The Home Medication Experience Questionnaire (HOME-Qv2) was developed to identify patient medication experience issues for pharmacist intervention. The study objectives were to (1) evaluate patient responses to the HOME-Qv2 medication experience questionnaire and (2) describe recommendations made by pharmacists in response to the identified medication experience issues and adoption of recommendations. The study sample was comprised of older adults, 55 years and above, who have one or more chronic illnesses for which they routinely take 4+ prescription medications. The HOME-Qv2 was administered to patients and a pharmacist made recommendations based on the responses. At 3 months, the research team followed up with participants via telephone, during which the HOME-Qv2 was again administered and participants shared their adoption of recommendations. Twenty-four patients completed the questionnaire, and twenty-one were available for follow-up. At 3 months, there was a significant decrease in patient self-reported HOME-Qv2 medication experience issues. There were 31 interventions/recommendations provided by the pharmacists, and 64.5% reported adopted. The HOME-Qv2 appears to facilitate patient disclosure of medication experience issues and informed targeted pharmacist recommendations. Medications, including over the counter (OTC) , dietaryHistorically, the focus of pharmacists has been on identifying and resolving medication-related problems by using their knowledge of pharmacotherapy . This apShoemaker et al. defined patient medication experience as “… an individual’s subjective experience of taking a medication in [their] daily life. It begins as an encounter with a chronic medication. It is an encounter that is given meaning before it happens and is often a reaction to the symbol that medication holds. The experience may include positive or negative bodily effects. The unremitting nature of a chronic medication often causes an individual to question the need for the medication. Subsequently, the individual may exert control by altering the way he takes the medication and often in part because of the gained expertise with the medication in his own body .” These Information about the patient’s medication experience cannot be revealed through either a pharmacotherapy-focused algorithm or a software suite which are better suited for identifying issues, such as out of range dosing and medication interactions . FurtherHowever, many patients do not actively engage with pharmacists and volunteer information without prompting . AdditioIn this study, a revised version, the HOME-Qv2 was developed based on several findings from the initial HOME-Q evaluation and trialed in a new sample of patients. The study objectives were to (1) evaluate patient responses to the HOME-Qv2 medication experience questionnaire  and (2) Approval was obtained for this study from the institutional review board (IRB) of the university (#201710776). Two independently owned community pharmacies were recruited into the study. The questionnaire was administered to a purposeful sample of pharmacy patrons aged 55 years or older who have one or more long-term illnesses  for which they take 4 or more chronic prescription medications. The rationale for this sample selection is to identify patients who are at higher risk for medication-related problems due to their complex treatment regimens.A revised version of the HOME-Q  was usedFour pharmacists, who were either residency trained or current residents at the time, partnered with the research team to obtain informed consent and deliver the HOME-Qv2 interventions. The participating pharmacists were trained on the study protocol and in the use of several communication techniques to facilitate an interactive environment where patients could comfortably disclose medication experience issues or beliefs about medicines.Study pharmacists used purposeful sampling to recruit patients during routine patient–pharmacist interactions that occurred while patients picked up their medicines at the pharmacy by asking them if they would be interested in taking a survey about their medication use experience in the home. Those who indicated interest received further information from the pharmacist about the study. After obtaining written consent, the pharmacists provided the patients with a study packet that contained a paper version of the HOME-Qv2. Patients had the option to complete the questionnaire in the pharmacy or take it home to complete. Those who took the questionnaire home received a postage-paid return envelope for them to mail the completed form back to the pharmacy.At a pre-arranged time, the pharmacists and patients met, either in-person or over the phone, to discuss patient responses to the HOME-Qv2, during which pharmacists made recommendations to address emergent medication experience issues. Patients received USD 10 compensation for taking the survey and meeting with the pharmacist, and another USD 10 for being part of the 3-month follow up call. The participating pharmacies were compensated USD 75 per patient recruited, consented, and met with for the intervention. One pharmacy used their own electronic records system to document pharmacist–patient encounters while the second pharmacy used a paper form provided by the research team.At 3 months, a follow-up telephone call was made by a member of the research team to study participants to re-administer the HOME-Qv2, and to assess if patients adopted and maintained the pharmacist’s recommendations. The research team considered 3 months between the first and second administration of the questionnaire to be enough time for patients to adopt recommendations while minimizing the risk of patients forgetting about their previous interaction with the pharmacist. Participants were asked to give feedback on the survey or their encounter with the pharmacist if they had any. The calls were not audio recorded, rather, handwritten notes were documented by the research assistant.Frequencies were calculated for each risk-factor item, and each best-practice item on the HOME-Qv2 at baseline (time 1) and at three months (time 2). Continuous variables were assessed for normality by examining histograms, skew, kurtosis, and by using the Shapiro–Wilk test. Medians and interquartile range (IQR) were calculated for continuous variables with non-normal distribution. HOME-Qv2 totals at baseline and at 3 months were calculated, and the differences were assessed using the Wilcoxon signed rank test with an a priori 0.05 level of significance.The HOME-Qv2 items are conceptualized as prompts intended to identify patients’ medication experience issues, concerns, and practices. A patient’s affirmative response to a risk factor phrased item (1–10) was expected to elicit a discussion, and for some, an intervention or recommendation from the pharmacist. For the purposes of the study, a “Yes” answer was assigned 1 point and “Sometimes” was assigned 0.5 points. A patient’s contradictory response to a best-practices phrased item (11–20) is similarly expected to elicit a discussion, and for some, an intervention or recommendation. For the purposes of the study, a “No” answer was assigned 1 point and “Somewhat” was assigned 0.5 points. The HOME-Qv2 totals , and medians for each subscale are calculated at baseline and after 3 months. The percentage contribution of the “Somewhat/Sometimes” response option to the identification of patient medication experience issues is determined. Two authors collaborated to count and descriptively code the pharmacist interventions into categories using a similar process used to evaluate the initial version of the HOME-Q.Twenty-four participants were enrolled and completed an initial encounter, but three patients could not be reached to complete a follow-up encounter at the 3-month call and were excluded from the final analysis. Twenty-one participants who had complete data for both rounds of the survey were included in this analysis. The average age of the participants was 70 (SD = 9.55), and 12 (57.1%) were men. The median number of oral medications was 6 (IQR = 3).At baseline, there were a total of 101 medication experience issues (45 yes/no at 1 pt ea and 56 sometimes at 0.5 pt ea) reported in response to the 20 HOME-Qv2 items . At follow up, there were 60 medication experience issues (35 yes/no at 1 pt ea and 25 sometimes at 0.5 pt ea) reported  and the difference was significant .The “sometimes” response option accounted for 53.4% (31/58) of the positive baseline responses on the risk-factor subscale and accounted for 36.5% (15.5/42.5) of the points. The per-respondent median for the risk-factor subscale was 3.0 (IQR = 2.375) at baseline and 2.0 (IQR = 0.75) at 3 months. On the best-practice subscale, , the “somewhat” response option accounted for 58.1% (25/43) of the positive baseline responses and accounted for 41.0% (12.5/30.5) of the points. The per-respondent best-practice subscale median was 2.5 (IQR = 2.625) at baseline and 1.0 (IQR = 1.0) at 3 months.Respondents reported a variety of risk-factor items on the baseline HOME-Qv2 , which iThere were 31 coded recommendations across seven categories . These cAll but two of the categories of recommendations had at least 50% acceptance. Recommendations to address cost concerns had 25% acceptance (2/8), and recommendations on organizing and planning medication use had 40% acceptance (2/5). Recommendations about proper medication use, proper disposal of medications no longer needed, and about diet and exercise reported 100% acceptance. The number of interventions per patient ranged from 0 to 4, and 20 of the interventions (64.5%) appeared to be adopted and/or maintained at the 3-month call.p = 0.008) which suggests some of the pharmacist recommendations may have resolved issues and concerns and promoted the adoption of medication experience best practices. These data also suggest the addition of a middle option and phrasing half of the items as best practices were beneficial changes.The HOME-Q was developed to facilitate patient disclosure of their home medication experience issues and practices, such that responses could lead to discussions and problem-solving with the pharmacist and prescribers when necessary . The revStudy data show a decrease in patient-reported concern about medication safety (“I wonder if my medicines are doing more harm than good”) and belief that they might be taking medicines that they do not need, at 3 months. This decrease suggests these concerns may have been addressed by the pharmacists’ counsel about the safety and proper use of medicines. Example actions in this study included educating a hypertensive patient about the unique value of lisinopril or advising on the prevention of adverse events for medications such as celecoxib. Studies suggest medication adherence largely relies on patients making intentional decisions ,26.Concerns related to the cost of medicines were the most self-reported medication experience issue, but related recommendations had the lowest acceptance rate. While how much medications cost is usually beyond the control of patients, pharmacists are sometimes, but not always, able to improve the medication experience by helping make medicines more affordable. For instance, one of the pharmacist’s interventions to address cost that was accepted was to evaluate cheaper alternatives for the patient, which is considered a viable approach to cost-saving ,28.For this study sample, when asked about their interest in having their medicines packaged, and the use of medication planning tools, some respondents indicated interest, but packaging solutions recommended by the pharmacists were generally not accepted. Patients may not be favorably disposed to adopting medication packaging solutions especially if there is added cost ,30. ThisWhile adherence packaging was not employed as a result of any of the encounters, patients did report adopting pharmacist recommendations to enlist the help of family members in developing and maintaining a convenient routine for using their medications as prescribed, with fewer reports of not taking medicines when there is a change in schedule recorded at the 3-month follow-up encounter. Such a recommendation could be particularly useful for older adults who might be experiencing some cognitive and physical limitations that add to the complexity of managing their medications daily .On the first version of the HOME-Q, patients did not seem to respond positively to an item that reported they keep old prescription medicines just in case. The item was revised because it was not associated with meaningful pharmacist actions. The HOME-Qv2 introduced an item about the disposal of medicines which seemed to resonate better as a best practice. An increase in awareness of how to properly dispose of medicines may reduce the potential for accidental poisoning and drug misuse by recommending a pathway to remove excess or unused over-the-counter and prescription medicines from the home .The pharmacist interventions provided in the study were personalized to the HOME-Qv2 responses of each patient who participated in the study. This case-specific approach to addressing medication experience issues may help further develop the relationship between the patient and the pharmacist . AnecdotThe revised version, HOME-Qv2, used in this study added middle response options “sometimes” and “somewhat” to the questionnaire. At baseline, when the patient and pharmacist were engaging in their HOME-Q intervention, these middle options were chosen more often than the full yes/no response for the two subscales . This suggests the middle response option gave patients a way to express themselves on issues where they would be conflicted to make a binary yes/no response. Since the goal of the HOME-Qv2 is to facilitate a discussion about medication experience, the sometimes option may have given the patient and pharmacist more medication experience topics to discuss. In addition, the HOME-Qv2 introduced items aimed at initiating discussions about best practices that could contribute to a better medication experience for the patient. Overall, the dual focus of the questionnaire may have enabled the pharmacist to explore the links between best practices, risk factors, and medication experience to provide interventions that address a variety of patient needs and perspectives.We posit that the revisions made to the HOME-Qv2 improved the value of the questionnaire. This is supported by the HOME-Qv2 yielding a higher proportion of adopted recommendations and a significant decrease in patient self-reported medication experience issues at the 3-month call, compared to no change with the initial version of the HOME-Q. However, there is a need for future work to further evaluate and validate this patient engagement tool.As the aim of the HOME-Qv2 is to help providers characterize and assess the home medication experience of patients, further studies are needed to validate if the HOME-Qv2 captures medication experience issues that would otherwise have been missed without the questionnaire, and to evaluate the interventions that derive from using the HOME-Qv2 for longer-term impact. Validation work also is needed to determine what point value should be assigned to the sometimes response or if all positive responses should be treated the same. Additionally, assessment of how the HOME-Qv2 impacts patient–provider discussions about patient experience with using and managing medications in the home, and whether using the HOME-Qv2 enables pharmacist–patient communication that facilitates trust-building and disclosure would provide valuable information about the questionnaire’s value ,16,36,37This study has several limitations. The small sample size and the limited diversity of patients and pharmacists who participated in the study diminish external validity. Additional trials with more diverse patient populations are needed to increase generalizability. Selection bias due to non-random sampling may limit internal validity. The study was not designed to differentiate between modes of administration (telephone and in-person) and patients may have responded differently when they knew their questionnaire was going to be viewed by the pharmacist as part of the visit and the 3-month call which was conducted by a member of the research team. Further, the questionnaire has not yet been tested for test–retest reliability.The HOME-Qv2 helped pharmacists identify medication experience issues and their frequency of occurrence in the study sample prompting discussions and recommendations from the pharmacists. A high proportion of the recommendations were adopted and there was a significant decrease in the reported medication experience issues after 3 months.


Due to concerns of carcinogenicity, it is necessary to assess long-term acrylamide exposure in individuals. Whether the available methods of estimating acrylamide intake can indicate long-term exposure remains unknown. We examined variations in the estimated dietary acrylamide intake of the Japanese population.The study included 240 participants aged 40–74 years who were a part of the Japan Public Health Center-based Prospective Study for the Next Generation (JPHC-NEXT). Twelve-day dietary records (DRs) were collected over a one-year period, and food frequency questionnaires (FFQs) were collected twice during the year. Dietary acrylamide intake was estimated from an acrylamide content database. Within-individual variations and between-individual variations were calculated using the random effects model. A linear regression analysis was performed to identify foods with large between-individual variations.The ratios of within-individual variance to between-individual variation were 3.2 for men and 4.3 for women. Days of DRs required to estimate the usual individual intake within 20% of the true mean intake with 95% confidence were 60 days for men and 66 days for women. Coffee/cocoa, potato, and green tea contributed to between-individual variations, in that order, and seven foods contributed to 93% of the between-individual variation.Estimating the acrylamide intake using DRs requires an extended data collection period to estimate the intragroup ranking and habitual intake of individuals. Long-term exposure assessments should be based on methods with less potential for measurement errors, such as the use of biomarkers. Acrylamide is a Group 2A probable human carcinogen  that is Methods such as dietary records (DRs), 24-h dietary recalls (24 h-Rs), duplicate methods (DMs), and total diet studies are used to assess dietary acrylamide exposure , 8–11. DEstimating the true mean intake of acrylamide is important for determining the accurate risk of acrylamide exposure from food. FFQs are well-suited for ranking individuals, but it is difficult to set a threshold because FFQs cannot sufficiently estimate the absolute intake of individuals. However, we can determine the number of days of DRs required to estimate the usual individual intake because long-term DRs allow for the estimation of the individual’s absolute value and variations. An estimation of the true mean intake can be a useful resource for risk assessment and assessing the association with diseases. The estimated value may lead to reference values that can be used for guidelines, as evidence of public health comments, and for individual dietary counseling. To the best of our knowledge, no reported studies have evaluated variations in the estimated within-individual and between-individual dietary acrylamide intakes or investigated the number of days of DRs needed to estimate the individual habitual intake and individual ranking in groups.FFQs are widely used in large-scale epidemiological studies to determine the effects of long-term dietary acrylamide intake , 13. As Details of the study design and participant characteristics have been described previously . ValidatWe used an established database of foods that contain acrylamide to estimate the acrylamide intake from the DRs and FFQs . The datThe study participants recorded menus, food and beverage names, and the amounts consumed (according to weight) in a food diary. Dieticians checked the DRs and coded each food using item numbers from the Standard Tables of Food Composition in Japan - 2010 (FCT 2010) . AcrylamAn FFQ was designed to estimate the habitual dietary intake for the previous 1 year . Of the The estimated acrylamide intake value for fried batter was calculated using the participants’ responses to the following question in the original FFQ: “How often do you consume deep-fried foods with batter?” Respondents chose their response from the following six frequency categories: almost never, 1–3 times/month, 1–2 times/week, 3–4 times/week, 5–6 times/week, and daily. Acrylamide intake from these foods was estimated by multiplying the frequency of fried-food consumption with batter by the value of daily acrylamide intake from fried batter calculated from the DRs of the JPHC study [Mean (with standard deviations [SDs]) and median with 5th and 95th percentile values of dietary acrylamide intake were calculated according to sex. Percentage differences in acrylamide intake according to the DR and FFQ methods were computed using the following formula:Data were analyzed using the random effects model:i is the random variable for variation among subjects; Season Xij, day Xijk represents the random effects of the season and day; and the error term (εijk) represents the random within-person variance. Using this calculation, acrylamide intake data (level 1) were nested for seasons (level 2), which were nested within participants (level 3). Estimates of within-individual variance where μ is the mean of acrylamide intake; participant a group .α is 1.96; CVw is the within-individual coefficient of variation; We also calculated the number of days of DRs needed to estimate an individual’s habitual intake with 95% confidence within a specified percentage deviation using the following formula , 27:\doThe number of days (D) needed to obtain a given unobservable correlation between the observed and true mean intake was calculated using the following formula :\documefication . We set A linear regression analysis with stepwise selection was used to identify foods that contributed to between-individual variations, with acrylamide intake from each food item as the explanatory variable and the total acrylamide intake according to the DRs as the response variables. A partial R-squared model value was developed for the selected food items.The following analysis was conducted to determine the validity and reproducibility of the FFQ and the contribution of each food to the total acrylamide intake. The results are summarized in Additional file 2 for men and 22.8 kg/m2 for women. The percentages of current smokers were 26.5% for men and 2.1% for women [Participants in this study had mean ages of 57.4 years (men) and 57.0 years (women). The mean body mass index (BMI) values were 23.7 kg/mor women . Particiw) and between-individual variance (CVb). In addition, Table Table Table The validity of the estimated acrylamide intake determined by using the FFQ indicated that the energy-adjusted correlation coefficients were 0.34 for men and 0.28 for women, and that the deattenuated correlation coefficients were 0.39 for men and 0.33 for women. The reproducibility of the FFQ indicated that the energy-adjusted correlation coefficients were 0.62 for men and 0.65 for women  . In the To the best of our knowledge, this is the first study to elucidate the within-individual variation and between-individual variation in dietary acrylamide intake. The key contribution of this study was that it assessed the within-individual variation of acrylamide intake, which must be considered as a measurement error that potentially contributes to erroneous findings. These findings highlight the need to use biomarkers to appropriately estimate long-term acrylamide exposure because biomarkers are thought to have fewer measurement errors than other measurement methods. The between-individual variation results of our study also show that it is possible to rank acrylamide intake based on several types of foods, thus implying that a simpler questionnaire could be developed that specifically targets the acrylamide intake of the Japanese population in the future.There were certain limitations to this study. First, the results of this study depended on the accuracy of acrylamide content database. The database includes approximately 50% of foods that may contain acrylamide. If there are large within-individual variations in foods that are not included in the database, then it may take more days to rank individuals and to estimate habitual intake from DRs. Second, the study did not include individuals 39 years or younger or individuals 75 years or older; therefore, the applicability of the results to all age groups remains uncertain. For other nutrients, within-individual variations tend to be larger in younger people than in older people , suggestOverall, the study results revealed that the estimation of habitual acrylamide intake and the ranking within the group according to DRs require an extended data collection period because of large within-individual variations. The estimation of acrylamide intake using short-term dietary surveys may lead to the misclassification of rankings and may not represent an individual’s habitual intake. Therefore, we conclude that assessing the long-term exposure based on methods that are unaffected by measurement error such as those associated with biomarkers is required, and that a repeated assessment of these biomarkers would be beneficial.Additional file 1 : Table S1. Evaluation of the relationships between dietary records, food frequency questionnaires used for the validation analysis, and food frequency questionnaires.Additional file 2 : Table S2. Contribution of food groups to total acrylamide intake estimated using dietary records.


In two animal models, immunization with a single dose of the -fcpA mutant was sufficient to induce a robust anti-protein antibodies response that promoted protection against infection with different pathogenic Leptospira species. Furthermore, characterization of the immune response identified a small repertoire of biologically relevant proteins that are highly conserved among pathogenic Leptospira species and potential correlates of cross-protective immunity.Leptospirosis is the leading zoonotic disease in terms of morbidity and mortality worldwide. Effective prevention is urgently needed as the drivers of disease transmission continue to intensify. The key challenge has been developing a widely applicable vaccine that protects against the >300 serovars that can cause leptospirosis. Live attenuated mutants are enticing vaccine candidates and poorly explored in the field. We evaluated a recently characterized motility-deficient mutant lacking the expression of a flagellar protein, FcpA. Although the  Leptospira. It is more common in warmer regions with high rainfall, especially in impoverished areas. The disease is spread in the urine of animals such as rodents, farm animals or dogs. Humans and other animals can get leptospirosis when they come in contact with urine-contaminated water and soil.Leptospirosis is a life-threatening disease with flu-like symptoms that is caused by bacteria known as Leptospira bacteria. It also fails to stop the bacteria from colonizing the kidneys of the infected animals, which means that vaccinated animals can still spread disease.Current measures to control leptospirosis are largely ineffective. Although a vaccine is available for animals, it only protects against a few types of the 300 disease-causing Leptospira bacteria to move, can stop them from infecting hamsters. Moreover, when these animals were exposed to the mutant bacteria, they did not get sick nor developed the disease. Here, Wunder et al. tested whether bacteria lacking the FcpA protein could be used as an attenuated vaccine. This form of vaccine contains live bacteria that have been modified to become harmless but are able to train the immune system to produce a long-lasting immune response against the invaders.Previous research has shown that inactivating a protein called FcpA, which is necessary for Leptospira and could stop them from colonizing mice kidneys. Moreover, Wunder et al. found that the immune system targeted specific proteins that were common to various types of Leptospira, which may explain the broad spectrum of protection the vaccine offered.The results showed that a single dose of the vaccine was enough to prevent hamsters and mice from dying of leptospirosis. It also worked against several types of Leptospira are safe to use in animals and potentially humans.Rapid urbanization and climate change are among the main drivers of leptospirosis. An effective vaccine for this disease would reduce the public health burden by providing protection against leptospirosis and by reducing the spread of the disease. A next step will be to ensure the mutant  Leptospirosis is caused by a genetically and antigenically diverse group of spirochetes of the Leptospira genus . CurrentAlthough Leptospira has a worldwide distribution, the large majority of the burden occurs in the world’s most impoverished populations , where tThe public health priority is therefore prevention of leptospirosis before severe complications develop. However, there is no effective control for leptospirosis and safe and efficacious vaccines are not available for human use . China aCurrent control measures have been uniformly ineffective in addressing the large human and animal global health burden due to leptospirosis, especially in developing countries. Given the limitations of the whole-cell vaccines available and the ineffective attempts to identify protein vaccine candidates , an attefcpA gene lost its ability to produce translational motility and to penetrate mucous membranes, resulting in loss of kidney colonization and lethality in the hamster model of leptospirosis. Although highly attenuated in the hamster model, a needle inoculation of the mutant produced a transient bacteremia prior to clearance by the host immune response . A mutant deficient in the response . In the fcpA- mutant caused a transient systemic infection, which was cleared 7 days after intraperitoneal inoculation of 108 leptospires in hamsters  and there was a significant difference in the magnitude of dissemination of the mutant in the blood compared to the wild type  that was essential for translational motility and thus for virulence . Despitehamsters . In thishe brain . These rd strain . We alsonfection . Althougnfection . Furtherild type . DNA sigsed here  is 100 l-fcpA mutant induces cross-protective responses, given previous findings  titers against heterologous serovars, both in hamsters , and there is a decrease on the OD for all doses when the Manilae antigen was treated with proteinase K  and mice (Mouse 107) respectively, immunized with a dose of 107 leptospires and a total of 13 protein targets  of proteins were widely represented in those targets , with at7 leptospires of the attenuated-vaccine  was inoculated subcutaneously with a dose of 107 leptospires in 0.5 mL of EMJH medium. A group of three animals was euthanized at 1, 4, 7, 14, and 21 days after infection. As a control, a group of nine animals was infected with Fiocruz L1-130 WT using the same route and dose, and animals were euthanized at days 1 and 4 after infection. The final group was euthanized at onset of disease. After euthanizing the animals, blood, kidney, liver, and brain were carefully removed, collected into cryotubes, and immediately placed into liquid nitrogen before being stored at −80°C until extraction of DNA. Kidney and blood were inoculated in EMJH for culture of leptospires when necessary.For the dissemination experiments with the Fiocruz L1-130 7, 105, 103, 102, and 101) and a control group with three animals was inoculated with Fiocruz L1-130 WT with a dose of 107 leptospires. Blood was collected by retro-orbital bleeding at 1, 4, 8, 13, 15, 18, and 21 days after infection.For the experiment in mice, groups of three 4-week-old female C57BL/6 mice (Jackson laboratory) were inoculated subcutaneously with different doses of the vaccine  in 500 and 200 μL of EMJH medium, respectively. The heat-killed vaccine was used in a single dose of 107 leptospires by subcutaneous route as a control group in hamster. In addition, groups of animals were injected with phosphate buffered saline (PBS) and served as unvaccinated controls. Blood samples were collected the day before and 20 days post-immunization by retro-orbital bleeding.All vaccination experiments  were per8 leptospires) of a range of serovars whose virulence has been well characterized in our laboratory (L. interrogans serovar Manilae L495 (108 leptospires)  . After e-fcpA mutant was generated by immunizing a group of ten 3-week-old male Golden Syrian hamsters using the same protocol, described above. A group of 10 animals injected with PBS was used to obtain control sera. Animals were euthanized at day 21 post-immunization by inhalation of CO. Blood was obtained by cardiac puncture, followed by separation of sera that was subsequently pooled as immune (hamster α-fcpA-) and control sera.Immune sera against Fiocruz L1-130 8 leptospires of serovar Manilae L495 (heterologous strain) by intraperitoneal and conjunctival route, respectively, as described above.Immune or control sera were passively transferred to groups of 5 naïve female mice and seven naïve male hamsters (6–7-week-old) in a dose of 0.5 and 2.0 mL, respectively, using the intraperitoneal route. After 24 hr mice and hamsters were challenged with 102. Before each blood collection animals were anesthetized by an open-drop method with a mixture of 20% v/v isoflurane in propylene glycol.All animal protocols were approved by the Institutional Committee for the Use of Experimental Animals, Yale University (protocol # 2017–11424). Hamsters and mice were monitored twice daily for endpoints including signs of disease and death, up to 28 days and 14 days post-infection, respectively. Surviving animals at the end of the experiment or moribund animals presenting with difficulty moving, breathing, or signs of bleeding or seizure were immediately sacrificed by inhalation of COPre- and post-vaccination sera were obtained by centrifugation of clotted blood at 1000 g for 15 min at room temperature. Sera samples were kept frozen at −20°C until analysis for the presence of antibodies against leptospires by MAT, ELISA, immunoblotting, and proteome array.MAT was performed using standard practices and as previously described . Serum wL. interrogans serovar Manilae L495 and Fiocruz L1-130 cultures (108 cells) at 12,000 rpm, 4°C for 20 min. The pellets were washed twice with PBS and resuspended in 500 µL of PBS. Resuspended cultures were sonicated in ice for 6 cycles at 30 kHz with a power output of 300 W. Lysates were quantitated by Bradford assay and employed as antigen at a concentration of 150 ng/well . Flat-bottomed polystyrene microtiter plates (Corning) were coated with Leptospira antigen and incubated overnight at 4°C. The plates were washed three times with PBS-0.05% (vol/vol) Tween 20 (PBST) and incubated with blocking solution  for 2 hr at 37°C. After four washes with PBST, wells were incubated with mouse immune sera, diluted 100-fold in 2% BSA, for 1 hr at 37°C. Secondary anti-mouse HRP conjugated antibody (Jackson ImmunoResearch) was used at a dilution of 50,000 (2% BSA) and incubated for 1 hr at 37°C. TMB SureBlue Reserve (SeraCare) was used for detection and the reaction was stopped by adding 100 μL of 2 N H2SO4. Absorbance (450 nm) was recorded by microplate reader (Biotek).For the ELISA, whole cell lysate was prepared by centrifugation of To evaluate the effect of proteolytic enzyme treatment on Leptospira antigen we used the protocol previously described . BrieflylipL32 primers and probe as described previously (DNA was extracted from blood and tissue samples using the Maxwell16 (Promega Corporation) instrument following the manufacturer’s instructions. Quantitative Real-time PCR assays were performed on hamster and mouse tissues using an ABI 7500 instrument (Applied Biosystems) and Platinum Quantitative PCR Supermix-UDG (Invitrogen Corporation) with eviously .fcpA- at dilution of 1:100. For subsequent detection, HRP goat anti-mouse or anti-hamster’s serum (Jackson ImmunoResearch) was employed at dilution of 1:100,000. Blots were analyzed using ChemiDoc Imager (Bio-Rad).Immunoblots with whole cell extract of Leptospira strains were performed as previously described . WesternLeptospira interrogans serovar Copenhageni strain Fiocruz L1-130 as previously described (E. coli HY system (5 PRIME) and synthesized crude proteins were printed on 3-pad nitrocellulose-coated AVID slides (Grace Bio-Labs) using a Gene Machine OmniGrid 100 microarray printer (Genomic Solutions). In addition to IVTT expressed proteins, each array contained no DNA control spots consisting of IVTT reactions without the addition of a plasmid, serial dilutions of purified IgG/spots.The full ORFeome was amplified from escribed . The ORFescribed  and usedE. coli lysate (Genscript) at a final concentration of 10 mg/mL to block anti-E. coli antibodies. The arrays were incubated overnight at 4°C with constant agitation. After the overnight incubation, the arrays were washed three times with T-TBS and then incubated for 45 min at RT with biotin-conjugated anti-human IgG secondary antibody (Jackson ImmunoResearch), diluted at 1/400 in array blocking buffer, followed by Qdot 800 streptavidin conjugate (ThermoFisher Scientific). The arrays were air dried after brief centrifugation. IgG signals were detected with ArrayCam 400 s Microarray Imaging System (Grace Bio-Labs) for Q800. The array signal intensities were quantified using QuantArray software. Mean pixel intensities are corrected for local background of all spots. Protein expression was validated by microarray using monoclonal anti-polyhistidine .The arrays were probed for IgG reactivity. For serum samples, the arrays were probed at 1/100 dilution in protein array blocking buffer (GVS) supplemented with In addition, 30 sera  of human patients from Salvador, Brazil with confirmed acute leptospirosis were probed in the array containing 660 proteins as described above. Patient samples were collected and selected as previously described .10 fold change (LFC) between pre- and post-vaccination proteome signal intensities. We subtracted the chip background and set the negative values to one (to avoid issues taking logarithms) before calculating the LFC. Analyses were conducted on three data sets: the hamster data, which used a single attenuated-vaccine dose of 107 leptospires (Hamster 107), the mouse dose–response data including all vaccine doses 101, 102, 103, 105, and 107 , and the subset of mice given a dose of 107 leptospires of the attenuated-vaccine (Mouse 107). The decision of those analysis group was made based on the fact that the high dose of the vaccine in mouse (Mouse 107) was the same as in the hamster experiments (Hamster 107) and was the only dose in mouse that gave 100% protection against death and colonization. Furthermore, the other doses  provided different levels of protection when combining death and colonization and the comparison analysis of all those three groups would increase our chances to identify potential protein targets with a role in those outcomes.We analyzed the logA as AV is an indicator variable for whether the animal A received the attenuated-vaccine or a control injection. These terms were included in all models. The LogDose term was only included in the analysis of the dose–response relationship in mice and is the logarithm of the dose . The indicator variable AV prevents LogDose = 0 for control animals from being treated as a true zero. Our statistic of interest was the t-statistic. For the Hamster and Mouse 107 models we interpreted the AV t-statistics, and for the Mouse All dose–response model we interpreted LogDose t-statistics. We used the Benjamini–Hochberg (BHp) correction . Clusters of orthologs groups (COGs), pSortB localization, transmembrane domains (TMhmm), and signal peptide  information were obtained from Genoscope platform (http://www.genoscope.cns.fr/agc/microscope/home/). p-Value for enrichment statistical analysis was calculated using Fisher’s exact test in the R environment (http://www.r-project.org).Prism 8 (GraphPad Software) was employed for all the statistical analysis of in vivo data. Fisher's exact test and analysis of variance (ANOVA) were applied to assess statistical differences between pairs of groups and multiple groups, respectively. A p-value of <0.05 was considered significant. A binomial proportion confidence interval was calculated to determine the efficiency of the vaccine in both hamster and mouse. Protein homologies of  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Leptospira species are important bacterial pathogens for humans and animals. The authors describe the first vaccine that provides cross protection against different species of Leptospira, reduces kidney colonization, but does not provide sterilizing immunity. They also identified potential candidate proteins that could be used for vaccines.Decision letter after peer review:Leptospira spp" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Jos van der Meer as the Senior and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal their identity: Yung-Fu Chang (Reviewer #1).Thank you for submitting your article "A Live Attenuated Vaccine Model Confers Cross-Protective Immunity Against Different Species of eLife.Below you will find a set of recommendations, which, if completed, would lead to publication in Summary:Leptospira species are important bacterial pathogens for humans and animals. The authors describe the first vaccine that provides cross protection against different species of Leptospira, reduces kidney colonization, but does not provide sterilizing immunity. They also identified potential candidate proteins that could be used for vaccines.Essential Revisions:fcpA- attenuated vaccine provides great cross protection against death, but only reduces kidney colonization.The single dose 1) The authors provided in Supplementary files 2 and 3 a summary of all animal experiments done, but a more detailed table with results of each individual experiment should be added, including control groups that receive PBS.2) We would like to see the detailed animal experiments in the supplementary data section.3) Figure 2 should mention that it shows the results of all combined experiments, and the control group should be added to the graphs. The legends of this figure should mention the route of infection for the challenge: SQ for hamsters and IP for mice.fcpA- vaccine? If so, that could be discussed in the Discussion.Only one dose of the heat-killed vaccine is used as a control, instead of 2 (subsection “Model for cross-protective immunity to leptospirosis”). Why was not the vaccine administered as commonly used ? Did the authors try two doses or more of the 4) In the proteome array, the authors should better explain the rationale for comparing all vaccine doses (mouse) to 10e7 dose (mouse), and not only 10e7 mouse to 10e7 hamster.5) The Discussion should not start with a new introduction but with the major findings. So start with the last sentence of the first paragraph. Essential Revisions:The single dose fcpA- attenuated vaccine provides great cross protection against death, but only reduces kidney colonization.1) The authors provided in Supplementary files 2 and 3 a summary of all animal experiments done, but a more detailed table with results of each individual experiment should be added, including control groups that receive PBS.We appreciate the suggestion from the reviewer and apologize for not having provided this information. We revised both Supplementary files 2 and 3 and made it more comprehensive, including details about individual experiments and information regarding the PBS control groups.2) We would like to see the detailed animal experiments in the supplementary data section.We appreciate the suggestion from the reviewer. As mentioned above, we made a thorough revision of both Supplementary files 2 and 3 to include all the details about individual experiments and information regarding the PBS control groups, but also keeping the overall efficacy of the vaccines.3) Figure 2 should mention that it shows the results of all combined experiments, and the control group should be added to the graphs. The legends of this figure should mention the route of infection for the challenge: SQ for hamsters and IP for mice.We thank the reviewer for the comment. We included in the legend of Figure 2 the route of challenged used in those experiments, for both hamsters and mice. We also included a sentence to make it clear that the analysis for efficacy was done using a combination of all experiments.With regards the request to include control group in the figure, we appreciate the suggestion. However, since the sections A to E of Figure 2 are analysis regarding the efficacy of the vaccine, which was calculated using the results obtained from the PBS control group, that information can’t be included on this figure without compromising its purpose. We hope that the information included in the revised Supplementary files 2 and 3 are sufficient to satisfy the reviewer.Only one dose of the heat-killed vaccine is used as a control, instead of 2 (subsection “Model for cross-protective immunity to leptospirosis”). Why was not the vaccine administered as commonly used ?We appreciate the question from the reviewer. Our goal in this study was to demonstrate the efficacy of the attenuated vaccine after a single dose, given that according to our hypothesis the strain was able to multiply in the host and stimulate the immune system, which would be enough to mount a strong immunity. For that reason and to avoid different doses comparisons we decided to keep a standard protocol of vaccination using only dose for all vaccines. To make this decision clear, we added to the Results section a brief explanation that hopefully will satisfy the reviewer and help the readers: “For the purpose of evaluating the efficacy of the attenuated vaccine after a single dose, we decided to keep a standard protocol for vaccination and thus using only one dose of the heat-killed vaccine as well”.Did the authors try two doses or more of the fcpA- vaccine? If so, that could be discussed in the Discussion.Indeed we never tried a boost dose of the attenuated vaccine after the promising results obtained with a single dose. But we acknowledge that for future experiments more doses of the vaccine should be evaluated. We included a sentence in the Discussion section: “Given the promising results obtained with a single dose of the attenuated vaccine, a boost dose was not evaluated. However, it should be considered for future experiments.”4) In the proteome array, the authors should better explain the rationale for comparing all vaccine doses (mouse) to 10e7 dose (mouse), and not only 10e7 mouse to 10e7 hamster.7) was the same as in the hamster experiments (Hamster 107) and was the only dose in mouse that gave 100% protection against death and colonization. Furthermore, the other doses  provided different levels of protection when combining death and colonization and the comparison analysis of all those three groups would increase our chances to identify potential protein targets with a role in those outcomes.”We appreciate the reviewers suggestion. We made the decision to consider the analysis of all vaccine doses in mouse given the observation that the protection either for colonization and death in the mouse model was related to the dose of the vaccine. With that analysis, we could identify protein candidates that were potentially involved on those outcomes when performing this combined analysis. We included in the analysis section of the text one sentence to justify the choice: “The decision of those analysis group was made based on the fact that the high dose of the vaccine in mouse (Mouse 105) The Discussion should not start with a new introduction but with the major findings. So start with the last sentence of the first paragraph.We appreciate the reviewer suggestion. We agree that the suggestion improves the message, and we shifted the order of the paragraph starting with the sentence mentioned by the reviewer.


Autophagy degrades the cytoplasmic contents engulfed by autophagosomes. Besides providing energy and building blocks during starvation via random degradation, autophagy selectively targets cytotoxic components to prevent a wide range of diseases. This preventive activity of autophagy is supported by many studies using animal models and reports identifying several mutations in autophagy-related genes that are associated with human genetic disorders, which have been published in the past decade. Here, we summarize the molecular mechanisms of autophagosome biogenesis involving the proteins responsible for these genetic disorders, demonstrating a role for autophagy in human health. These findings will help elucidate the underlying mechanisms of autophagy-related diseases and develop future medications. This nonselective degradation system is essential for survival during starved conditions as it provides cells with buck-up energy and building blocks required to retain cellular functions. Moreover, a low level of basal autophagy constitutively maintains the turnover of self-components even without any exogenous stresses. Furthermore, autophagy has been shown to target invading bacteria in a highly specific manner to support the viability of host cells, suggesting that autophagy can selectively degrade harmful substances that cannot be digested by other pathways such as proteasomal degradation pathway. The selective autophagy eliminates damaged organelles, such as mitochondria and lysosomes or protein aggregates, that play a causative role in a range of diseases, including neurodegeneration. Recent studies have shown that the number of the type of targets is larger than previously anticipated, calling attention to a role of autophagy as a biological defense mechanism2. Although autophagosome observation by electron microscope and biochemical analysis had been suggested for basic topology and workflow of autophagy since the 1960s, the underlying molecular mechanisms were not elucidated for many years due to the difficulty of analysis. Ohsumi et al. carried out a large-scale genetic screening of Saccharomyces cerevisiae mutants and identified a series of genes essential for autophagy, some of which are overlapped with genes independently identified by Thumm’s lab and Klionsky’s lab3. These results led to powerful genetic analyses and striking mechanistic findings and an establishment of basic mechanism of autophagy. These reports were followed by subsequent research on autophagy in mammals, which led to the identification of the role for autophagy in human health; this discovery was awarded the 2016 Nobel Prize in Physiology or Medicine.Macroautophagy (hereafter autophagy) is a bulk intracellular degradation system that digests self-components, as its name is derived from the Greek words auto (self) and phagy (eating). Autophagy degrades the cytoplasmic contents engulfed by a characteristic double-membrane structure called an autophagosome, which is ~1 µm4.Our current understanding of autophagy in physiology is strongly supported by studies using model experimental systems. Moreover, many human hereditary diseases caused by autophagy-related genes have been reported in the last decade, contributing to the elucidation of the role of autophagy in human physiology. In this paper, we summarize the molecular mechanism of mammalian autophagy and describe the current understanding of human hereditary disorders to elucidate the physiological significance of autophagy in humans for future studies. Although not mentioned in detail here, microautophagy, which directly delivers substrates into vacuoles or lysosomes, and chaperone-mediated autophagy (CMA), which transports substrates to lysosomes via activity of chaperones, are also involved in maintenance of cellular homeostasisThe most characteristic feature of autophagy is the engulfment of cytoplasmic components by a double-membrane structure called an autophagosome. Unlike other organelles such as mitochondria, autophagosomes are generated de novo upon induction by stresses such as starvation. First, an isolation membrane (phagophore) is generated at the autophagosome formation site, followed by elongation and closure of the edge of the membranes to form autophagosomes. By the subsequent fusion with lysosomes containing various hydrolases, autophagosomes mature to autolysosomes so that the contents with the inner membranes are degraded Fig. . In an e6. Concomitantly, de-phosphorylation of ULK1 by phosphatase PP2A promotes activation of ULK1 complex8. In yeast, the phosphorylation changes the complex formation as phosphorylation of Atg13 by TOR inhibits the binding of Atg1 with Atg139. In mammalian cells, the phosphorylation does not halt the formation of the ULK1-ATG13-FIP200 complex but instead inhibits the kinase activity of ULK1 complex via regulating the binding of ULK1 complex with AMPK10. mTORC1 also regulates autophagy through transcription factors. Under normal nutrient conditions, the transcription factor TFEB is phosphorylated by mTORC1 that facilitates its localization in the cytoplasm through binding to the 14-3-3 protein. When this phosphorylation decreases during starvation, TFEB is transferred to the nucleus, which can induce the transcription of autophagy- and lysosome-related genes12. In addition to TFEB, another transcription factor, TFE3, also translocates to the nucleus in response to starvation to activate autophagy13. In summary, the mTOR pathway plays a central role in the control of autophagy. However, some pathways regulate autophagy independent of mTOR; and thus, the mechanism of autophagic activation is not always the same and varies in response to different kinds of cues14.Nutrient starvation is the best-characterized stressor inducing autophagy. The release from negative regulation by the mTOR complex triggers autophagic induction. Under normal nutrient conditions, ULK1 complex is inactivated due to its constitutive phosphorylation by the mTOR complex. The high amino acid concentration in the lysosome keeps the mTOR complex activated in a manner dependent on lysosomal proton pump v-ATPase complex, which promotes the binding of Rag GTPase with mTORC1. When cells are exposed to starvation conditions, decreased amino acid levels in the lysosomes makes mTORC1 to be released from the lysosomes, resulting in reduced phosphorylation of its targets15. It is generated from an extended ER membrane in a form of Ω-shaped structure called omegasome16. The shaping of omegasome and autophagosomes is supported by an assembly of actin inside these structures17. Consistent with the fact that mitochondria can also be a source, autophagosomes are formed at the contact sites between the ER and the mitochondria, as shown by simultaneous imaging of three organelle markers19. Other materials, such as cell membranes and lipid droplets, are also considered sources for autophagosomes22. ATG9 vesicles and the ER-Golgi intermediate compartment (ERGIC) are believed to be involved in supplying materials for isolation membranes26. To facilitate the source supply and the shaping of autophagosome, two upstream components play a major role: ULK1 complex and ATG9 recruit downstream factors and coordinate the events as described below.The source of autophagosomes has been a hot topic in the field of autophagy. The best-known source is the endoplasmic reticulum (ER). Autophagosomes have been proposed to be formed by extending the isolation membrane along with the ER, which is called the “cradle model”. This hypothesis is supported the three-dimensional structure generated by electron tomography27. The mechanism of their action was investigated by intracellular localization analysis of Atg proteins with fluorescent tags in the absence of each gene28. Based on this model, the hierarchy of the molecular actions of mammalian autophagy-related proteins was established. Mammalian cells do not form a clear PAS like yeast cells, but each autophagy-related protein accumulates at distinct sites where the isolation membrane is generated. These sites are observed by the formation of dots under fluorescence microscopy. As described above, phosphorylation of ULK1 complex is the most upstream target for autophagic induction and is located upstream of the cascade29. ATG9 is the only membrane protein in the core ATG proteins and is contained in a small membrane structure of ~30–60 nm called ATG9 vesicles. To initiate autophagosome nucleation, both ULK1 and ATG9 are localized at autophagosome formation sites that are supposed to be contact sites of ER and other organelles such as mitochondria30. Under the nutrient-rich conditions, ATG9 travels through plasma membranes, early endosomes, recycling endosomes and trans-Golgi network (TGN). Upon induction, a portion of it localizes to autophagosome formation site from recycling endosomes or TGN. It depends on TRAPPIII complex that mediates retrograde trafficking from endosomes to Golgi33. Although ATG9 vesicle itself does not become a membrane source, it facilitates the delivery of membrane sources for autophagosomes23. A recent study using a proteomics approach identified contents of ATG9 vesicles, showing that it is enriched in BAR-domain-containing proteins including Arfaptin 234. It supports the distribution of ATG9 vesicles and the delivery of PI4K and PI4KIIIβ to autophagosome formation sites.In yeast, the pre-autophagosomal structure (PAS) is formed at the proximal area of the vacuole, where ATG proteins accumulate to generate isolation membranes23, and that ATG9 and ULK1 complex are independently targeted to depolarized mitochondria for Parkin-dependent mitophagy35, ULK1 and ATG9 are not essential for their initial recruitment to the site of autophagosome formation each other. However, ULK1 complex is needed for the trafficking of ATG9 vesicles as shown by the retention of ATG9 on TGN in the absence of ULK1 even in starved conditions36. It is mediated by phosphorylation of ATG9 on Ser14 by ULK1 upon starvation signal as is the case with yeast showing that Atg1 phosphorylates Atg9 to facilitate recruitment of downstream components to PAS38. Interestingly, knockdown of PI4KIIIβ leads to a reduction in dot formation of ATG13, a component of ULK1 complex, in starved condition, suggesting that ATG9 supports recruitment of ULK1 complex by promoting PI4P enrichment on the site of autophagosome formation34. Thus, it is most likely that those two major factors are required for their full activity to facilitate autophagy. It is not only at the step of initiation of isolation membrane formation but also elongation of the membrane. It has been known that ATG2A, which is required for supplying lipid to autophagosome membrane to facilitate the elongation40, works to establish ATG9 localization on mitochondria-associated ER membranes42. It is also the case of ULK1 complex because this complex phosphorylates a wide variety of substrates related to autophagosome biogenesis that are involved in the initiation step to the fusion step as well43.Given that knockdown of ATG9 results in only a slight reduction in ULK1 dot formation44. As ATG9 is embedded in ATG9 vesicles that only transiently localize to the vicinity of autophagosome45, one can speculate that ATG9 vesicles exist as a distinct component from the liquid droplet, and ATG9 on the membrane might temporally contact with ATG2A, ULK1 or other autophagy-related proteins in the liquid droplet. Thus, it is important to understand a topological basis which ensures ATG9 vesicles to access to other ATG proteins in the liquid droplet and promote a range of events described above.Recently, it has been clear that PAS is a liquid-like condensate of Atg proteins. Upon starvation signal, the de-phosphorylation of Atg13 results in an establishment of Atg1 complex and formation of a liquid droplet of early PAS. This phase separation ensures Atg1 kinase activation and subsequent recruitment of other Atg proteins to make matured PASTaken together, coordinated actions of ATG9 and ULK1 complex are inevitable for autophagy through regulating membrane source supply and recruitment of downstream components, promoting proper autophagosome maturation.48. While the PI3K with UVRAG is involved in the autolysosome formation, that with ATG14 promotes both formation of isolation membrane and the later steps as well49. PI3P promotes the localization of proteins with PI3P-binding motifs such as FYVE domains to the membrane where PI3P is embedded. Isolation membranes require the recruitment of four ATG18 homologs, WIPI1-4 that interact with PI3P through FRRG motif for WIPI1 and WIPI2 or LRRG motif for WIPI3 and WIPI443. Since these paralogs may have not only a functionally redundant role as shown by localization of all of four paralogs on nascent autophagosomes upon starvation, but also specific roles as well, since phenotypes caused by the loss of each gene could not be fully compensated by the presence of the other paralogs. WIPI2 acts as a scaffold to recruit ATG16 to isolation membrane, WIPI3 binds to TSC complex and FIP200, and WIPI4 interacts with ATG2A to facilitate autophagosome formation51. Consistent with this idea, mutations in the WIPI4 (WDR45) gene cause static encephalopathy of childhood with neurodegeneration in adulthood (SENDA), a human genetic disease involving neurodegeneration52. WIPI2 gene is also known to be responsible for a hereditary disease showing neurological disorders (described below)53. FYVE domain-containing protein DFCP1 is often used as a marker of isolation membranes but is not functionally involved in autophagy itself54. The function of PI3K in the formation of the isolation membrane depends on ULK1 and ATG9 since the formation of ATG14, DFCP1 or WIPI2 dots decrease with deficiencies in the components of ULK1 complex or ATG929. By these actions, omegasome is formed from the ER, from which a bag-shaped isolation membrane is developed16.Phosphatidylinositol 3-phosphate (PI3P), generated by PI3K, is a phospholipid that plays an important role in the control of membrane trafficking. While yeast has only one PI3K (Vps34), mammalian cells have several PI3Ks, of which class III PI3K is involved in autophagy. This is composed of the complex containing ATG14 or UVRAG in addition to VPS34, VPS15, and Beclin 1 (VPS30 homolog)57(reviewed by N. Mizushima in detail58). This reaction is carried out via two pathways. The first pathway is a modification of the ubiquitin-like protein ATG12, which is covalently bound to ATG5 via the action of the E1-like enzyme ATG7 and the E2-like enzyme ATG10. Most ATG12 proteins exist in a form bound to ATG5. The ATG5-12 complex binds to ATG16. The ATG5-ATG12-ATG16 complex functions as an E3-like enzyme in the last step of another ubiquitin-like reaction to facilitate modification of LC3 with PE. The ubiquitin-like protein LC3, a homolog of the budding yeast Atg8, is first cleaved by the ATG4 protein at the C-terminus to expose the Gly residue. Through sequential reactions of ATG7 (E1), ATG3 (E2), and the ATG5-ATG12-ATG16 complex (E3), PE is added to the C-terminal glycine residue of LC3 to target it to the isolation membrane.The regulation of autophagosome formation and the subsequent events are governed by a characteristic reaction involving the post-translational modification of the Atg8 homolog LC3 with phosphatidylethanolamine (PE). It is mediated by a series of ubiquitin-like modification enzymes60, movement of the autophagosomes64, fusion with the lysosomes and degradation of the inner membrane of the autophagosomes68. This molecule is also needed for the binding to adapter proteins in selective autophagy. LC3 homologs are mainly classified into the LC3 subfamily and GABARAP subfamily. Knockdown of the Atg8 homologs of each subfamily with siRNA reduced autophagic activity, which suggests some functional segregation69. Moreover, it is recently shown that LC3 subfamily and GABARAP subfamily have opposite roles for regulation of autophagosome formation via binding to ULK1 and ATG1370.Membrane-bound LC3 with PE functions as a key molecule that controls critical steps in the molecular mechanism of autophagy: autophagosome closure71. LC3 is the best-known marker used to monitor the formation of autophagosomes by microscopic observation as well72. The use of fluorescent-tagged LC3 enables the observation of autophagosomes in living cells. Specifically, a tandem-tagged LC3 with both GFP and RFP has been developed to distinguish autophagosomes from autolysosomes, which is critical to determine the state of autophagic induction or inhibition. In the case of lysosomal inhibition, LC3 dots accumulate, which cannot be distinguished from elevated levels of LC3 due to autophagic induction. Because the GFP signal is quenched in lysosomes due to its pH-sensitive nature, RFP-GFP-LC3 shows green plus red for autophagosomes and red with no green signal for autolysosomes73 . Thus, a series of methods using LC3 as a marker for autophagosomes has substantially advanced research on mammalian autophagy71.While other ATG proteins are located outside the isolation membrane and move away from the membrane after autophagosome formation, LC3 is located both outside and inside the isolation membrane and stays within the autophagosome. Therefore, LC3 itself is degraded by autophagy, making it a useful substrate to measure autophagic activity. As LC3-I (without PE) and LC3-II (LC3-PE) can be easily separated from each other by SDS-PAGE, an autophagic flux assay using LC3 is widely used as a standard method to measure autophagic activity61. It is mediated by a binding of LC3 with FYVE and coiled-coil domain containing protein (FYCO1), which tethers autophagosomes with kinesin and microtubules to facilitate the plus-end direction of movement 64. The movement of autophagosomes in the perinuclear direction is mediated by the binding of autophagosomes with dynactin and dynein via Rab7, ORP1L, and RILP63. Interestingly, a lack of ORP1L caused decreased recruitment of the components required for the following fusion event described below to late endosomes or lysosomes63, suggesting that the movement and the fusion are not separated from each other but are instead regulated as a sequential event. To date, researchers have shown that SNARE complexes containing Stx17 and the HOPS complex are required for the tethering of autophagosomes with lysosomes to facilitate the formation of autolysosomes74. This process is also under the control of the abovementioned PI3P production by PI3K. Beclin 1/VPS34/VPS15 with UVRAG functions as a mammalian PI3K complex working at the fusion step. Interestingly, PI3K with UVRAG is controlled by binding to a protein called Rubicon75. Rubicon knock-down results in an upregulated autophagic flux and a promotion of the fusion process, suggesting that Rubicon is a negative regulator of the fusion step. Rubicon itself is regulated by other factors, including mTOR, indicating that it is a key regulatory factor of autophagy whose deregulation may cause deleterious effects in pathology78. Indeed, Rubicon protein showed excessive accumulation in fatty liver induced by a high-fat diet, and a genetic abrogation of Rubicon suppressed diet-induced liver diseases, suggesting that abnormal accumulation of Rubicon may be a cause of these diseases79. The tethering and fusion of autophagosomes and lysosomes are mediated by two regulatory factors, EPG5 and INPP5E, which are known to be responsible for two human hereditary disorders, Vici syndrome and Joubert syndrome, respectively. EPG5 is present in late endosomes and lysosomes, facilitating the fusion of both by promoting the assembly of the SNARE complex, which is required for autophagosome-lysosome fusion80. INPP5E is a phosphatase that converts PIP2 to PI3P. The downregulation of INPP5E resulted in a reduction in the autophagic flux associated with an accumulation of LC3 dots and reduced colocalization of LC3 with lysosomes, showing that INPP5E facilitates autophagy via regulation of autophagosome-lysosome fusion82. Consistent with the fact that actin polymerization facilitates autophagy17, PIP2 regulates the endosomal actin dynamics by antagonizing cortactin binding to actin filaments83, and reduction in cortactin decreased autophagosome-lysosome fusion84, downregulation of INPP5E resulted in reduced activation of cortactin on lysosomes. This evidence suggests that the conversion of PIP2 to PI3P by INPP5E on lysosomes may increase the stability of actin filaments in a cortactin-dependent manner, promoting the fusion of autophagosomes with lysosomes.Not only autophagosome formation but also the subsequent fusion with lysosomes is controlled in a highly orchestrated manner. The fusion event requires physical contact of both organelles, and autophagosomes dynamically travel throughout the cytosol because lysosomes are predominantly generated at a peripheral region of the nuclei, while autophagosome formation sites are not restricted to the area where lysosomes are present. Inhibition of LC3 by injection of an LC3 antibody prevented the movement of autophagosome, suggesting that LC3 is needed for this event85. To date, over 150 types of tissue-specific autophagy-related gene knockout mouse models have been generated, suggesting the important roles of these genes in maintaining homeostasis in each tissue. Moreover, studies on mice with an Atg5 homozygous deletion in a mosaic throughout the body86, tamoxifen-induced knockout mice after birth87, and mice that express Atg5 only in the nervous system88 suggest a comprehensive role for autophagy throughout the body. These findings emphasized that autophagy ensures the normal development of tissues and the maintenance of homeostasis of differentiated tissues, and the failure of this process leads to dysfunction of each organ in mammals.The physiological significance of autophagy has been investigated using various model animals, including mice, zebrafish, fruit flies and nematodes. In the case of mice, these studies are mainly performed using models in which the genes essential for autophagy are specifically knocked out for each tissue because knockout mice with a systemic homozygous deletion in these genes show embryonically lethal or postnatal lethalityWhat about its role in human beings? Direct evidence has been provided by human hereditary diseases. These diseases are caused by hypomorphic mutations in the genes essential for autophagy or loss-of-function mutations in genes related to but not essential for autophagy. No genetic disease linked to a lack of both alleles of genes essential for autophagy has been reported to date. This phenomenon is most likely because autophagic deficiency affects the survival of human individuals, as shown in the results of embryonic lethality or postnatal lethality in mice with systemic homozygous deletions in these genes. Here, we will summarize the features of each disease and discuss the physiological role of autophagy in human health.89. Most SENDA is sporadic, and thus, de novo mutations are expected to be responsible for its symptoms. Saitsu et al. conducted an exome analysis of two SENDA families and identified mutations in WDR45 (WIPI4), a human homolog of Atg18, one of the core genes essential for yeast autophagy52 , the accumulation of protein aggregates and the multi-layered membrane-like structures were observed, suggesting the role of WIPI4 in autophagosome formation in vivo92. A recent study showed that Wdr45 knockout mice have increased neuronal apoptosis due to an accumulation of ER proteins, which was associated with an increase in the unfolded protein response (UPR)93. An investigation of the link between ER stress and autophagy indicated that ER stress can be one of the cues that induces autophagy94.Neurodegeneration with brain iron accumulation (NBIA) is a rare degenerative disease  characterized by iron deposition in the brain, and SENDA is one of its subtypes. Psychomotor development is delayed early in childhood. Motor function develops to some extent, but dystonia, parkinsonism, and dementia progress rapidly in adolescence or early adulthoodWIPI2 results in a defect in LC3 lipidation and the associated autophagosome formation, WIPI2 may have a distinct function in autophagy among human Atg18 paralogs54. Recently, individuals with a homozygous mutation in WIPI2 were reported to show developmental abnormalities and mental retardation53. Exome sequencing of two affected individuals identified the G745A mutation in exon 9 of the WIPI2 gene, which leads to a V249M amino acid change in the gene product. This mutation causes reduced binding activity of WIPI2 with ATG16L1, which is known to facilitate recruitment of ATG16L1 to the site of autophagosome formation and thus promote LC3 lipidation and autophagosome biogenesis50. The affected individuals analyzed for exome sequencing were aged 43 years and may have residual autophagosome biogenesis activity, as fibroblasts from the patient had mild but still induced LC3 lipidation activity in response to a starvation signal. The individuals with the mutation showed a complex developmental disorder including mental retardation, speech and language impairment, and cardiac, neurological, thyroid and skeletal abnormalities. Characteristic features of the disease are severe mental retardation associated with global brain volume loss and short stature. Cerebellar dysfunction is suggested, as fine/gross motor activities are delayed in patients. Cardiac abnormalities shown by electrocardiograms were observed53. The clinical features were similar at some degree in the patients with the WIPI2 mutation and those with SENDA although the patient with the WIPI2 mutation did not show iron deposition, a characteristic feature of SENDA.Since a loss of ATG5E122D mutation. Exome analysis of two families with congenital ataxia (ataxia), intellectual disability, and growth retardation identified the chr6: 106,727,648 T > A mutation, a missense mutation that causes an E122D change in the ATG5 protein95. ATG5 is covalently bound to ATG12 to form a heterodimer of a ternary complex with ATG16, which promotes lipidation of LC358. Since the ATG5E122D mutation weakens the binding to ATG12 and decreases PE formation of LC3, basal autophagic activity was reduced in the LCLs derived from patients, and accumulation of p62 was observed95. These data showed that ATG5E122D is a hypomorphic allele whose homozygous mutation results in reduced but residual autophagic activity. The reduction in autophagy is most likely sufficient to support survival after birth but insufficient to maintain homeostasis in the CNS and suppress the symptoms of this condition. In contrast to SENDA, this disease did not show significant cerebral atrophy. Intriguingly, while cerebellar atrophy is a characteristic feature of the patients ATG5E122D, it is not a major feature of SENDA as nearly one third of the patients of SENDA show the symptom89. This controversy might be explained by the tissue-specific function of the WDR45 gene or genetic diversity (see below)96. No iron deposition in the cerebral area of the patient was reported.Mutations in the genes essential for autophagy among the core autophagy-related genes have not been shown to be related to human diseases until the identification of the EPG5 gene, which has a regulatory role in autophagy at the fusion step between autophagosomes and lysosomes97. This condition affects not only the corpus callosum but also a wide range of organs and can include cardiomyopathy, cataracts, depigmentation, and combined immunodeficiency. Ectopic P-Granules Autophagy Protein 5 (EPG5) is a metazoan protein identified by screening Caenorhabditis elegans, and its homolog has not been found in yeast98. Systemic knockout mice that lack both alleles of Epg5 are born according to Mendelian inheritance and survive after birth99. This phenotype is clearly distinguished from those caused by the core autophagy genes, such as Atg5 and Atg7, which include neonatal lethality due to defects in neurological activity related to sacking defects and survival with starvation after birth101. This difference is probably due to the weak remaining autophagic activity that mitigates the phenotype in the Epg5 knockout mice99. The knockout mice began to show motor dysfunction after approximately 4 months and then showed symptoms similar to amyotrophic lateral sclerosis (ALS), such as muscle fiber atrophy and progressive paralysis99. Notably, the Epg5−/− mice showed only some features that are observed in patients with Vici syndrome. The mice showed agenesis of the corpus callosum and muscle atrophy but lacked several key symptoms of Vici syndrome, including facial dysmorphism, cataracts, and hypopigmentation. Additionally, Epg5 knockout mice can grow and reach sexual maturity in contrast to those with Vici syndrome, in whom death occurs at a median age of 42 months102. This observation that abrogation of EPG5 causes a less severe phenotype in mice than human support a speculative idea that human might depends on autophagy more than mice to maintain tissue homeostasis.Vici syndrome is an autosomal recessive disorder caused by mutations in the 80  loci have mutations in the INPP5E gene104. INPP5E is an enzyme that generates PI3P by dephosphorylating the 5-position of PI P2, which is a kind of inositol phospholipid (PIP). As described above, PI3P plays an important role in autophagosome formation and autophagosome maturation. Screening for factors related to autophagy among the factors involved in the metabolism of PIPs showed that INPP5E was required for autophagosome maturation82  gene-specific roles other than autophagy, (2) genetic diversity and (3) tissue specificity of each gene.The characteristics of symptoms in the CNS also vary. SENDA is characterized by iron deposition in the CNS, which is not found in the other four diseases. While agenesis of the corpus callosum is typically found, and atrophy of cerebrum is not a major feature of the patients with Vici syndrome, patients with the 108. Deletion of FIG4, which facilitates the production of PIP2, resulted in a depletion of PIP2 and developmental defects including neurological abnormalities. Its homozygous mutation has been identified as a cause of Yunis-Varon syndrome, which is characterized by structural brain abnormalities and skeletal abnormalities109. Mutation of FIG4 was also found in mutant mice called “tremor mice” that show a severe tremor and abnormal gait, as well as in patients with the hereditary condition Charcot-Marie-Tooth disorder110. Therefore, it is likely that a defect in endocytosis may cause an additive effect on brain development, resulting in the complex symptoms found in Vici syndrome and Joubert syndrome. This phenomenon may also be the cause of symptoms in organs other than the brain, such as the immunological abnormalities in Vici syndrome and the liver disease in Joubert syndrome that are not found in SENDA or patients with ATG5E122D mutations. Notably, ATG5 also has roles in processes other than autophagy, such as cell death and pathogen resistance112, which could contribute to the symptoms of patients with the ATG5E122D mutation to some degree.Regarding (1) the gene-specific roles other than autophagy, the Vici syndrome-related protein EPG5 has a role in the regulation of endocytosis, which has a critical function in neural development and neurons. Similarly, the Joubert syndrome-associated protein INPP5E catalyzes the synthesis of PI3P from PIP2, as PIP2 and PI3P have physiological functions other than autophagy, including the regulation of endosomal function113 and the formation of isolation membranes19, is known to be mutated in Charcot-Marie-Tooth neuropathy type 2A114. Multiple genetic variations in this wide range of factors may contribute to the differences in severity and tissue specificity. We expect that genetic variation in other genes may be related to the symptoms but independent of autophagy. For example, since genetic loci of BECN1 (encoding beclin 1) is close to a well-known tumor suppressor BRCA1, large deletions including both loci are found in the cancer genome115.Regarding the #2 possibility, genetic diversity including quantitative trait loci (QTLs) is expected to contribute to variations in symptoms because autophagy is a multistep event requiring several different kinds of biological pathways. Membrane trafficking, including endocytosis, is a major contributor to autophagy because it regulates the maturation of autophagosomes. Genes involved in mitochondrial biogenesis, lysosomal biogenesis, and hormonal regulation are required for the proper regulation of autophagy. For instance, MFN2, a mitochondrial dynamin-related protein that is required for the tethering of the mitochondria with the ERWdr45 and Wdr45b showed neonatal lethality, while each single knockout mouse was born normally, suggesting functional overlap between the genes96. Double knockout mice showed an increased accumulation of p62 aggregates in many brain regions and neurons, which was associated with suckling defects similar to those of Atg7 knockout mice100. Interestingly, these accumulations of the p62 aggregates were not detected in the liver and kidney in double knockout mice, suggesting that both genes may not be essential for autophagy in these tissues96. Therefore, the functional contribution of each autophagy-related gene may vary in different tissues.For the #3 possibility, the tissue specificity of each gene may also contribute to the symptoms. Mice with double systemic homozygous deletions in both In summary, the shared feature of dysfunction in the CNS in various diseases highlights the critical role for autophagy in the maintenance of homeostasis in neurons for CNS function. However, the possibilities shown above  suggest that some abnormalities found in Vici syndrome, such as cataracts and severe immunological abnormalities, and Joubert syndrome, such as renal disease and liver disease, might be due to combined defects in autophagy and endocytosis, as well as other unidentified genetic factors(s). A comprehensive study using humanized mouse models for each disease with a defined genetic background may help elucidate the role of autophagy in the suppression of these diseases and human physiology as well.117. Due to the prosurvival effect of autophagy on tumor cells, tumors arising from these mice usually do not develop into malignant tumors. However, these findings do not necessarily indicate that autophagy is dispensable for tumor suppression. Rather, an irreversible genetic abrogation of the genes essential for autophagy in these model systems clearly show both side of autophagy in tumorigenesis118. Nonetheless, the human genetic disorders with mutations in autophagy-related genes described here do not show a significant increase in any sign of neoplasm formation. Do these findings indicate that autophagy may not be important for tumor suppression in humans? Evidence from cell biology using human cells clearly shows that autophagy is required to suppress the genomic alterations that promote tumorigenesis. Autophagy promotes genomic integrity through the control of cellular senescence via both positive and negative regulation. Promoting cellular senescence in response to oncogenic stress119, autophagy also suppresses senescence by selective degradation of the transcription factor GATA4 in response to DNA damage120. Autophagy also maintains stemness of muscle stem cells by suppression of senescence caused by excess ROS production121. Alternatively, autophagy triggers cell death in response to telomeric DNA damage during replicative crisis122. These lines of evidence suggest that autophagy suppresses tumor-promoting genomic alterations in various processes. Therefore, we hypothesize that the controversy between human symptoms and the phenotypes of mice with tissue-specific, irreversible deletions in autophagy genes is most likely due to a major contribution of autophagy to the maintenance of the CNS in humans, which is vital for survival. Simply, a reduction in only a fraction of the CNS may immediately cause a serious defect, in contrast to carcinogenesis, which requires a long period to acquire multiple mutations for the development of a malignant tumor. Indeed, a mouse model with temporal systemic inhibition of autophagy followed by its restoration supports this idea. The mice with reduced expression of Atg5 by dox-inducible siRNA after birth expressed reduced viability (~6 months) and various signs of accelerated aging due to systemic degradation of tissue homeostasis. Interestingly, the restoration of autophagy in mice partially restored the shortened lifespan but accelerated the formation of spontaneous tumors123. We speculate that a systemic decrease in autophagy becomes a serious threat to survival, especially due to a defect in the CNS that precedes the formation of neoplasms, but a temporal but severe decrease in the autophagy of cells in a limited area, such as the damaged liver, may cause genomic alterations that facilitate tumorigenesis.Several studies of tissue-specific knockout mice have identified a tumor-suppressive role for autophagy in many tissues, including the liver, lung, and pancreas124, indicating that autophagic activity in the host supports cancer cell viability125. As circulating arginine is degraded by the arginase ARG1, which is released into blood flow due to liver damage in autophagy-deficient host mice, tumorigenesis in mice with autophagy knockout in each tissue may differ from mice with systemic loss of autophagy. Alternatively, autophagy in loser cells supports their elimination during cell competition126, which may cause selection pressure toward an expanded population of autophagy-deficient cells in a microenvironment involving heterogeneous autophagic activity, including model mice generated by an approach involving Cre recombinase. These non-cell autonomous factors need to be taken into account to determine the effect of systemic changes in autophagy in future studies.A complex tumor environment and the heterogeneity of cancer cells must be considered for the role of autophagy in tumorigenesis. Autophagy not only suppresses genomic instability or conversely supports cancer cell survival in a cell-autonomous manner but also controls tumor cell status in a non-cell autonomous manner. Autophagic activity in host mice is required for the maintenance of circulating arginine, which supports the growth of allografted cancer cellsIn summary, autophagy suppresses tumorigenesis but, once cancer has developed, it promotes tumor malignancy. Thus, to interpret the effect of autophagy deficiency in animal models and pathology, it is essential to consider the effect of autophagy defect in non-cancerous and cancer cells independently. On top of that, it should be taken to account that there exists a non-cell autonomous effect of autophagy in multiple organs and tumor microenvironment.67. These structures could be fused with a lysosome. Thus, it is suggested that the low residual autophagic activity observed in mice with knockout of the genes involved in LC3 lipidation could be sufficient to support embryonic development, although complete defects in autophagy result in embryonic lethality85. Therefore, hypomorphic alleles of the upstream factors could be identified as the cause of a hereditary disease with severe symptoms. Alternatively, similar to becn1+/− mice, which exhibit a high carcinogenic phenotype128, those with heterozygous mutations with haploinsufficiency may show some aspects of autophagy deficiency.Knowledge of the human genetics of autophagy is rapidly expanding as hereditary disorders with autophagy deficiency have been identified in the past decade, and we expect that many hereditary diseases for which other autophagy-related genes are responsible remain to be identified. We expect that the genes identified in the future will likely be biased. At present, genes encoding essential upstream regulatory factors for autophagy, such as FIP200 and ATG9, have not been associated with human hereditary diseases. This result could be partially due to functions specific to each gene, but data from cell biology have supported other possibilities. FIP200, ATG9, and the PI3K complex have essential roles in the formation of isolation membranes. However, cells deficient in the genes involved in LC3 lipidation, such as ATG5 and ATG12, show a small number of autophagosome-like structuresOPTN is known as one of the causative genes of ALS129. To date, the role of OPTN in autophagy is focused on the autophagy receptor, which helps sequester ubiquitinated substrates by isolation membranes via binding to both LC3 and ubiquitin131. However, recent reports have indicated that OPTN knockout cells have decreased autophagosome formation induced by starvation132. SQSTM1, a gene encoding p62, is one of the best-investigated substrates for autophagy. While it functions as a receptor protein to tether LC3 with targets for selective autophagy, several studies have suggested a possible involvement of p62 in autophagosome formation itself as well133. Interestingly, homozygous mutations in SQSTM1 were identified as a cause of human neurodegeneration with ataxia and other associated symptoms134. Of course, symptoms of the patients or phenotypes of model mice are expected to result from a combination of autophagy deficiency and aberrations other than autophagy, so it is requisite to carefully estimate the contribution of autophagy for suppression of each disease. To date, there are several powerful tools to estimate autophagy in vivo such as GFP-LC3 mice, GFP-LC3-RFP-LC3ΔG mice, and mt-keima mice137. However, it remains technically difficult to estimate autophagy activity in human. Moreover, a cavity of the model systems is that markers of autophagosome do not always necessarily indicate autophagy in vivo; LC3 is related to other events such as LC3-associated phagocytosis (LAP)138. Therefore, interpretation using both the animal models and the approach of cell biology is necessary to understand the role of autophagy in pathology.Since the molecular mechanism of autophagy has not been fully clarified, it is predicted that there are many unidentified factors related to autophagy. In future studies, the factors related to hereditary disorders could be recognized as autophagy-related disorders. One example is optineurin (OPTN), which is involved in selective autophagy. 79. In addition, autophagic activity gradually decreases as aging progresses and increased basal autophagy extends lifespan and healthspan in many organisms including mice141. Our group recently showed that the Rubicon protein level increases with aging and that Rubicon knockout reduces various diseases associated with aging142. In addition to the genetic diseases outlined here, determination of the pathology and molecular mechanisms of the changes in autophagic status in lifestyle-related diseases and the aging process is needed to develop novel strategies for human healthcare. In addition to basic research to elucidate the mechanisms of autophagy, further studies on these topics are needed in the future.The expansion of autophagy-related diseases may increase the demand for therapy. More strikingly, there are many lifestyle-related diseases with autophagic abnormalities. For example, autophagy is decreased in the fatty liver caused by excessive nutrition. This phenomenon is mainly due to the accumulation of Rubicon protein, which is a negative regulator of autophagy, and Rubicon knockout mice showed reduced hepatocyte cell death and fat accumulation associated with fatty liver