Haemorhous mexicanus) showed increased infection from a common coccidian endoparasite (Isospora spp.) when feeders were left uncleaned and that daily cleaning (with diluted bleach solution) over a 5-week period successfully decreased parasite burden. Moreover, this pattern in rural finches was true for males but not females. These experimental results reveal habitat- and sex-specific harmful effects of bird feeder use . Our study is the first to directly indicate to humans who maintain feeders for granivorous birds that routine cleaning can be critical for ensuring the health and viability of visiting avian species.Animals inhabiting urban areas often experience elevated disease threats, putatively due to factors such as increased population density and horizontal transmission or decreased immunity . However, for animals that take advantage of human food subsidies, like feeder-visiting birds, an additional mechanism may include exposure to contaminated feeders as fomites. There are some published associations between bird feeder presence/density and avian disease, but to date no experimental study has tested the hypothesis that feeder contamination can directly impact disease status of visiting birds, especially in relation to the population of origin . Here we used a field, feeder-cleaning experimental design to show that rural, but not urban, house finches ( And despite the fact that some human activities may have apparent benefits to either humans or wildlife 2, in some cases they carry hidden costs, which may outweigh the benefits.Humans impact wildlife in many indirect and direct ways, and these effects can be both positive and negative. Given the recent extent and rate of human expansion across the globe, many are concerned about the range and severity of harmful anthropogenic effects, including pollution, habitat alteration, and climate change4. Human resource subsidies in gardens and yards provide animals with abundant, predictable foods5 and deepen the wildlife knowledge and appreciation of many residents who feed them6. However, these food supplies  may also elevate predation risk8  or alter nutritional/energy balance10. Disease susceptibility is also touted as a putative elevated risk of animal feeding11, although the mechanism(s) by which this may occur are uncertain12. Horizontal disease transmission may increase if feeding stations elevate population density and/or individual-to-individual contact rates13. Alternatively, feeders may serve as fomites14 . Animals can leave disease-contaminated materials  on feeder surfaces that, if not cleaned, can foster and exacerbate transmission of blood-borne pathogens, endoparasites, and other microbes.Supplemental feeding of wildlife may be one such activity15, but outside of mentions in guidelines for feeding birds 16, data supporting the sanitary value of cleaning wild bird feeders  have been virtually absent from the literature to date17. Here we undertake, to our knowledge, the first field experiment to determine if feeder soiling or cleaning has direct impacts on avian disease. We performed this experiment with house finches (Haemorhous mexicanus), a popular backyard-bird subject for the study of avian disease dynamics19. Because bird feeders are typically much more concentrated in urban areas20, and urban animals can have higher pathogen and parasite burdens21 (including in our study species22), we also assessed the extent to which feeder soiling or cleaning treatments might differentially impact bird disease in urban v. rural areas. We conducted a 10-week experiment, in which we offset 5-week experimental rotations of active feeder cleaning (i.e. daily with a dilute bleach solution) and soiling (i.e. not cleaned) at an urban study site and a natural desert site and measured changes in a common avian disease—coccidiosis—due to its fecal–oral transmission route on soiled surfaces22. We predicted that disease levels would rise during periods when feeders were not cleaned, would fall during sanitization periods, and that, because of comparatively rare presence of bird feeders in rural areas, rural birds may be relatively more impacted by the extent of feeder soiling.This raises the prospects of whether or not extensive soiling of feeders is responsible for elevating incidence or severity of certain pathogens and parasites, and whether or not routine feeder cleaning may be an effective means of reducing disease risk at feeding sites. Hummingbird feeders notoriously require regular sanitization due to sugar spoilage22. In our previous work at these representative sites, we have shown consistent urban–rural phenotypic differences in these finch subpopulations, especially for plumage color expression24 and disease prevalence/severity22, although we recognize that studies at additional urban and rural sites would permit broader urban-ecological generalizations to be made.We studied house finches at one urban (Arizona State University—Tempe campus) and one rural  site. These habitat categorizations were determined previously based on land-use and land-cover attributes as well as human population density within 1 km of the trapping siteOn 15 June 2017 , we placed 5 plastic yellow 1.8 kg bird feeders  filled with black oil sunflower seeds at each site. Thereafter, we conducted a 10-week “feeder-cleanliness” experiment, which we broke into two 5-week periods of dirty or clean feeders. Specifically, at our urban study site, for the first 5 weeks we cleaned the feeders daily with a 10% bleach solution before rinsing, drying, and re-hanging in the field; in the last 5 weeks of the study, we left the urban feeders uncleaned. At our rural study site, we implemented the same treatments but in the opposite order; we left feeders uncleaned for 5 weeks and then cleaned them as above for the last 5 weeks. We employed this offset rotation to remove any seasonal confounds, although without a true control/reference sample, where we examined seasonal changes in coccidia per se, we cannot rule out the possibility that there were temporal confounds that affect the interpretation of our findings. Feeders were filled every other day; all feeders contained seeds at all times.25, banded each bird for individual identification, determined age and sex when possible using plumage characters26, measured body mass , and transported them in small brown paper bags within an air-conditioned vehicle to obtain fecal samples (see below). We trapped on 1–3 days per week, with the aim of capturing up to 10 newly captured (i.e. unbanded) and sexable birds/site/week. Finches were housed in an indoor vivarium at the Arizona State University—Tempe campus upon return from the field site until 1600 h., at which point we began fecal collection (due to the diel cycle of oocyst shedding by Isospora spp.27). This caging procedure is standard for fecal coccidian analyses in wild birds28, and it is not expected that very brief captive housing alters parasite oocyst shedding (i.e. time course of coccidia changes is on the order of days30), although this has not been tested directly in house finches. Birds were housed individually in small wire cages with ad libitum access to sunflower seed and water in the same indoor rooms (kept at 25 °C). At 1600 h we replaced paper at the bottom of the cage with fresh sheets, and at 1700 h, we returned to the room to retrieve the paper and scraped fresh fecal samples from each bird into a solution of 2.2% potassium dichromate22. The birds were then placed into their brown paper bags and released at their capture site before sunset. Methods for oocyst isolation and microscopic analyses follow those in Brawner et al.27; briefly, we transferred the sample contents into a fresh 9 mL culture tube and filled it to the top with Sheather’s sugar solution . We placed a microscope coverslip atop the convex meniscus of the solution on the tube mouth and centrifuged for 5 min at 3000 RPM. We then transferred the microscope slip to a microscope slide, and two scorers examined the slide under a light microscope (Olympus BX60) at × 40 magnification with constant  microscope settings and ranked oocyst load (i.e. degree of infection) based on a 0–5 scale27. We thus had individual-level values of infection (i.e. coccidia scores31) that we could use to analyze changes in both the presence and severity (degree of infection) of coccidiosis over time. We did not include birds in this study who showed signs of poxvirus infection, and birds in our study populations do not have mycoplasmal conjunctivitis32.We captured finches (from 0500 to 0900 h) using standard walk-in basket traps33, with α = 0.05. We developed models that allowed us to test, within each study site: (1) if and how coccidia infections changed over time when birds were held on each of the 5-week clean- or dirty-feeder treatments, and, (2) because of potential sex differences in disease34, if and how males and females responded differently to these treatments. We focused on temporal change because our focal prediction was that daily sanitization during the ‘clean’ treatment, or accumulated soiling during the ‘dirty’ treatment, would progressively improve or reduce, respectively, bird health state. Specifically, we built generalized linear models35 , separately for our rural and our urban site and for our two measures of coccidiosis (severity and presence), that contained sex, feeder treatment, and time since the feeder treatment began as our main effects and included all possible interaction terms. For the coccidia-presence models, we used logistic regression with a binary response variable (presence/absence of coccidia). We conducted post-hoc tests when variables were statistically significant, with special attention to higher-order terms containing time since treatment began; when a term with this variable was significant (e.g. sex × treatment type × treatment duration), we used either univariate generalized linear models (for coccidia-severity models) or univariate logistic regression (for coccidia-presence models) to test the hypothesis that the slope of the line differed from zero. We also ran Tukey’s post-hoc tests to compare line slopes between the sexes and within the treatments, to determine if disease state changed differently over time when males and females fed from clean v. dirty feeders. We initially included age (hatch-year v. after-hatch-year) as a variable in the models, but no terms containing age (i.e. either as a main effect or in interaction terms) were significant, so we excluded age as a variable in final analyses , which is likely due to adjustments for family-wide error rates.We also found significant effects of the sex × treatment and sex × treatment × time interactions on disease presence in urban birds Table . The slo37, urban and rural birds differ significantly in their exposure to and use of supplemental feeders, we predicted that rural birds would be comparatively more impacted by the introduction of soiled and cleaned feeders. We found intriguing habitat- and sex-specific effects of a daily bleach feeder cleaning treatment on coccidiosis in house finches. Namely, rural males became significantly more infected with endoparasitic coccidians when feeding from uncleaned feeders and significantly less infected when feeding from clean feeders; however, we found no such effects in urban males or in females from either site. Overall, these results support the health-beneficial effects of routine bird feeder cleaning but only in a specific cohort of birds .We performed the first field experimental test of the effectiveness of bird-feeder cleaning on avian disease transmission. Using a widespread backyard bird species from North America (the house finch), we also considered differential effects of feeding cleanliness on disease in urban v. rural birds. Based on the premise that, among many other traits38 and that feeders themselves can serve as active fomites for disease transmission . Also, various organizations  recommend on their websites that feeders be cleaned in some fashion  and with frequencies ranging from ‘regularly’ to biweekly. However, it has largely been unclear how effective these methods are for any particular infectious agent, bird species, sex or age class, or feeder surface/location. To our knowledge, there are only two studies that have tested effectiveness of bird feeder cleaning methods on microbial communities. Boyd et al.39 found that 10% bleach wipes reduced numbers of aerobic, but not gram-negative, bacterial colonies on feeder surfaces, but that such cleaning had little impact on rates of seed consumed by birds from those feeders . Feliciano et al.17 found that several forms of single-instance cleaning  were effective at reducing Salmonella colonies on feeders, but that more thorough cleaning procedures  are needed when debris has accumulated on a feeder. However, in both instances, the efficacy of the cleaning methods was not directly tested by monitoring disease state in feeder-visiting birds. Here we used a daily bleach-scrub and water-rinse protocol to clean feeders and effectively reduce gut coccidian loads in rural male house finches. We chose to use this frequent, more liberal method in order to ensure daily cleanliness and broad anti-microbial effectiveness, but it will be interesting now to see if different  or more conservative (i.e. weekly) procedures could also be effective in combating disease in feeder-visiting birds.There are many literature suggestions about the value of cleaning bird feeders, but none to date have come with rigorous experimental support. For example, extensive work on mycoplasmal conjunctivitis in house finches has shown that birds who visit feeders are more likely to be infectedlower coccidia levels as they fed more from a soiled feeder. Why might disease state in the two sexes have responded differently to feeder cleanliness? We originally suspected a stronger effect of our treatments in comparatively naïve rural birds (i.e. which may not commonly feed from feeders and that permit them to aggregate in high density), but one idea for why females were not affected may be attributed to female social dominance in this species. Female house finches displace males at feeders40 and thus presumably gain access to preferred feeding locations. Moreover, because house finches from their native range show significant aversions to feeding near sick conspecifics41, it is possible that females also choose to feed at feeder microsites that reduce their disease risk . This would leave them comparatively less impacted by feeder cleaning efforts, and instead males would be more likely to be left to feed on riskier, soiled surfaces. It will be interesting now to test whether females do in fact show such clean-microsite feeding preferences .The fact that the effect of feeder cleaning on disease was limited to just rural males was surprising. In fact, in one instance, we found that urban females had significantly 24. However, it was recently found that urban female house finches are less severely infected by coccidia than rural females42. This opposing pattern between males and females suggests complex disease dynamics in urban areas , such that urban females do not necessarily spread or acquire more coccidia from feeders simply because, compared to rural birds, they feed more from feeders. Immune responsiveness in songbirds, for example, can differ between sexes43 and in relation to urbanization44. Last, because a portion of our experimental study overlapped with the late breeding season of house finches in Arizona and because we did not conduct routine feeder observations of bird visits, we also cannot rule out the possibility that males and females visited feeders differently  due to their separate breeding roles. For example, male house finches provide greater nutritional provisions to older nestlings and fledglings45, including accompanying them at feeders . Surprisingly, to date, there is little information across feeder-visiting bird species whether the sexes differ significantly in feeder use, but consistent with this some have found (including in house finches) that males tend to visit feeders more than females46, although females tend to have longer visitation bouts47. This now seems like a profitable avenue to pursue, both as it relates to our disease findings here but also generally to improve our understanding of the sex-specific foraging ecology, nutritional physiology, and even predation risks7 of feeder-visiting species.An alternative to this environmental/behavioral explanation is one centered on physiological/immunological differences. In prior work on these finch populations, researchers have shown that males are consistently more colorful and healthier (including having less coccidiosis) in rural compared to urban areas14. Our study also has broad implications for wildlife health and management, where bird feeder cleaning has been recommended by many but without clear guidance on protocols or expected effects. We uncovered no evidence for benefits of feeder cleaning in urban areas, suggesting possible adaptive co-evolution of birds with feeders and feeder-transmitted disease and that feeder cleaning here may not be as critical, although further work is needed to broadly test this idea among cities, species, seasons, and perhaps including community-wide feeder-cleaning intiatives. We urge others to also consider this conservation practice as it relates to their wild animal model, the pertinent diseases, and other transmission-risk factors, such as feeder-surface type, geographic location, and time of year.In sum, we show conditional  field experimental evidence that feeder soiling and cleaning can have significant impacts on endoparasitism in a common desert and urban passerine species. This result is consistent with suggestions from prior studies on feeder transmission of a different bacterial disease (mycoplasmosis) in house finches, such that feeder surfaces themselves can play a key role in disease transmission


Phylogenetic analysis showed that HTLV-1 samples belonged to the Cosmopolitan subtype Transcontinental subgroup. From a cohort of 1,000 individuals, no new case of HTLV infection was detected. Although the prevalence rate of HTLV infection found in this study was similar to that observed in the Brazilian general population, the lack of access to preventive interventions and harm reduction measures all contribute to increasing the risk of HTLV transmission and acquisition among this key and vulnerable population.Human T-lymphotropic virus (HTLV) infection is endemic in some group populations. Prisoners are at high risk of acquiring HTLV infection mainly due to the environment of closed penal institutions, socioeconomic conditions, and risk behaviors. This study aimed to investigate the prevalence, the occurrence of new cases of HTLV infection after a 1-year follow-up, and circulating subtypes of HTLV-1 among prisoners from twelve closed prisons in Mato Grosso do Sul state, Central Brazil. A total of 1,507 prisoners were randomly enrolled in the cross-sectional study. Out of the 1,507 participants, 1,000 prisoners, susceptible to HTLV infection, were included in the prospective cohort study. In the cross-sectional study, serological evidence of HTLV infection was 0.4% (CI 95%: 0.1-0.7), tested for anti-HTLV antibodies by enzyme-linked immunosorbent assay (ELISA). Three samples were positive for HTLV-1, two samples were positive for HTLV-2, and 1 sample was indeterminate by the Western blot method. The presence of HTLV proviral DNA was detected in all positive samples by amplification of the HTLV  Inmates represent a highly vulnerable population to several infectious diseases compared with the general population, particularly blood, and sexually transmittable infections (STI) . Use of Brazil has the third-largest prison population in the world, with approximately 702,000 inmates in 2020 . Heath cThe Human T-lymphotropic virus (HTLV) is a retrovirus that has been found throughout the world . In BrazConsidering the relevance of HTLV-1/2 infection and limited data to this infection among the prison population, this study aimed to investigate the prevalence, the occurrence of new cases of HTLV infection after a 1-year follow-up, and circulating subtypes of HTLV-1 infection among prisoners from twelve closed penal institutions in Central Brazil.The study population consisted of inmates from 12 of the 21 closed penal institutions of five different cities of Mato Grosso do Sul State, Central Brazil, as follows: Campo Grande, Corumbá, Ponta Porã, Dourados, and Três Lagoas . ParticiAccording to the sample size calculation based on the estimated prevalence for anti-HTLV-1/2 of 1.05% (mean) for anti-HTLV found among Brazilian inmate studies , a signiTo investigate the occurrence of new cases of HTLV infection after a 1-year follow-up, 1,000 from the 1,507 participants were included in the prospective cohort study. The inclusion criteria were (a) having participated in the prevalence study; (b) not leaving prisons (for any reason) during this period; (c) having samples stored in sufficient quantity to perform the analyses proposed; and (d) being susceptible to HTLV infection (anti-HTLV-1/2 negative).The selected serum samples were screened by an enzyme-linked immunosorbent assay (ELISA) commercial kit for the presence of anti-HTLV-1/2 antibodies (GOLD ELISA HTLV-I/II – REM), following the manufacturer’s instructions. Positive samples were repeatedly tested and confirmed by HTLV-1/2 Western Blot (WB) assay (MP Diagnostics HTLV BLOT 2.4–Singapore).The samples reactive by screening and positive by WB were considered positive for HTLV-1 or 2 infections. The selected serum samples were also tested by ELISA method for anti-HIV-1/2 , anti-HCV , and for the presence of HBV serological markers   as described before by Tax gene of both HTLV-1 and -2 was performed to confirm the HTLV-1/2 infection as previously described (Reactive samples (anti-HTLV-1/2 positive) were also confirmed by nested polymerase chain reaction (PCR). To this end, a whole blood collection from anti-HTLV positive individuals was performed and DNA was extracted from these samples using the QIAamp DNA Blood mini kit (QIAgen), according to the manufacturer’s instructions. The amplification of a 218 bp fragment of the HTLV escribed . Furtherescribed , and to All anti-HTLV-1 infected individuals were recalled for a new blood collection to confirm the result. On this occasion, all anti-HTLV-1 infected individuals were submitted to the first clinical screening by an infectious disease specialist of our group. Hematologic tests and clinical manifestations of this disease such as slow and progressive paraplegia of the lower extremities, spasticity, hyperreflexia, bladder and bowel dysfunction, lower back pain, and sphincter disorders were investigated.This study was approved by the Federal University of Mato Grosso do Sul Ethics Committee on Human Research, under protocol number 1.250.132, CAAE: 49361715.8.0000.0021.Statistical analyses were performed using STATA 13.0 software . The prevalence of HTLV infection, estimated with 95% confidence intervals, and the categorical variables were presented by absolute and percentage frequency. The chi-square test was used to evaluate differences between proportions. Continuous variables were expressed as mean, standard deviation, median, and range. The sample size was calculated using BioEstat 5.0.A total of 1,507 prisoners were included in the cross-sectional study. The median age of participants was 29 ± 10 years (range 18-76), including 1,307 males (86.73%) and 200 females (13.27%). The majority reported less than 9 years of formal education (73.39%) and was from Mato Grosso do Sul State (67.00%). Regarding ethnicity, most of them declared themselves as multiracial (52.60%).Low frequency (1.16%) of inmates reported a history of illicit injection drug use (IDU). History of drug use such as marihuana, cocaine, and crack was reported by 29.40%, 18.50%, and 4.20% of prisoners, respectively. Sharing sharp objects was reported by 3.30% of the participants and was more frequent among males (76.00%). History of blood transfusion was informed by 12.64% of subjects, of whom 33 reported that received a blood transfusion before 1993 when the screening for anti-HTLV became mandatory in Brazilian blood banks. The presence of tattoos was reported by 66.95%.Regarding risk characteristics related to sexual transmission, prisoners reported a high frequency of irregular condom use (67.18%). History of STI was reported by 12.32% of participants. Most of the prisoners declared themselves as heterosexual (97.46%), however, 8.00% reported that they had already had homosexual intercourse. 51.70% of individuals reported less than 10 sexual partners in the past 5 years. Anti-HIV and anti-HCV were detected in 22 (1.46%) and 37 (2.46%) samples of the 1,507 prisoners. Anti-HTLV was detected in six prisoners [0.40% (CI 95%: 0.1-0.7)] in the cross-sectional study . All ELIn = 6) by amplification of the HTLV Tax gene through nested PCR. Out of 6 confirmed samples, only 2 (33.33%) amplified HTLV-1 5′LTR region and were successfully sequenced by the Sanger method. The nucleotide sequencing of the isolates from the HTLV-1 5′LTR region was subjected to an analysis in the BLAST and the 2 isolates were classified as Cosmopolitan (1a) subtype and Transcontinental (A) subgroup. Nucleotide sequences were registered into the GenBank database under accession numbers OK247616 and OK247617.The presence of HTLV proviral DNA was detected in all positive samples  between anti-HTLV positive and non-HTLV-positive prisoners.Those participants infected with HTLV-1 were subsequently referred to a clinical assessment by the prison infectious disease specialist. After clinical evaluations, none of the anti-HTLV-1 positive subjects showed clinical signs compatible with HAM/TSP or ATL. Only ID-3270 had already been tested for anti-HTLV and had a positive HTLV-2 result in his medical record. He reported injecting drug use with needle sharing, was breastfed for approximately 1 year, and had unsafe sexual behavior. It is noteworthy that half of the anti-HTLV positive individuals were co-infected with HCV and 1 of them was co-infected with HIV. There was a significant difference of positivity for anti-HCV , sharing needle over the last year (1.30%), injecting drug use in prison (0.11%), and history of blood transfusion over the last year (1.41%).This is the first survey on HTLV-1/2 infection prevalence carried out among inmates in Central Brazil, involving the five major closed penal institutions of Mato Grosso do Sul State . The higThe HTLV prevalence found in this study [0.40% (CI 95%: 0.1–0.7)] is similar to the prevalence observed in the general population from Mato Grosso do Sul such as blood donors and pregnant, 0.2% and 0.1%, respectively . In addiAdditionally, when compared to international studies, the prevalence of HTLV infection found in our study was lower than that reported among imprisoned immigrants from Northern Italy that found HTLV infection prevalence of 1.1%, corresponding to 1.4% of HTLV-1 infection and 0.8% of HTLV-2 infection . A studyFew studies assessed higher HTLV infection prevalence in prisoners, most of them included inmates with a history of injection drug use or with HIV infection, which may justify a higher number of infectious diseases in these studies compared to our findings .The presence of HTLV-2 infection among prisoners studied was an important observation coming out of this survey. Most cases of HTLV-2 infection described in Brazil have occurred among people who inject drugs (PWID) and the indigenous population . There wIt was observed that only one anti-HTLV positive individual was co-infected with HIV. Although there is strong evidence that HTLV and HIV may be transmitted to susceptible individuals by the same routes, in this case, risk behaviors such as a history of STI, injecting drug use, multiple sexual partners, and sharing needles were not reported by him. However, he reported irregular condom use, non-injected drug use, and a history of treating tuberculosis. Conclusions regarding routes of transmission cannot be drawn from this single case of HTLV/HIV co-infection .The distribution of HTLV-1/2 infection by gender differs from previous reports which found a higher prevalence of HTLV infection in female prisoners, possibly reflecting the high-risk behavior observed among male prisoners .Tax gene through nested-PCR. The finding of indeterminate results in the WB for HTLV and positive by PCR has already been reported in other studies (One sample (ID-573) was HTLV indeterminate in the western blot test, but anti-HTLV was detected by ELISA method and the presence of HTLV proviral DNA was detected by amplification of the HTLV  studies . About t studies . TherefoThe HTLV transmission could occur through blood transfusion until 1993 when serological screening for HTLV-1/2 became mandatory in Brazilian blood banks. History of blood transfusion was found in one-third of HTLV infected individuals , but it occurred after 1993. However, both individuals reported other risk factors, such as irregular condom use  and injecting drug use (ID-3270).It is known that a greater number of sexual partners is a high-risk behavior factor for acquiring sexually transmitted infections, such as HTLV . HoweverThe positive anti-HTLV-1 samples ID-452 and ID-2545 were classified as belonging to the Transcontinental (A) subgroup of the Cosmopolitan (a) subtype. HTLV-1aA is considered the most prevalent in Brazil .Those participants infected with HTLV-1 were subsequently referred to a clinical assessment by the prison infectious disease physician and an investigation of symptoms was carried out. No sensory, motor, and hematological changes were observed. Furthermore, no deficits were observed in the general and neurological examination.Although new cases of HCV and HIV infections were found by our group in cohort studies conducted in the same population , no new This study has limitations that should be considered. Regarding the veracity of the participant’s responses and the possibility of some behavior risks that may have been under-reported due to discrimination and stigma, leading to underestimating potential risk factors associated with HTLV infection. Despite these limitations, our study, having a large sample size, has results that would contribute to a clearer scenario of the circulation of HTLV-1/2 in the inmate population. It is important to emphasize that HTLV infection carriers are potential disseminators, although most of them remain asymptomatic.Cross-sectional and cohort surveys are important to study the dynamics of this retrovirus in low prevalence areas. This study indicates the circulation of HTLV-1/2 and the concomitant infections by HIV and HCV in the prison population of Mato Grosso do Sul, Central Brazil, representing a serious reality to be considered by the Public Health authorities.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Federal University of Mato Grosso do Sul Ethics Committee on Human Research Protocol number 1.250.132, CAAE: 49361715.8.0000.0021. The patients/participants provided their written informed consent to participate in this study.LMB, JC, and ARCM-C: conceptualization. LMB, MAMP, JC, MAP, and ARCM-C: data curation. LMB, MAMP, CA, and ARCM-C: formal analysis. JC and ARCM-C: funding acquisition. LMB, MAMP, CA, GRR, MAP, and ARCM-C: investigation and methodology. LMB and ARCM-C: project administration. LMB, MAMP, CA, GRR, JC, MAP, and ARCM-C: writing – original draft and writing – review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Organochlorine pesticides, such as DDT, methoxychlor, and their metabolites, have been characterized as endocrine disrupting chemicals (EDCs); suggesting that their modes of action involve interaction with or abrogation of endogenous endocrine function. This study examined whether embryonic thymocyte death and alteration of differentiation induced by the primary metabolite of methoxychlor, HPTE, rely upon estrogen receptor binding and concurrent T cell receptor signaling. Estrogen receptor inhibition of ERα or GPER did not rescue embryonic thymocyte death induced by HPTE or the model estrogen diethylstilbestrol (DES). Moreover, adverse effects induced by HPTE or DES were worsened by concurrent TCR and CD2 differentiation signaling, compared with EDC exposure post-signaling. Together, these data suggest that HPTE- and DES-induced adverse effects on embryonic thymocytes do not rely solely on ER alpha or GPER but may require both. These results also provide evidence of a potential collaborative signaling mechanism between TCR and estrogen receptors to mediate adverse effects on embryonic thymocytes, as well as highlight a window of sensitivity that modulates EDC exposure severity. Endocrine disrupting chemicals (EDCs) are environmental contaminants that perturb hormonal systems  by mimicThe immune system evolved to mount protective responses against harmful pathogens or their biological products . The immThe thymus plays an integral part in the developmental process, primarily due to its facilitative role in thymocyte differentiation and maturation. Therefore, any disturbances of the thymus during the critical periods of pre-natal development can result in later life clinical disorders, such as autoimmune disorders ,15 or ab−/low DP thymocytes. Thus, the cumulative results of these studies offer evidence for the role of ERα and GPER in the modulation of thymic development through estrogen signaling.It is clear that estrogen receptors are critical to normal mouse thymic development and are involved in (at least) estrogen-induced atrophy and alteration of development. A study by  using esintermediate stage  that trigger the early steps of maturation and induce partial differentiation of thymocytes from the DP stage to the CD4+CD8low; ). Prior +CD8low; . In thisp-value cutoff of 0.0125 were anti-BOTH, DMSO in ER alpha inhibition, and GPER ligation. The block variable was significant across the analyses . Since DES by itself showed no significant difference from DES plus ICI at increasing concentrations, it appears that ICI did not rescue in vitro differentiated thymocytes from DES-induced death for any antibody condition. In vitro differentiated thymocytes treated with ICI by itself  were exposed to DES, the DES treatment still induced death  or 25 µM HPTE plus 10 µM ICI  were statistically significantly different under all antibody conditions E–G. Howep ≥ 0.05).We also wished to determine whether ICI would selectively rescue any thymocyte subpopulations  involved in the EDC-induced death. However, none of the subpopulations were rescued by ICI pre-treatment . The combination of 20 µM G15 and HPTE . Interestingly, similar to HPTE plus ICI at 10µM, addition of the GPER/GPR30 receptor antagonist by itself at 20 µM reduced viability of embryonic thymocytes compared with the control under all antibody conditions .To investigate whether the cell death induced by HPTE was due to its interaction with the nonclassical estrogen receptor GPER/GPR30, the selective antagonist G15  was addepCD2 = <0.0001; pTCR = < 0.0001; pBOTH = 0.0021) and comparable to embryonic thymocytes exposed to 25 µM DES under all antibody conditions .The reduction in death observed following G15 pre-treatment and exposure to HPTE, though not statistically significant, suggested that GPER may be involved in EDC-mediated death of embryonic thymocytes. To address whether activation of GPER would induce death, embryonic thymocytes were placed in the in vitro differentiation culture with the GPER agonist G1. As seen in intermediate cells were [Prior work had shown that DN embryonic thymocyte numbers were not significantly decreased when subjected to DES exposure under anti-CD2 or anti-TCR conditions, whereas DP and CD4lls were . In the As expected, 25 µM DES-treated DP mean cell number differences were statistically significant compared with the medium control under all antibody conditions , but not in cultures treated with 25 µM DES (intermediate—row 1). Moreover, mean cell numbers of CD4intermediate in 25 µM DES-treated or 2 µM G1-treated were statistically significantly different under the anti-CD2 antibody condition (intermediate—row 3). CD4intermediate mean cell numbers from 2 µM G1-treated cultures were not statistically significantly different from 25 µM DES-treated cultures under anti-TCR and anti-BOTH conditions , but were statistically significantly different from medium control .Generation of CD4The embryonic thymocytes were harvested between gestational days 16 and 18. Therefore, the majority of the cells in the CD8 subpopulation represented immature thymocytes prior to the DP stage and were small in number. CD8 mean cell numbers from 2 µM G1-treated cultures were not statistically significantly different from 25 µM DES-treated cultures under any antibody condition  and diluent  controls. When the EDCs were added to recovery culture  and HPTE compared with the diluent control (pBOTH/recovery = 0.0497), but not for HPTE compared with the medium control (pBOTH/recovery = 0.0847). In addition, there was a statistically significant difference in cell numbers between cells that were exposed to EDCs during the signaling culture compared with cells exposed during the recovery culture . Fewer cells were lost when EDC exposure occurred in the recovery culture, i.e., after the cells had received the TCR signal. These results suggest that concurrent TCR signaling and EDC exposure led to maximal cell death, whereas prior TCR signaling was somewhat protective for subsequent exposure to DES or HPTE.When the EDCs were added to signaling culture  revealed differences in cell numbers. In DES-treated cultures, there was no statistically significant loss of DN thymocytes in cultures exposed during recovery phase compared with the medium and diluent controls . Fewer DP cells were lost when EDC exposure occurred in the recovery culture, a difference that was statistically significant .A statistically significant loss of CD4CD8 thymocyte losses occurred in cultures exposed to DES during signaling phase compared with controls and to cultures exposed to DES during recovery phase (high peak compared with top trace) and reduced TCR (high peak compared with top trace) on their surface.To further probe the differences in survival of embryonic thymocytes exposed to EDCs at different stages of in vitro differentiation culture, cells were stained post-culture for surface markers that identify subpopulations. Surviving DN and DP embryonic thymocytes exposed during signaling culture to 50 µM DES or HPTE exhibited reduced CD5 and reduced TCR expression on their surface compared with controls . Survivihigh expression (high peak compared with top trace) and reduced TCR expression (high peak compared with top trace) on their surface compared with controls  DP and CD4intermediate thymocyte populations, key players in the selective processes of thymocyte development. In the current study, we aimed to elucidate whether the estrogen receptors ERα and GPER played an active role in EDC-induced adverse effects on differentiating embryonic thymocytes, and whether differentiation-associated signaling exacerbated these effects. Neither ICI 182,780  nor G15 (GPER antagonist) rescued differentiating embryonic thymocytes. In fact, at the highest concentration used, ICI seemed to worsen cell death induced by HPTE. When ICI or G15 was added by itself to the differentiating thymocytes, neither ER inhibitor induced death on their own, except at the highest dose of G15 under some conditions. When G1 (GPER agonist) was added to differentiating embryonic thymocytes, similar declines in cell number and thymocyte subpopulations were observed compared with DES exposure. Lastly, this study was the first to demonstrate significant differences between differentiating embryonic thymocytes exposed to HPTE or DES during signaling phase versus recovery phase.In our prior work , we showintermediate thymocytes) similar to that of DES, supports a role for GPER in EDC-induced adverse effects on thymocyte development. In addition, our observation that neither ER inhibitor (ICI or G15) by itself rescued adverse effects of HPTE or DES exposure on differentiating embryonic thymocyte supports the idea that ERα and GPER may be working in concert to trigger events in embryonic thymocytes leading to amplification of cell death and alteration of differentiation. Our results agree with ERKO and GPER studies [Our finding that G1 induces embryonic thymocyte death and subpopulation distribution alterations  has been shown to have GPER agonist activity  that tri2, thus binding of estrogen to GPER induces rapid non-genomic signaling [HPTE-induced apoptosis was observed as early as after 4 h in our prior work , a findiignaling . Non-genignaling , crosstaignaling , and colignaling ,50. Membignaling ,59,61. Gignaling  through ignaling . Anotherignaling .high, TCRhigh cells appeared to survive. Moreover, it has been observed that signaling through TCR and CD2 results in upregulation of expression of Bcl-2, an anti-apoptotic protein, in the in vitro differentiation assay [Signaling through the T cell receptor utilizes many of the same signal transduction molecules observed to be modulated by membrane ER ligation . Moreoveon assay , and Bclon assay . These rIn conclusion, this study indicates that, while ERα or GPER may be critical to thymocyte development and EDC exposure, they do not appear to be acting alone. To further test this hypothesis, it will be informative to use ERα and GPER inhibitors together in the differentiation assay to determine whether in combination, they can abrogate HPTE or DES adverse effects. Additionally, our study is the first to demonstrate that signaling through estrogen receptors appears to work in concert with differentiation signaling to exacerbate EDC effects, perhaps mimicking the established collaborations between membrane ER and other non-ER receptors observed in other cell types ,36,50. FAll experiments were approved and conducted under the supervision of the Institution’s Animal Care and Use Committee  of the University of La Verne in accordance with federal guidelines for the care and use of live animals in research . C57BL/6The following antibodies  were titrated and used to stain freshly isolated and cultured thymocytes: APC anti-CD4 , FITC anti-CD8 , PE anti-CD5 , anti-T cell receptor . Cells were stained with fluorochrome-conjugated antibodies for 30 min then washed with PBS containing 1% FBS.A minimum of 10,000 events were collected per sample on the Accuri C6 system (BD Biosciences). Flow cytometry data files were analyzed using the FlowJo  software. Live gates were set on FSC vs. SSC plots and thymocyte population quad gates determined using single stains and FMO controls. Mean live cell numbers were determined by FlowJo software based on gating parameters.intermediate8low thymocytes, a post-positive selection intermediate population in T cell development [l-glutamine, 50 mM beta-mercaptoethanol, 1% penicillin/streptomycin), and 4.0 × 105 cells were added per well. Thymocytes were cultured in the absence of phenol red to avoid confounding effects of phenol red estrogenicity [All 96-well plates were prepared and incubated 24 h at 4 °C with purified antibodies  against CD2  and the T cell receptor , alone or in combination. The differentiation assay has two stages: signaling culture (stimulation of antigen receptor and/or adhesion molecule by antibodies) and recovery culture . The combination of antibodies has been shown to induce differentiation of DP cells into CD4elopment . Embryongenicity . ThymocyIn order to determine whether DES or HPTE mediated their induction of death by nongenomic amplification of antigen receptor signaling or some other mechanism related to the TCR signaling during differentiation, we set up two parallel in vitro differentiation assays. The original exposure regimen called for the addition of the EDC during the signaling culture, such that TCR signaling and EDC exposure were concurrent. The second regimen entailed adding the EDC to the recovery culture, after the embryonic thymocytes had been removed from the antibody-coated plates and washed.Endocrine disruptors, diethylstilbestrol , and HPTE -1,1,1-trichloroethane, purity 97%; metabolite of methoxychlor) were purchased from Sigma–Aldrich Chemical . MXC is normally metabolized in the liver  to form 50 = 0.29 nM), shown to lack any partial agonism both in vitro and in vivo. It is also a high affinity agonist of the membrane estrogen receptor GPER/GPR30. For estrogen receptor alpha inhibition experiments, the inhibitor, ICI 182,780 , was added at various concentrations  to thymocyte-seeded signaling cultures and incubated for one hour prior to EDC addition at 5% CO2, 37 °C, and 96% humidity. Pre-treatment was performed to ensure ICI blocking efficiency. After one hour, the EDCs were added and thymocytes incubated between 18 and 24 h in the signaling culture. Thymocytes were then washed and transferred to recovery culture.ICI 182,780 is high affinity estrogen receptor antagonist (ICi = 20 nM). At ≥10 μM, G15 has affinity for Erα and Erβ. For GPR30 inhibition, the cells were placed in a 12-well plate and treated with various concentrations  of the GPR30 inhibitor G15  or cRPMI and incubated for one hour at 37 °C, 5% CO2, and 95% humidity. Pre-treatment was performed to ensure G15 blocking efficiency. After exposure, the cells were transferred to the signaling culture and EDCs or control solutions were added. The plate was incubated between 18 and 24 h in the signaling culture. Thymocytes were then washed and transferred to recovery culture.G15 is a high affinity and selective GPER/GPR30 receptor antagonist . The identification of independent (explanatory)/dependent (response) variables, blocked design, and most assumptions were applied similarly across analyses. The dependent variable , which represented the outcome of the experiment, was log transformed when it did not follow a normal distribution. The first independent variable was either the antibody condition (referred to as condition) or the experimental treatment (referred to as treatment). The second independent variable is a block designed to control for variation introduced by treating the cell populations at different times, different lengths of gestation (range was 16–18 days), and any other differences arising during treatment. Tukey Honest Significant Difference (HSD) post hoc tests, with adjustments for multiple comparisons, were performed to identify what groups were different from others by comparing the conditions/treatments to one another within each experiment.p-value cut off for some ANOVA tests in order to reduce the chance of a Type I error. The Bonferroni corrected p-value cutoff used for ER alpha inhibition, GPER inhibition, and GPER ligation experiments is 0.0125 (α = 0.05/4 cell populations), while the concurrent versus sequential exposure experiment did not require a correction (α = 0.05). The p-values for Tukey HSD post hocs were evaluated at α = 0.05 as the p-values reported are adjusted for multiple comparisons. All analyses were completed in R [A Bonferroni correction ,74 was ated in R  and grap


Protein Kinase A (PKA) is an essential kinase that is conserved across eukaryotes and plays fundamental roles in a wide range of organismal processes, including growth control, learning and memory, cardiovascular health, and development. PKA mediates these responses through the direct phosphorylation of hundreds of proteins–however, which proteins are phosphorylated can vary widely across cell types and environmental cues, even within the same organism. A major question is how cells enact specificity and precision in PKA activity to mount the proper response, especially during environmental changes in which only a subset of PKA-controlled processes must respond. Research over the years has uncovered multiple strategies that cells use to modulate PKA activity and specificity. This review highlights recent advances in our understanding of PKA signaling control including subcellular targeting, phase separation, feedback control, and standing waves of allosteric regulation. We discuss how the complex inputs and outputs to the PKA network simultaneously pose challenges and solutions in signaling integration and insulation. PKA serves as a model for how the same regulatory factors can serve broad pleiotropic functions but maintain specificity in localized control. Protein kinase A (PKA) is among the best studied eukaryotic kinases, owing in part to its essential function in many cellular processes. The kinase can phosphorylate hundreds of proteins, including enzymes linked to metabolism, cellular machinery involved in transcription and translation, structural proteins in the cytoskeleton, and other kinases and regulators that amplify its effects . Which pA major question is how the PKA kinase can be involved in so many processes yet maintain specificity in coordinating the correct response for the conditions at hand. Part of the solution emerges from the complexities of the broader PKA signaling network. As summarized below, the network includes multiple upstream activation branches, numerous control points modulating allosteric regulation, and the action of combinatorial network interactions that result in modularity. In addition to the complex network of regulatory players, cell also utilize dynamic regulation to control where, when, and how PKA activity is enacted within the cell.Saccharomyces cerevisiae and mammalian systems. Yeast cells maintain a simplified PKA network with fewer players than mammalian cells, but it remains one of the best characterized systems for understanding signaling control. Comparing and contrasting yeast and mammalian systems underscores several cases in which signaling principles are conserved, even when the regulatory players have evolved. Below, we present an overview of the PKA regulatory network in yeast and mammals before discussing mechanisms by which cells spatially and temporally regulate PKA signaling, including a focus on newly emerging mechanisms of PKA control.Here we focus on models of PKA signaling insulation and specificity during cellular responses, using examples from budding yeast The PKA kinase fits into a broader network with many different levels of control, including multiple input branches and extensive regulatory checkpoints ; Table 1S. cerevisiae encodes three paralogous catalytic subunits, Tpk1, Tpk2, and Tpk3, with a single regulatory subunit, Bcy1  subunits and two regulatory (R) subunits. it, Bcy1 , while mit, Bcy1 .While PKA C subunits encompass the catalytic kinase domain, the R subunits serve as a major point of PKA control. R subunits bind the allosteric second messenger cAMP, which induces conformational changes that alter R-C interactions. One long-stranding model is that this conformation change causes R subunit release, allowing C subunits to phosphorylate substrates. Recent evidence in mammalian cells suggests that disassociation of R subunits may be short-lived , and oneSince cAMP is a key regulator of R-C interactions, cells sensitively control its abundance. Multiple upstream regulatory branches converge on adenylate cyclase (AC), which catalyzes the conversion of adenosine triphosphate (ATP) to cAMP and pyrophosphate. Yeast harbors a single AC , while t2+/calmodulin-dependent kinase . Once again, complexity varies from yeast with two PDEs  to mammat kinase . The larHoloenzyme and regulatory points described above serve as hubs for signaling input and integration. Numerous upstream regulatory pathways feed into these hubs to control PKA activity in response to the appropriate cues. Among the best studied are RAS-GTPase and G-protein coupled receptors (GPCRs). In yeast, Ras is one of the primary PKA regulatory inputs regulating growth control, consistent with the classical implication of RAS in human cancers. Ras proteins are stimulated by GTP hydrolysis to activate downstream targets including AC and, therefore, PKA signaling  in mammals that participate in tethering via multi-protein interactions. AKAPs are defined by three domains: a PKA-anchoring domain, a unique cellular localization signal, and domains that organize other proteins to assemble , a PKA substrate that mediates calcium efflux and is critical for proper cardiac muscle excitation and contraction . Mutant Beyond bridging PKA with specific targets, AKAPs also bring together PKA regulators including ACs, PDEs, phosphatases, and other kinases to produce highly localized microenvironments known as signalosomes -based reporters have enabled the quantification of localized cAMP abundance and PKA activity . FRET ocalosomes . Other falosomes . These talosomes . Severalalosomes , includialosomes . One elealosomes . Underscelopment .An under-explored mechanism of PKA control just beginning to emerge is phase separation. Under the appropriate conditions, some proteins can form biomolecular condensates, also known as membraneless organelles. Kinases can phase separate into these condensates in a way that affects their activity. For example, in yeast experiencing nutrient starvation or heat shock, PKA subunits can be sequestered in phase-separated granules including P-bodies (PB) and stress granules (SG) . These gWhile less well studied, PKA can phase separate in mammalian cells as well. A recent study by Feedback and feedforward signaling play important roles in signaling dynamics, sensitization, and buffering . In the Dictyostelium discoideum (2+ waves in developing retinal neurons, and these waves in turn feedback to regulate PKA signaling (Interestingly, specific patterns of feedback signaling can in other cases produce propagating waves of second messenger. One example is seen in the formation of propagating waves of cAMP as measured in the slime mold scoideum . Starvatscoideum . There iignaling . While tignaling .The intricate mechanisms that specify PKA activity–including differences in holoenzyme composition, subcellular localization, signalosome organization, cAMP microenvironments, and feedback regulation–provide a model for how PKA can participate in such a breadth of cellular processes and responses yet provoke distinct and precise downstream outputs. Ultimately, much of PKA activity is controlled in a highly localized manner. Thus, thinking of the pathway as generally “on” or “off” in cells is likely inappropriate. In budding yeast PKA suppresses the environmental stress response, leading to the general model that PKA must be globally inhibited during stress to mount defense systems . Indeed,Another important feature giving breadth to PKA responses may be how the kinase recognizes its targets. Whereas many other kinases recognize docking sequences shared among their target proteins , PKA’s cThe modularity of PKA signaling presents opportunities and weakness for the cell. On the one hand, the complexity in network organization enables PKA to respond to many different upstream signals and environmental inputs while producing unique cellular outputs. It also imparts systems-level properties, including buffering or sensitizing cells to input signals, producing oscillatory or pulsatile dynamics via feedback loops, and enabling bistability . On the


Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents. Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic  Giardia spp. are flagellated protozoan parasites of vertebrates and a frequent cause of gastrointestinal disease in wild and domestic animals and humans [Giardia duodenalis is the most widespread species in mammals and subdivided into eight genetic subgroups (assemblages A-H) [Giardia muris and Giardia microti [G. microti and, in particular, for G. muris, distinct morphological characteristics in comparison to G. duodenalis have been described [d humans . Giardiages A-H) . These sges A-H) ; assemblges A-H) . Small r microti ,3,4. Forescribed .G. duodenalis assemblages A and B are genetically very distinct, and each group can be further subclassified based on genetic information determined by multi-locus sequence typing (MLST) [The zoonotic g (MLST) . Within g (MLST) . The subg (MLST) .Giardia spp., and the potential zoonotic risk has been highlighted by several studies [Giardia-positive beavers (Castor canadensis) has been estimated at 13–30%, and zoonotic transmission has been implicated by several studies [G. duodenalis assemblages A and B [Chinchilla lanigera, where mainly G. duodenalis assemblage B-type infections have been recognized [Rodents are frequently parasitized by  studies ,11,12,13 studies ,15,16. Acognized ,17,18.G. muris, G. microti and/or G. duodenalis assemblage G, respectively [The roles of zoonotic transmission by smaller rodents commonly found in natural and human habitats, such as mice, voles and rats, are not well-resolved. The few studies providing molecular typing data revealed that these genera are mainly infected by ectively ,21,22,23ectively ,25,26,27ectively ,28,29. Giardia prevalence in wild rodents often lack species information or genotyping data, so that the zoonotic risk cannot be estimated from those studies. One likely reason for the lacking data is that Giardia species determination and assemblage typing mainly relays on a cumbersome test principle, including amplification by the (nested) PCR and sequencing of genetic marker genes, such as small subunit rRNA gene (SSU), triosephosphate isomerase (TPI), glutamate dehydrogenase (GDH) and beta giardin (BG) [Giardia species is the SSU locus.Studies reporting the din (BG) ,32,33,34G. muris, G. microti and zoonotic G. duodenalis assemblage types in rodent samples. These protocols will help in future studies to better determine the potential risk for zoonotic Giardia transmission from wild rodents.The aim of the study was to provide a new workflow based on the available and newly developed real-time PCR protocols to determine the G. duodenalis assemblages A  and B  were derived from axenic in vitro cultures using standard culture procedures [G. muris (Thompson isolate) were commercially obtained . Fecal samples of G. muris and G. duodenalis-infected mice were obtained from experimental laboratory infections done in a different context. These procedures were approved by the local authorities, the Landesamt für Gesundheit und Soziales .Trophozoites of ocedures . Cysts oBalamuthia mandrillaris, Toxoplasma gondii and Leishmania donovani were previously extracted from routine in vitro cultures and available from archived materials. DNA of Entamoeba histolytica axenic cultures was a kind gift from Prof. Iris Bruchhaus . DNA samples of wild rodents are described elsewhere . DNA of Giardia species-specific oligonucleotides: G. muris (X65063 and AF113895), G. microti (AF006676 and AF006676), G. duodenalis assemblage A (M54878 and AF199446), G. duodenalis assemblage B (AF199447 and AF3898), G. duodenalis assemblage C (AF199449), G. duodenalis assemblage D (AF199443), G. duodenalis assemblage E (AF199448), G. duodenalis assemblage F (AF199444) and G. duodenalis assemblage G (AF199450). These references were supplemented with the sequence information of G. muris and G. microti derived from a screen in wild rodents (KY114167-KY114486) [Giardia-specific nested PCR and sequencing approach, as described earlier [Giardia isolates in the public databases, including all three mammalian Giardia species and assemblage types. In addition, due to its comparably high copy number in the genome, the ribosomal gene loci has been widely used for genotyping and phylogenetic approaches, as it promises supposedly higher PCR detection sensitivity [G. muris, G. microti and G. duodenalis was done using the Geneious software tool .The following reference sequences (GenBank accession numbers) of the SSU gene locus were used to design  earlier ,36. Currsitivity . The desG. duodenalis assemblage types A and B, we used a previously described protocol that detects the single-copy target gene 4E1-HP [For determination of the e 4E1-HP .n = 3) with 95% confidence intervals. DNA extraction of samples was done with the QIAamp Fast DNA Stool Mini Kit  according to the manufacturer’s protocols. Real-time PCR assays were standardized and performed using single PCR approach as the multiplexing reduced assay performance (data not shown). Conditions were as follows using the 2X Maxima Probe/ROX qPCR Master Mix  in a 25 µL PCR reaction: 12.5 µL reaction buffer, 1 µL of each forward and reverse primer (from 3.3-µM stock), 0.5 µL of probe (from 3.3-µM stock), 1–5 µL DNA template and add water for the final volume. Reactions were started at 95 °C for 10 min, followed by 45 cycles of 95 °C denaturation for 15 s, 60 °C annealing for 30 s and 72 °C extension for 30 s. Real-time PCR and analysis was performed using a CFX Maestro PCR machine and the respective software tools . Sequences of the primers and probes used in the study are presented in SSU genome fragment (see Methods for details) was used for the primer design. At this locus, a well-established and robust real-time PCR to diagnose G. duodenalis in humans has been previously described [G. muris on the one hand and G. duodenalis and G. microti on the other hand allowed for the design of a G. muris-specific primer and probe combination  indicated a high sensitivity reporting 1 fg DNA with a cq value of 36.3 ± 0.65. This equals approximately 0.1 genome equivalents for this species (estimated genome size of G. muris is 9.8 MB [The assay designed for the detection of s 9.8 MB ). G. duodenalis assemblages A and B were equally well-detected in the G. duodenalis/G. microti (Gmi/Gd PCR) approach, with a sensitivity of 10 fg  and, in the G. duodenalis-only (Gd PCR) approach, with a sensitivity of about 1 fg DNA , which equals about 0.07 genome equivalents  or approximately 7 genome equivalents per PCR reaction were detected. The lower sensitivity was expected due to the single-copy target versus multi-copy target in the former PCRs. Sensitivity of the Giardia-positive samples from a previous project [G. muris, G. microti or G. duodenalis, respectively, as previously determined by nested PCR and the sequencing of a fragment at the SSU locus [Giardia species could not be determined in the previous study. To test the applicability of the new PCR approach, we reanalyzed 38  project   does not allow to formally exclude an additional underlying G. microti infection. However, in this case, a lower cq value in the Gd/Gmi combi-PCR compared to the Gd-only PCR would be expected, which was not the case.All nine G. microti samples, 12 were confirmed by our new PCR workflow. Unexpectedly, two samples exhibited a signal in the G. muris-specific PCR with relatively low cq values of 31, but no signal was recorded in the Gd/Gmi-PCR. We can only speculate on the reason and favor the possibility of an underlying double infection with a lower G. microti load that may have been amplified in the previous nested PCR approach. The nested PCR tends to amplify G. microti  sequences with higher sensitivity than that of G. muris .Of the 14 G. duodenalis samples were also confirmed by our assay; however, two samples were not confirmed, likely due to lower sensitivity of the real-time assay compared to the nested PCR approach used in the previous study . One sample was further confirmed as a zoonotic assemblage type, whereas the other sample  was not confirmed. Again, the reason is most likely lower sensitivity of the assemblage-specific PCR that targets single-copy genes in comparison to the PCR at the multi-copy SSU locus.Two of the four G. muris real-time PCR, which indicates a possible underestimation of the G. muris prevalence in the previous work [Gd-specific PCR; however, as the Gmi/Gd PCR was negative, we do not consider this result as reliable.Four of the 11 samples that could not be typed in our previous study displayed a positive signal in the ous work . One samG. muris, G. microti and G. duodenalis infections in small rodents, including the detection of zoonotic G. duodenalis assemblage types A and B. The workflow provided an improved time-to-results procedure for the analysis of rodent fecal samples to determine the Giardia species and for an assessment of the potential zoonotic risk.Here, we provided a real-time PCR workflow for the detection of G. duodenalis assemblages A and B, and the zoonotic transmission has been described [Giardia species. Pet chinchillas harbor zoonotic G. duodenalis (mostly assemblage B) and non-zoonotic assemblages C, D and E [Two rodent species are well-described to harbor the potential zoonotic escribed ,43. One escribed . The seq D and E ,43,44,45Giardia in their analyses were based on the microscopic detection of cysts and rarely included molecular tools to determine the Giardia species or assemblage types. The potential relevance for zoonotic transmissions was nevertheless often discussed [Giardia species and may help to assess the potential relevance of rodent species for zoonotic transmissions of Giardia infections. For ubiquitous small mammals such as mice and voles, the potential relevance of zoonotic transmission is largely unknown. Most of the larger screens for zoonotic pathogens that included iscussed ,46. HereG. microti cannot be detected directly. We based the design of the primers and probes on previously acquired sequence information at the SSU locus of 106 unique G. microti and 10 unique G. muris sequences [G. microti, the sequence analysis revealed a broad variety of genotypes even at the usually highly conserved SSU gene locus [G. microti-specific PCR and needed to rely on PCR signals obtained with primers detecting G. microti/G. duodenalis or solely G. duodenalis, respectively. Notably, a previously described real-time PCR for human diagnostic purposes [G. micoti genotypes (data not shown), hence providing little benefit to the current study. The results presented here indicated that our workflow was sensitive and robust for such purposes. However, our method also had clear limitations. The most prominent limitation was that equences . To our ne locus . Due to purposes  partiallG. microti/G. duodenalis-specific real-time PCR is less sensitive than G. duodenalis PCR in particular, because the same region is covered. Possibly, conformational hindrance at the gene locus may influence the binding affinity of the primer and probe. For the G. duodenalis assemblage-specific PCR, the lower sensitivity was likely due to the single-copy target gene used for detection. The lower sensitivity compared to real-time PCR at the SSU locus has been also described previously [The analysis revealed different sensitivities for the various PCR approaches, which was important for the interpretation of the results. However, all PCRs detected one or less genome equivalents at the SSU locus and about 10 genome equivalents at the 4E1-HP locus. This was in good concordance with previously published results using these loci ,48. It ieviously . SSU sequences derived from wild rodents revealed only five G. duodenalis-positive samples, and these samples were not typable at the single locus genes TPI, GDH and BG. Four of these samples were included in the present study and revealed either very low cq values for G. duodenalis qPCR or G. duodenalis was undetectable with the presented PCR methods. Only one sample revealed detectable amounts of G. duodenalis assemblage B DNA, confirming our previous typing results. Overall, this implied a very low zoonotic risk in the studied German rodent population. Whether the low-level detection of pathogens in real-time PCR assays, as indicated by high cq values, are indeed relevant for zoonotic transmissions should be carefully evaluated in future screening studies. For example, in our original study, an analysis of 358 Giardia species populating small rodents, including the zoonotic G. duodenalis assemblage types. This approach could be used in future studies to estimate the possible relevance for zoonotic transmissions by these animals.In conclusion, we provided a processive and robust workflow for the detection of all


Genetic deletion of TPCs not only affects endocytosis, recycling, and degradation of various surface receptors but also uptake and impact of bacterial protein toxins and entry and intracellular processing of some types of viruses. This review points to important examples of these trafficking defects on one part but mainly focuses on the resulting impact of the TPC inactivation on receptor expression and receptor signaling. Thus, a detailed RNA sequencing analysis using TPC1-deficient fibroblasts uncovered a multitude of changes in the expression levels of surface receptors and their pathway-related signaling proteins. We refer to several classes of receptors such as EGF, TGF, and insulin as well as proteins involved in endocytosis.Two-pore channels (TPCs) constitute a small family of ion channels within membranes of intracellular acidic compartments, such as endosomes and lysosomes. They were shown to provide transient and locally restricted Ca The first 3D architecture was determined for a TPC from Arabidopsis thaliana using X-ray crystallography [Two-pore channels (TPCs) constitute a small family of cation channels within the membranes of endolysosomal organelles. In mice and humans, only two TPCs were identified, whereas several other species express three or even more functional tpc-genes. The diversity of tpc-genes, exon-intron structures, transcripts, and proteins and phylogenetic relationships was recently described ,2,3. Thelography ,5. Laterlography ,7. Functlography ,7.2+ mobilizing messenger and was identified first in sea urchin eggs in 1995 [2+ release from small acidic organelles, photoaffinity labeling studies failed to demonstrate a direct binding between NAADP and TPCs [2+ release mechanisms.Initially, TPCs were identified as the long-sought class of ion channels opened by the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) . NAADP iand TPCs . Instead2+ signal. Two other teams, Gunaratne with colleagues and Roggenkamp and coworkers, converged on JPT2 (Jupiter microtubule-associated homolog 2) as an NAADP binding protein [2+ release [2+ pathway and suggests an important role of the RyR/JPT2-complex for the first seconds of T cell activation [Recently, several groups presented their efforts in identifying NAADP receptors and introduced two promising candidates. Yan and Guan identified LSM12 as a putative NAADP interactor by showing that recombinant LSM12 bound to immobilized NAADP but not to NADP. These data were confirmed by using LSM12 knockout cells that demonstrated an abolished interaction between NAADP and TPCs and a reduced Ca protein ,12. The  release . The sec2 was described as an endogenous activator of TPCs. A considerable number of studies characterized TPCs initially as non-selective Ca2+ -permeable channels activated by NAADP [2 [2+ signals and non-selective cation currents, whereas the other one induced only modest Ca2+ signals but a Na+ selective current. It turned out that the two agonists mimic the actions of NAADP and PIP2, causing different ion permeability and selectivity of TPC2.Besides NAADP, the phospholipid PIPby NAADP ,13,14,15NAADP [2 ,17,18,19NAADP [2 ,21. They2+ from these vesicles into the cytoplasm and thereby to a locally and temporally restricted increase of Ca2+, likely necessary for fusion and/or fission of endolysosomal membranes. This forms the basis for vesicle trafficking and sorting as a consequence for endocytic, recycling, and degradation processes [2+ channels [Mammalian TPC1 and TPC2 are found in intracellular membranes of the endolysosomal system but not in the plasma membrane. Within these compartments, they show a smooth transition from TPC1 predominantly found in early and recycling endosomes to TPC2 mainly expressed in late endosomes and lysosomes ,23,24,25rocesses ,27. Recechannels . In phag2+ in endolysosomal compartments and the filling of endoplasmic reticulum (ER) Ca2+ stores. Data from exocytosis experiments using mast cells suggested that TPC1 controls intracellular Ca2+ homeostasis and Ca2+ balance between the ER and the endolysosomes [2+ homeostasis and granular histamine content and causes an altered filling of ER and organellar Ca2+ stores. In vivo, TPC1 knockout mice have fewer mast cells but are characterized by an enhanced exocytosis and mediator release due to an increased immunoglobulin-triggered Ca2+ signal [TPCs also contribute to the uptake of Caysosomes . Thus, T+ signal .In view of the importance of TPCs for intracellular trafficking, one might expect more severe phenotypes in existing knockout models. However, a closer inspection of these mouse models suggests that phenotypes come to light mostly under stimulated but not under basal conditions. This is evident in liver cells from TPC2 deficient mice; here, only mice fed with a high cholesterol diet demonstrated an accumulation of cholesterol in late endosomes, probably due to an impairment of endolysosomal fusion processes. Furthermore, TPC2 deficient mice were susceptible to cholesterol overload and liver damage only under this diet . AnotherThe question arises of the role of TPCs beyond involvement in endocytosis, recycling, and degradation. There is a huge number of cell surface receptors and transporters that are regulated via endo- or exocytosis or recycling and degradation. Furthermore, some receptors are still signaling even after endocytosis and as long as intracellular parts have access to the cytosol. Given the role of TPCs as general regulators of intracellular trafficking, one might expect effects of TPCs on surface expression and receptor signaling. These consequences should emerge in TPC-deletion studies, such as for TPC1, TPC2, and even more prominently in TPC1/2-double knockout cell lines. In this review, we first describe the role of TPCs for intracellular trafficking but mainly focus on the functions beyond those aspects. In particular, we used RNA sequence analysis from wildtype and TPC1-deficient fibroblasts to gain insight into the expression levels of surface receptors and their pathway-related signaling proteins.2+ sources, i.e., classes of ion channels for determining individual endosomal trafficking routes [Bacterial protein toxins were established as a tool to uncover the specific roles of different Cag routes . These tg routes .The application of the “short trip” toxins to appropriate cell lines resulted in a delayed toxin uptake and reduction of target protein modification (PMT and DT). The cytotoxic effect of LF/PA in a TPC1-knockout macrophage cell line was also delayed. The result from the latter was confirmed by inhibition of TPC channels with tetrandrine, which decreased the effect of LF/PA in a dose-dependent manner. CT serving as example for a “long trip” toxin showed very little quantitative differences in cAMP accumulation in wildtype and TPC1-deficient cells within 3 h of toxin treatment . However2+ channels and identified TPCs as key players [Another excellent model for investigating the roles of TPC1 and TPC2 for vesicle trafficking is based on endocytosis and endolysosomal processing of Ebola virus . The vir players . Thus, d2+ channels was without any impact [TPCs were also shown to be involved in controlling the life cycle of HIV . A pharmy impact .2+ release promoted the activity of furin, a proprotein convertase necessary for internalization, trafficking, and release of MERS-CoV into the cytoplasm [More recently, there is accumulating evidence that the TPC complex is of critical importance for the infectivity of coronaviruses (MERS and SARS). For instance, an NAADP-mediated Caytoplasm . Later oytoplasm . Viral tIn summary, above mentioned studies indicate that the development of pharmacological modulators of TPCs might be promising for the treatment of a range of viral infections.There is a huge number of transmembrane receptors whose signaling is regulated or terminated via receptor endocytosis, recycling, and/or lysosomal degradation. Among these, the epidermal growth factor receptor (EGFR) seems to be an ideal candidate to study the role of TPCs during these processes . AlthougThe deletion of TPCs in MEF cell lines resulted in an increased uptake of the EGF–EGFR complex and altered trafficking when cells were incubated with high concentrations of EGF. TPC1/2 double knockout cells accumulated even more EGF than single knockouts. Interestingly, the use of Rab5 as a marker of early endosomes uncovered that TPC2 and TPC1/2 double knockouts showed numerous examples of co-localization of Rab5- and EGF-positive vesicles, whereas wildtype and TPC1-deficient MEF cells exhibited only minor co-localization. These observations suggest that EGFR trafficking is more delayed in TPC2 knockout cells, which may be caused by a longer retention time of EGFR in late endolysosomal vesicles. Those data were collected using fluorescence microscopy and FACS-based approaches . A similTo further substantiate the role of TPCs for the EGFR network, the amount of total and surface accessible EGFR was quantified by Western blot analysis and EGF binding studies. For both approaches, the amount of EGFR was significantly increased in TPC single knockout cells but was highest in TPC1/2 double knockout cells. Additional experiments using cycloheximide as a protein synthesis inhibitor demonstrated that there were no significant differences in EGFR degradation kinetics between all genotypes and suggested that the higher expression levels of EGFR in TPC knockout cells cannot be attributed to an altered degradation process . A similFurther studies were designed to discriminate between the two major regenerative mechanisms of EGFR availability, recycling, and de novo protein synthesis. As a result, from regenerative binding studies and quantitative RNA analysis, only EGFR de novo synthesis remains as the main mechanism responsible for the increased levels of surface accessible EGFR .The results from the above mentioned experiments directly lead to a central question: what are the direct consequences of an increased EGFR expression for EGFR pathway related proteins? Initial evidence for quantitative changes in gene expression of EGFR pathway proteins following deletion of TPCs was obtained by an RNA sequencing analysis comparing wildtype and TPC1-deficient cells. The corresponding dataset indicated numerous quantitative changes in the gene expression levels of EGFR pathway proteins, whereby up- and downregulation were observed .Based on these data, we propose a close link between function of TPCs and EGFR trafficking and signaling. EGFR dimerization and activation are initiated by EGF binding; subsequently, EGFR recruits downstream signaling complexes and triggers specific cellular signaling cascades. An important element is the ongoing EGFR signaling in intracellular organelles as long as the receptor-kinase domain is accessible from the cytosolic side ,42. Duric-Jun was shown to be a major factor for Egfr gene transcription, it is a promising candidate [JNK signaling is a major factor that contributes to the high EGFR expression found in TPC-deficient cells. We hypothesize that a prolonged EGFR signaling in endolysosomal signaling platforms caused by deletion of TPCs results in increased JNK signaling, which, in turn, leads to increased Egfr expression [The question arises if further signaling pathways might be affected in the same way by inactivation of TPCs. Since andidate ,44,45. Apression .The massive effects of a TPC gene inactivation for the EGFR transportation network and for EGFR signaling lead directly to the investigation of EGFR-unrelated receptors and pathways. To achieve an unbiased approach, a detailed RNA sequencing analysis was performed as previously described . Three iGene ontology (GO) enrichment analysis enables the search for over-represented GO terms in large biological data sets, for example, differential gene expression analysis results. Each gene product is annotated to one or more GO terms and can thereby be classified with respect to its molecular function , its cellular component , and its biological processes  . By screGene ontology enrichment analysis using ClueGO  receptor was described for a continuous signaling in endosomal compartments . The TGF-β receptor is a cell surface receptor kinase and is activated by a plethora of secreted polypeptides of the transforming growth factor beta superfamily. Members of the TGF-β superfamily are, among others, activins, bone morphogenetic proteins (BMPs), Nodal, and TGF-β1, -β2, and -β3. TGF-β signaling is involved in embryonic and adult control of proliferation, differentiation, and motility of cells or tissue. Genetic mutation, malfunction, or dysregulation of members of this pathway are associated with chronic inflammatory diseases, cancer, and fibrotic disorders ,53.Using RNA sequencing data , we studThus far, we focused on receptors characterized by two features: (1) an altered expression in TPC1-deficient MEF cells and (2) an ongoing signaling in endosomal compartments following activation at the cell membrane. In contrast, the insulin receptor was not differentially expressed , and signaling in intracellular organelles is not described. Therefore, the insulin receptor is an ideal candidate to study the consequences of a TPC1 deletion for pathway-related proteins independent of the receptor expression level. In total, the expression of 55 transcripts related to insulin signaling was significantly changed in TPC1 knockout cells: 22 were up- and 33 were downregulated . DownstrTPCs are important regulators of intracellular trafficking. Within the endolysosomal system, they show a differential distribution with TPC1 predominantly found in early and recycling endosomes and TPC2 mainly expressed in late endosomes and lysosomes ,23,24,25Due to the dominant expression of TPC1 in early endosomes, the number of affected genes was highest in this compartment . In the context of clathrin-mediated endocytosis, five proteins were changed, and for clathrin-independent endocytosis, nine proteins were affected . The latTPCs are main regulators of intracellular trafficking. TPC1 is predominantly found in early and recycling endosomes, and TPC2 is mainly expressed in late endosomes and lysosomes. Therefore, genetic deletion of either TPC1 or TPC2 causes trafficking defects at various steps such as endocytosis, phagocytosis, recycling, or lysosomal degradation. These consequences emerge in all model systems investigated thus far: uptake of bacterial protein toxins, macropinocytosis and processing of some classes of virus, and endocytosis, recycling, and degradation of surface receptors in general. In this article, we present evidence that these defects are not restricted to trafficking only but go far beyond that. Of note, the use of cell lines in our studies restricts this general statement, but the concordant results provide an incentive to continue the work with appropriate in vivo models.JNK signaling was identified as a major factor that contributes to the high EGFR expression found in TPC-deficient cells. Significant changes were also found for clathrin dependent and independent endocytosis in TPC1 deficient cells, further corroborating an important role for TPC in different types of endocytosis.We focused on receptor signaling and on receptor-linked pathways by investigating the transcript levels of receptors and pathway related proteins. A detailed RNA sequencing of TPC1 deficient MEF cells uncovered massive changes in transcript levels when compared with wildtype cells. In particular, we observed transcriptional changes in pathway related proteins for receptors that are characterized by an ongoing endosomal signaling but also for receptors that are active exclusively at the plasma membrane. For the latter, we supposed that cross-linking of pathways explained the observed alterations. In the context of EGF receptor expression,