Socs36E Controls Niche Competition by Repressing MAPK Signaling in the 2) Amoyel M, Simons BD, Bach EA. Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo. EMBO J. 2014;33(20):2295-313. 10.15252/embj.201387500The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The reviewers appreciate that the authors addressed most of concerns raised during the first round of the review. Reviewer #3 raised a few points that other reviewers agreed to be important points. These points can be simply addressed by textual changes, and we would like the authors to edit the text to reflect on those points. After that, the reviewing editor should be able to editorially accept the manuscript.Reviewer #3:The authors have addressed many of my previous concerns. I have just a couple of points that need further clarification.The authors state: "Prior work has shown that the number of GSCs decreases slightly during aging under normal laboratory conditions, and this has led to the model that dedifferentiation provides a means to offset normal GSC loss during lifespan . If this hypothesis is correct, we would expect a further reduction in GSC number after bam mis-expression by bam-Gal4.". They then present data that shows GSC numbers do not drop further when dedifferentiation is blocked, opposite from their expectations.Previous results from the Matunis lab have shown that GSCs can undergo symmetric renewal. Perhaps blocking dedifferentiation forces the testis to use this alternative mechanism for maintaining GSC numbers. This does not necessarily mean that dedifferentiation does not play a role in maintaining GSC numbers in older flies. By blocking dedifferentiation, one may be shifting the equilibrium towards this and other potential mechanisms to maintain GSC numbers.bam-lineage dedifferentiation is inhibited. We write “Additionally, prior work has shown that symmetric renewal, whereby a gonialblast swivels to gain direct access to the niche, occurs at low levels in testes from wild type males . It is possible that by blocking bam-lineage dedifferentiation, we are shifting the equilibrium towards symmetric renewal, but live imaging will be needed to test this hypothesis.”We thank reviewer 3 for pointing this out. In this second revision we now directly acknowledge this possible shift in equilibrium towards increased symmetric renewal when The authors go on to write "Although unexpected and contrary to our predictions, these results strongly suggest an important role for dedifferentiation of the bam-Gal4 lineage in maintaining the GSC pool during aging under normal laboratory conditions." This conclusion seems at odds with the data that precedes it. Indeed, in the abstract the authors state "blocking bam-lineage dedifferentiation under normal conditions in virgin males has no impact on the GSC pool." Perhaps the authors could rephrase their concluding sentence in the Results section and mention the aforementioned alternative mechanisms.bam-lineage dedifferentiation does not play a critical role in maintaining the GSC pool under normal laboratory conditions. However, we forgot the negative. In the revised manuscript we now clearly state our original intention. “Nevertheless, although unexpected and contrary to our predictions, our results strongly suggest that dedifferentiation of the bam-lineage does not play an important role in maintaining the GSC pool during aging under normal laboratory conditions.”We are extremely grateful to reviewer 3 for pointing this out. In fact, we made a terrible omission in this sentence in the original revision. We had intended to state that DN testes compared with bam>LacZ controls . Taken together, these results indicate that JNK pathway activity is autonomously necessary for germline cells to undergo dedifferentiation."The paper is entitled "JNK triggers dedifferentiation during chronic stress to maintain the germline stem cell pool in the Drosophila testis" The authors state in the final paragraph of the Results section- "To functionally test the relevance of JNK activity in spermatogonial dedifferentiation, we blocked its activity in the bam-Gal4 lineage by mis-expressing the JNK inhibitor puc or a dominant negative form of the JNK basket (bsk). Concomitantly, we lineage traced bam-Gal4 cells. If JNK activity is necessary for dedifferentiation, we predict that blocking its activity would reduce dedifferentiation upon refeeding. Indeed, after one cycle of starvation and refeeding, the increase in bam-lineage GSCs was no longer detectable in bam>puc or bam>bskDN reduces germ cell viability, which could interfere with the interpretation of the data.Can the authors clarify whether cells expressing puc or basketDN survive and continue to differentiate? I do see bam-lineage positive cyst in Figure 6J, which partially speaks to this point. The worry is that over-expression of puc or bskJNK may be negatively impacting the health of germ cells. To address this concern, we have examined at least 15 testes of the bam>puc or DNbam>bsk genotypes. In all cases, we found evidence for robust germ cell differentiation into pre-meiotic stages. Representative examples are included in a new supplementary figure . Additionally, we write “We note that neither mis-expression of puc nor DNbsk appears to adversely affect germ cell differentiation as robust spermatogonia and spermatocytes expressing puc or DNbsk survive and continue to progress towards meiotic stages .” We have also provided a legend for the new figure supplement.Reviewer 3 has raised an important point, that inhibition of