Furthermore, TLE MOIs were significantly shorter than ETE  and presented higher jerking levels . Our newly implemented 3D approach is faster by 87.5% in extracting body motion trajectories when compared to a 2D frame by frame tracking procedure. We conclude that this new approach provides a more comfortable , simpler, faster and lower-cost procedure than previous approaches, therefore providing a reliable tool to quantitatively analyze MOI patterns of epileptic seizures in the routine of EMUs around the world. We hope this study encourages other EMUs to adopt similar approaches so that more quantitative information is used to improve epilepsy diagnosis.Epilepsy is a common neurological disorder which affects 0.5–1% of the world population. Its diagnosis relies both on Electroencephalogram (EEG) findings and characteristic seizure−induced body movements − called seizure semiology. Thus, synchronous EEG and (2D)video recording systems (known as Video−EEG) are the most accurate tools for epilepsy diagnosis. Despite the establishment of several quantitative methods for EEG analysis, seizure semiology is still analyzed by visual inspection, based on epileptologists’ subjective interpretation of the movements of interest (MOIs) that occur during recorded seizures. In this contribution, we present NeuroKinect, a low-cost, easy to setup and operate solution for a novel 3Dvideo-EEG system. It is based on a RGB-D sensor (Microsoft Kinect camera) and performs 24/7 monitoring of an Epilepsy Monitoring Unit (EMU) bed. It does not require the attachment of any reflectors or sensors to the patient’s body and has a very low maintenance load. To evaluate its performance and usability, we mounted a state-of-the-art 6-camera motion-capture system and our low-cost solution over the same EMU bed. A comparative study of seizure-simulated MOIs showed an average correlation of the resulting 3D motion trajectories of 84.2%. Then, we used our system on the routine of an EMU and collected 9 different seizures where we could perform 3D kinematic analysis of 42 MOIs arising from the temporal (TLE) (n = 19) and extratemporal (ETE) brain regions (n = 23). The obtained results showed that movement displacement and movement extent discriminated both seizure MOI groups with statistically significant levels (mean = 0.15 m vs. 0.44 m,  Epilepsy is a common neurological disorder which affects 0.5–1% of the world population. The main symptom of epilepsy are epileptic seizures which typically occur unexpectedly. In between seizures most individuals with epilepsy function normally. Video-EEG monitoring is the most accurate tool for diagnosis and differential diagnosis of epilepsy. The seizure semiology and its relations to changes in the electroencephalogram (EEG) are the cornerstone of this diagnostic method  that that4] tThe limitation of the 2D approaches were obvious: the difficulty of tracking the erratic MOIs, frequent marker occlusions and instability of the attached reflectors or sensors due to violent seizure associated movements. Eventually, the lack of precision when MOIs are not performed parallely to the image plane distorts the analysis. These limitations asked for a 3Dvideo-EEG system.An evolution in this direction, based on the integration of a high-speed (200Hz) 6 infrared motion tracking camera system  with a commercial 64/128 channels video-EEG system , has beNon-camera approaches for motion quantification in epileptic seizures have been presented relying on wearable inertial sensors such as gyroscopes, accelerometers or magnetometers. These approaches were used for seizure detection based on filtering  16], pr, pr16], Newly available RGB-D cameras (color and depth streams) such as the Microsoft Kinect , ASUS Xtp<0.05 level. Finally, we compare the time needed to perform the presented kinematics analysis using our 3D tracking method vs. using a corresponding 2D frame by frame tracking procedure we have used in the past in similar studies  [32 [3231] [The initial step of the tracking process consists in calculating the optical flow velocities over the RGB frames using the Horn-Schunk OF method  12] [31 [3112] [Pn be the centroid of the ellipsoid mask in frame n. The centroid in frame n+1 is given byv stands for the OF velocity vector in both horizontal (Hx) and vertical (Vy) components.Let Once the ellipsoid mask is defined, the median over the highest velocities vectors is calculated and then used to shift the centroid in the calculated proportion. The median was considered after performing the Jarque-Bera test  over 10 Zn and Zn+1 be the depth values associated with the points  and  in frames n and n + 1, respectively. Once the new centroid is calculated, the following criteria is applied:Let α—set by the user) when compared to the previous position, the centroid is labeled as valid and the mask is shifted automatically as defined by So, if the shifted mask centroid is within a certain depth range  into a custom made Matlab program that reads KiMA exported files. 3D quantification is performed after mapping the resulting 2D tracking information to the 3D Kinect coordinate system, using the available mapping method [Once movement tracking is completed, 3D quantification is performed by feeding the MOIs tracking time series  of velocity, acceleration and jerk (second derivative of velocity) . MovemenTo these 17 measures, we added a new parameter—3D movement extent (ME)–that is a generalization for 3D of a previous 2D measure we have been using for our 2D studies [Laterality is the maximum distance of a MOI trajectory in relation to the median planes (XY and YZ)—left/right; down/up and back/front, respectively. A criterion was also applied to assess whether there was a clear tendency of the MOI to travel in a certain direction. In such case, the distance between the MOI position and the medial plane is calculated .s stands for seizure and m represents the number of MOIs in that seizure. The same analogy is applied for the remaining variables.Finally, we introduce several new seizure quantification parameters—Seizure Movement Extent (SME); Seizure Covered Distance (SCD); Seizure Movement Displacement (SMD); Seizure Aggregate Velocity/Acceleration/Jerk , which correspond to 18 seizure quantification parameters. SME is the aggregation of the MEs of all MOIs involved on a given seizure. For example, if a seizure presents hand automatisms and head rotations, these parameters aggregates the ME of both hands’ MOIs and the head MOI resulting in the overall volume covered by that seizure MOIs, as defined in Given the dataset characteristics  we testep<0.05.All 56 + 23 seizure quantitative parameters for the two different seizure pattern groups  were compared using the Wilcoxon signed rank test . In addiMOIs were acquired at 200 Hz by the Vicon system and 20 Hz by KiT. To perform a comparison, the Vicon motion signals were down-sampled to 20 Hz. For all stimulated MOIs, signals were low-pass filtered and then compared visually and through a 3D correlation analysis using a Matlab custom-made program. The overall average correlation was 84.2% considering all 3 axes. Details of this study can be found in p<0.01). Significant differences were also found in the MOIs jerk, indicating that TLEs presented higher jerking than ETEs . Movement displacement also separated both seizure MOI groups .The results of the performed statistical analysis are summarized from Tables 3 m3, p<0.05).Additionally, the analysis of the movement extent separated TLE MOIs from ETE MOIs, as it was greater in the extratemporal than in the temporal seizures  and Y  direction, but also in the Z axis, namely in the labeled front movements .Based on the performed classification, we then evaluated whether there were any significant differences between TLE and ETE movements in the same direction. It can be seen  that thep > 0.05).We also evaluated whether there is any significant asymmetry between ictal movements of TLE and ETE seizures. For that purpose, three 2x2 contingency tables were designed (using the information available in p > 0.05) showed a trend of being longer than that of the TLE seizures. Also, the same trend is seen for the seizure movement extent, movement displacement and covered distance (p > 0.05).Finally, considering the seizure quantification parameters, presented in Comparing our semi-automatic approach with a state-of-the-art 2D motion analysis software, MaxTRAQ , that weThis feasibility study shows that our low cost 3D movement analysis system is easily usable in the clinical routine of a standard EMU setting and is capable to track and identify sets of MOIs which allow the discrimination between seizures arising from temporal and extratemporal brain areas. This system allows to overcome major limitations of our previous 2D marker-based system using a monochrome camera and infrared markers attached to the patient’s body .The direct comparison to a high cost multicamera system  shows thThe parallel use of a RGB-D camera with a conventional video-EEG recording allows full synchronization of the signals. This is particularly important for the evaluation of ictal EEG localization with regard to seizure related movements. It is well known that spread of epileptic activity over the brain leads to changes of characteristic seizure associated movements .Analysis of epileptic seizure semiology relies basically on qualitative criteria which makes it prone to inter-observer discrepancy . It has Our 3D analysis shows that TLE seizure related movements are faster and more confined in space than ETE. On the other hand, the ETE patterns are longer and cover a higher tri-dimensional volume. The ETE movement occurs at lower velocities and is progressive in space. For the purpose of this paper, we focused on the statistical analysis of the above mentioned movement parameters. However, other approaches would also be of interest such as pattern recognition and machine learning. MOI pattern analysis did not reach statistical significance to discriminate TLE and ETE seizures in this study, probably due to the low number of seizures included in this pilot feasibility study.http://brain.inesctec.pt/) was one of the first 500 groups being granted with this new sensor, in early 2014. The sensor has been re-engineered with wider field-of-view, higher resolution of the color data (1920 × 1080 High-Definition), higher depth fidelity due to the use of time-of-flight technology, more stable body tracking algorithm and overall SDK improvements [Kinect for Windows Developer Program Preview  enabled ovements .Kinect v2 sensor was recently integrated into the 3Dvideo-EEG system. From the acquisitions carried out so far, Kinect v2 seems to provide better information than the Kinect v1 sensor used in the present study, as it can be seen in In this contribution, we introduce a novel 3D-video-EEG system, based on a low-cost markerless motion capture system that has been integrated into a routine EMU setting, allowing 24/7 patient monitoring. The system has been in operation for approximately one year and is producing valuable data every day now. To our knowledge, we are gathering the only 3D-video-EEG database in the world used in a routine clinical epileptology department. The present paper is a report of the first version of this system and the first dataset extracted form our multimedia clinical database, approaching the methods behind the acquisition and processing of seizure semiology. A new version of the system is already producing data which is now equipped with the new Kinect v2 sensor. It is providing more accurate information than the previous sensor, and we are also able to relate the quantified motion information here reported with EEG events occurring in the course of the seizure. Automation methods are not covered in this paper but we intend to do so in the coming publications. Seizure types or psychogenic vs. real seizure separation and seizure automated detection are in the horizon of the next challenges we are already tackling.We also believe that the low-cost aspect of the system has the potential and the strengths to be spread and deployed in the routine of multiple epilepsy units around the world. We are open to receive requests from other EMUs around the world so that our development is used for the benefit of the larger number of patients possible.To the best of the authors’ knowledge, the results here reported constitute the first usage of a Kinect sensor (both v1 and v2) in a real hospital environment, in the context of seizure semiology analysis.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(MP4)Click here for additional data file.


Plasmodium falciparum circumsporozoite protein (CSP) from the 3D7 reference strain and has shown modest efficacy against clinical disease in African children. It remains unclear what aspect(s) of the immune response elicited by this vaccine are protective. The goals of this study were to measure diversity in immunogenic regions of CSP, and to identify associations between polymorphism in CSP and the risk of P. falciparum infection and clinical disease. The present study includes data and samples from a prospective cohort study designed to measure incidence of malaria infection and disease in children in Bandiagara, Mali. A total of 769 parasite-positive blood samples corresponding to both acute clinical malaria episodes and asymptomatic infections experienced by 100 children were included in the study. Non-synonymous SNP data were generated by 454 sequencing for the T-cell epitopes, and repeat length data were generated for the B-cell epitopes of the cs gene. Cox proportional hazards models were used to determine the effect of sequence variation in consecutive infections occurring within individuals on the time to new infection and new clinical malaria episode. Diversity in the T-cell epitope-encoding regions Th2R and Th3R remained stable throughout seasons, between age groups and between clinical and asymptomatic infections with the exception of a higher proportion of 3D7 haplotypes found in the oldest age group. No associations between sequence variation and hazard of infection or clinical malaria were detected. The lack of association between sequence variation and hazard of infection or clinical malaria suggests that naturally acquired immunity to CSP may not be allele-specific.The malaria vaccine candidate RTS,S/AS01 is based on immunogenic regions of  Plasmodium falciparum blood stage antigens found that immune responses to these antigens may be allele-specific, and a field trial of a vaccine based on one of these antigens reported allele-specific efficacy To date, malaria vaccine development and testing has generally not been informed by molecular epidemiologic evaluations of how genetic diversity in vaccine antigens in parasite populations may affect vaccine efficacy. For example, vaccines that confer allele-specific protection may not be effective in a parasite population in which the vaccine allele is rare, and may create a selective advantage favoring non-vaccine alleles, compromising vaccine efficacy P. falciparum circumsporozoite protein (CSP), encoded by the cs gene. The cs gene is polymorphic, with diversity in regions that encode epitopes recognized by the human immune system. The central repeat region of the cs gene contains tetrameric repeats that vary in both the sequence and number of tetramers. This region codes for epitopes recognized by anti-CSP antibodies cs gene, Th2R and Th3R, encode epitopes that are recognized by CD8+ and CD4+ T-cells The malaria vaccine RTS,S/AS01 targets specific immunogenic epitopes of the Follow-up studies to Phase 2 trials of the vaccine evaluated evidence of selection of non-vaccine strains in vaccinated vs. non-vaccinated study participants. In a Phase 2 trial of RTS,S in children in Mozambique no evidence of selection favoring non-vaccine strains was found cs gene which encodes the B-cell epitopes of CSP and which is also included in the vaccine, was not considered.Both of these studies used direct sequencing to detect polymorphism in the regions coding for the T-cell epitopes, Th2R and Th3R, and excluded samples that could not be resolved into predominant alleles from the analysis. Moreover, diversity in the central repeat region of the P. falciparum antigens, were associated with a higher risk of clinical malaria To design more effective malaria vaccines and to help interpret efficacy data from vaccine trials, an understanding of the dynamics of polymorphism in vaccine antigens and the factors that are driving that polymorphism is necessary. With the aid of next generation sequencing to help circumvent some of the issues encountered by previous studies of genetic diversity in CSP, this study has examined both the population level and within-host dynamics of polymorphism in this vaccine antigen. We have previously shown that in consecutive infections experienced by Malian children followed in a prospective cohort study, changes at specific polymorphic sites, but not at other polymorphic sites, within two blood-stage The study was approved by the institutional review boards of the University of Sciences, Techniques and Technology Faculty of Medicine, Pharmacy and Dentistry in Bamako, Mali and the University of Maryland, Baltimore. Informed consent was obtained from all study participants or their guardians. The trial was conducted in compliance with the International Conference on Harmonization of Good Clinical Practices, the Declaration of Helsinki, and regulatory requirements of Mali.falciparum malaria is intense with the peak coinciding with the rainy season from July–October. Children aged less than 10 years experience, on average, two clinical episodes a year, and the prevalence of parasitemia at the beginning of the study before the onset of malaria transmission was 17% The study was conducted in Bandiagara, a rural town of approximately 13,000 inhabitants located in central Mali. Transmission of A complete population census was conducted in Bandiagara before study initiation. Study participants were sampled in proportion to the population size in each of the eight districts comprising the town of Bandiagara. Study subjects were aged ≥3 months to 20 years. Recruitment of study subjects took place in randomly selected households until the target number of subjects in each age group was achieved.P. falciparum asexual parasites and symptoms consistent with malaria including, fever, anemia, headache, body aches, cough, diarrhea, or abdominal pain, in the absence of any other obvious cause of symptoms. Infections were defined as the presence of falciparum parasites in the blood, with or without symptoms The study was conducted prospectively during the years 1999, 2000, and 2001. From July to January of each year, blood samples were collected on 3 MM Whatman filter paper monthly and at every episode of clinical malaria. Clinical malaria episodes were detected both by passive surveillance through provision of around-the-clock free clinical care, as well as by active weekly follow-up of the children in the study by physicians working at the study site. Clinical episodes were defined as blood smears positive for One hundred children with at least two years of follow up during the malaria incidence study were chosen GTTGAGGCCTTTTCCAGGAATACCAG and GTACAACTCAAACTAAGATGTGTTC. These primers were designed to amplify the region of the cs gene that contained both the repeat region and the Th regions. Primary PCR conditions are as follows: 30 cycles of 95°C for 30 s, 52°C for 30 s, 72°C for 1 min. The secondary forward and reverse PCR primers for the repeat region were TGGGAAACAGGAAAATTGG and GCACTGTTGGCATTAGCATTT. Secondary PCR conditions for the repeat regions and ThRs were 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min 30 s, and 25 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min respectively. PCR products were amplified using HotStar Taq . Secondary PCR primers for the ThRs contained specific adapters necessary for the emPCR CGTATCGCCTCCCTCGCGCCATCAG and CTATGCGCCTTGCCAGCCCGCTCAG. Sequence specific primers for the Th and repeat regions were previously reported in a methods paper The primary forward and reverse PCR primers were The concentration of each PCR product containing Th2R and Th3R was determined by band intensity measurements taken on the Qiaxcel capillary gel imaging system , and 100 ng of each product was then pooled. Identical barcodes were used to tag more than one sample. PCR products were separated into sixteen separate pools containing approximately 45 samples each ensuring that no pool contained more than one sample with the same barcode. Each pool was physically separated within a 454 sequencing run. Pooled PCR products were sequenced at the University of Maryland School of Medicine Genomic Resource Center at the Institute of Genome Sciences on the GS FLX Titanium 454 Platform . Sequences were aligned using gsAmplicon  software. For samples containing more than one allele at a polymorphic site, predominance was determined if the majority allele was present in 71% or more of all reads obtained for that sample. This cut-off was determined in the methods validation study performed by the same authors comparing 454 and Sanger sequencing The methods validation study established the comparability of these methods with direct sequencing as well as comparable majority/minority allele cut-off values. The results of that study indicated that 454 was more sensitive at detecting minor alleles and more accurate in the quantitation of these alleles than direct sequencing To determine the length of the repeat region, PCR products were run on a high resolution gel cartridge on a Qiaxcel capillary gel imaging system  using the OM500 analysis method capable of resolving size within 10 base pairs (bp). To validate this method, a 96-well plate containing 15 ul of repeat region PCR product amplified from 100 ng/ul 3D7 genomic DNA per well, was run using the method listed above. A systematic underestimation of 10 bp was observed in the experimental data. A 10 bp correction was therefore applied to length values obtained for PCR products from study samples.Repeat region PCR products for samples that were determined to be ‘single clone’  with respect to Th2R and Th3R, were subjected to Sanger sequencing. Once amplification was verified by gel electrophoresis, PCR Products were purified by vacuum filtration in Excela Pure  96-well plates. Purified PCR product was then sequenced on an ABI3730 xl at the University of Maryland School of Medicine Biopolymer Lab.Sequences were viewed in Sequencher , and then copied to Transeq web software Mixed infections with respect to the ThRs were defined as samples with no clear majority allele at a given polymorphic site. Mixed infections with respect to the repeat regions were defined as samples which contained more than one clear band in high-resolution gel analysis.Fisher’s exact tests were used to compare haplotype frequencies between seasons, age groups, and between clinical and non-clinical episodes. Cox proportional hazards models were used to infer the relationship between diversity in Th2R and Th3R and the development of naturally acquired immunity in the context of symptomatic  and asymptomatic parasitemia (infection). In the clinical model, the association between changes at polymorphic sites in Th2R and Th3R which occurred between consecutive clinical episodes and the hazard of clinical disease was examined. In the infection model, the association between these changes which occurred between a clinical episode and a consecutive asymptomatic infection was examined. Intervals starting with an asymptomatic episode were included in the model if there was an intermediate time point at which the study participant was parasite-negative verified by microscopy. To account for the possibility of treatment failure and to allow for time for allele-specific antibodies to the first of two paired consecutive infections to be present by the time of the second infection, time intervals two weeks or less between consecutive episodes were excluded from the analyses. A logistic regression analysis was performed to determine the odds of a change in the predominant amino acid at polymorphic residues in intervals including an asymptomatic episode followed by a symptomatic one, to intervals including consecutive asymptomatic episodes. Age and time between consecutive intervals were included as covariates in this model.Of the 769 parasite-positive samples, 63 were missing or did not have enough material for successful PCR amplification. With respect to the Th regions, DNA was successfully amplified and sequenced from 684 (97%) of the samples. Repeat region PCR products and fragment length data were successfully generated for 504 (74%) of the samples. Of these 504 samples, 157 were successfully sequenced via Sanger sequencing.A total of 31 and 14 nonsynonymous SNPs were detected by 454 sequencing with an average coverage of ∼600x  in Th2R and Th3R, respectively . There wThe number of unique haplotypes found in Th2R and Th3R respectively was 87 and 23 . GenbankFor the purposes of this study, polyclonal infections were defined as samples that contained no clear majority allele . With respect to Th2R, 34.9% of samples were mixed, and with respect to Th3R, 29.8% of samples were mixed. Haplotype distributions did not vary significantly by age group, study year, or presence of clinical symptom for either Th2R or Th3R, with the exception of the Th2R 3D7 haplotype, which had a significantly greater prevalence in the oldest age group compared to the youngest age group p<0.03,  and 2.The prevalence of different repeat region sizes did not vary significantly by age group, study year, or presence of clinical symptoms . The mosAmong the 157 samples for which Sanger sequencing data were available, 19 unique haplotypes were detected, of which six were detected only once in the three year study period. Genbank accession numbers for the nucleotide sequence of these haplotypes are JQ868451–JQ868469. The majority of samples (67.5%) had one of three most prevalent haplotypes . When SaThere was no significant change in hazard of infection or clinical disease when a change occurred at any of the 17 polymorphic amino acid positions identified in Th2R and Th3R, or when a change occurred in the size of the repeat region  and 6. TThere was no significant difference in the odds of a change occurring between an asymptomatic episode followed by a symptomatic one compared to consecutive asymptomatic episodes, taking into account age and length of interval between episodes, for either the Th regions or the repeat region . Four ofMolecular epidemiological studies are an important tool for evaluating the potential of polymorphic malaria vaccine antigens to provide strain-transcending protective efficacy By using 454, this study was able to include samples containing more than one parasite clone, circumventing a problem that previous studies evaluating the allele-specificity of immunity to CSP have faced. Complete sequence reads were available for each parasite type detected by 454 so haplotypes could be constructed. In 684 samples, 87 haplotypes were found in Th2R, whereas in earlier studies using Sanger sequencing on samples from other West African settings with similar malaria epidemiology, 24 haplotypes were found in 44 Gambian samples Repeat region size determination was very accurate, with 94% agreement between lengths obtained by direct sequencing and those determined by high-resolution gel analysis. Using this method made it possible to examine the association between repeat region size and increased risk of infection and disease. Although the number of samples successfully sequenced was limited, results from one-third of parasite positive samples showed that 67% of successfully sequenced samples had one of three haplotypes with different lengths. This finding suggests that length polymorphism may serve as a reasonable surrogate for haplotype in the Cox and logistic regression models.This study had some limitations which may have affected the results. Due to a large degree of polymorphism in the ThRs and a limited sample size, smaller changes in the hazard ratios between consecutive episodes in which there was a change versus no change for both clinical disease and infection could have been missed. For seven of the 23 polymorphic sites that were modeled there may not have been enough observations to fully examine associations with infection and disease. Additionally, effects of sequence variation on hazard of clinical disease or infection were not examined with respect to the repeat region due to the limited number of sequences generated. Finally, determination of polyclonal infections for the repeat region was done by capillary gel analysis, which may have been less sensitive than sequence analysis.cs gene being under diversifying selection The large number of SNPs and haplotypes in our study is consistent with this region of the falciparum parasites. Combined molecular-immuno-epidemiological studies may help to assess these possibilities.If the diversity in immunogenic regions in CSP is not the target of allele-specific immune responses, the question of what is driving this diversity remains to be answered. It has been proposed that diversity in the repeat region might have evolved as an immune evasion mechanism by the parasite in which the host mounts a non-protective immune response against this region while the parasite escapes into the liver


There has been no population-based study on human papillomavirus (HPV) prevalence or its genotypes in Bangladesh; a country eligible for GAVI funding for HPV vaccine.We used baseline survey data of a prospective cohort study that was conducted in one urban and one rural area of Bangladesh. A total of 997 urban and 905 rural married women, aged 13 to 64 years, were enrolled in the baseline during July-December, 2011. Information was collected on socio-demographic characteristics and potential risk factors for HPV infection followed by gynecological examination and collection of endocervical samples using the cervical cytobrush . HPV DNA testing was done by Polymerase Chain Reaction (PCR) using a consensus primer set.Prevalence of any HPV infection was 7.7% with no significant difference between urban and rural women. Most common high-risk genotypes were HPV16, HPV66, HPV18, HPV45, HPV31 and HPV53. Urban women working as housemaids or garment workers were at higher risk of any HPV infection  compared to housewives. Rural women whose husband lived overseas were almost two times more likely to have any HPV infection  compared to women whose husbands lived with them.The prevalence of HPV infection among Bangladeshi women is similar to other regions of Asia. However, type-specific patterns are different. The study findings will inform the formulation of HPV vaccination policies in Bangladesh, monitoring the impact of vaccination programmes, and the identification of target populations for screening. Cervical cancer is a major public health problem worldwide. It is the fourth most common cancer in women with an estimated 527,624 new cases and 265,653 deaths in 2012 Human papillomavirus (HPV) is one of the most commonly acquired sexually transmitted infections (STIs) and a significant source of morbidity and mortality The prevalence of HPV infection has shown to vary by region, country and within a country by population sub-groups The literature identifies several risk factors for the acquisition and persistence of HPV infection. Age is a strong predictor, although there are notable differences in age curves of HPV infection across regions While some epidemiological research on HPV has been conducted in the South Asian region The baseline survey was conducted between July and December 2011 and was implemented in two areas of Bangladesh; one rural and one urban . The urbThe rural site of the study is located in Mirzapur, a sub-district of Tangail district, which is 56 kilometers northwest of Dhaka. In Mirzapur, icddr,b has been running a demographic surveillance since 2007 covering a population of approximately 240,000 in eight unions . We purposely selected two of the eight unions  that comUsing systematic random sampling, every third woman in the sampling frame in both urban and rural areas was approached and asked to participate in the study. In the field, the selected women were identified by their name and address recorded in the listing database (urban site) or in the surveillance data base . In both the urban and rural areas, the clusters and villages under study were purposely selected for feasibility issues considering proximity to icddr,b’s field offices, logistics and cost-related issues.The study protocol was approved by the Ethical Review Committee (ERC) of icddr,b. Informed written consent was taken from women aged 18–64 years whereas informed written assent was taken from women aged 13–17 years. For the later group, the assent form was read out to these women in the presence of their parents/legal guardians. If agreed, the assent form was then signed by the participants in the presence of their parents/guardians. The parents/guardians signed the same form.In both study areas, women were first approached at their home and eligibility was determined by trained female research staff. They were briefed about the study background and purpose, and the potential roles they would have in the study. Women who were eligible and willing to participate were sent to a study field office for interview, clinical examination and sample collection. In the case of a refusal, the next eligible woman on the list was approached.Upon arrival at the study field office, a female study physician provided them with detailed information about the study process including the process for collecting the cervical sample. On agreeing to participate in the study, the women were further screened for eligibility criteria by the physician; upon meeting these criteria they were then taken to the next room for interview. Women who were pregnant, or post-partum, or with a history of hysterectomy, cervical cancer or uterine prolapse were excluded from the study. Women with major psychological/psychiatric problems were also excluded.After informed, voluntary, written consent or assent, six trained female research assistants collected information on socio-demographic characteristics , sexual and reproductive history  and other risk factors, such as use of oral contraceptives, tobacco and condom use. Two trained female research physicians performed clinical examinations, including visual examination of the vulva. Following this, the speculum was inserted and the vagina and cervix were examined for any discharge, erosion or lesions. The physician then collected cervical samples using the cervical cytobrush . Following sample collection slides for cervical cytology were prepared The head of the brush was then detached in 10 ml BD SurePath  and stored at 4°C. The specimens (including slides) were then transported to the virology laboratory of icddr,b maintaining appropriate temperature and stored at 4°C until tested. While HPV DNA testing was performed at icddr,b, the cervical slides were sent to the pathology department of Bangabandhu Sheikh Mujib Medical University (BSMMU) for testing. The results of both the HPV DNA test and cervical cytology were provided to the study participants within 2–4 weeks of sample collection. Women presenting the possibility of precancerous or cancerous changes were referred to BSMMU for further assessment and treatment. The details of cervical cytology will be published elsewhere.A 6ml aliquot of cervical swab sample was centrifuged at 5000rpm for 15 min. After decanting the supernatant, the pellet was resuspended in 500 µl phosphate buffer saline. DNA extraction was carried out with a 200 µl aliquot of this concentrated sample using a DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions. The extracted DNA was stored at −20°C until tested.HPV genotyping was carried out in three steps: 1) Detection of HPV L1 gene using PCR; 2) Genotyping using nucleotide sequencing, and 3) Identification of co-infection and genotypes of a small number of HPV positive cases for which typing could not be done in step 2.The first round of PCR amplification was carried out in a 25 µl reaction volume containing 10 µl extracted DNA as a template, 2.5 µl Qiagen 10× PCR buffer, 0.25 µl Hotstar Taq polymerase  (5 U/µl), and 20 pmol papillomavirus specific consensus primers MY09/MY11 HPV positive PCR products either visible in the first or second PCR round were sequenced using respective PCR-product primers with a Big-Dye Xterminator ready reaction kit v3.1  in an ABI 3500xL genetic analyser . Genotyping of HPV was inferred by comparing the resulting sequence for each sample to the GenBank database using BLAST . This method identified HPV types in all samples except six.To identify co-infections, all HPV positive samples were reassessed with a commercially available real time PCR kit . The test specifically identifies HPV16 and HPV18 while concurrently detecting the rest of the 10 high risk types  and two possible high-risk type  in a pooled result (other HR-HPV other than 16/18) and any of the 5 LR-HPV types . The reassessment also assisted us to identify genotypes of six HPV positive cases for which typing could not be done using PCR sequencing in step 2.5′-CAACTTCATCCACGTTCACC-3′), PC04 (5′-GAAGAGCCAAGGACAG GTAC-3′) and HMBB01 (5′-GCGACCCAATGCAAATTGGT-3′) To verify the presence of human genomic DNA in samples, a 268 bp fragment of the β-globin housekeeping gene was amplified using primers GH20  agreed to participate; interviews were completed and samples collected for 1,113 (97%) women. Out of these, 997 women were currently married and having an active sexual relationship with their husband. Similarly, in the rural area, a total of 1,499 eligible women were approached, of which 1,159 (77%) agreed to participate. Interviews were completed and samples collected from 924 (80%) women, of whom 905 women were currently married and having an active sexual relationship with their husband. The rest of the women who were approached but not included in this study either refused to take part , did not show up in the project office on the scheduled day of interview , or were found ineligible after examination by the study physicians . Of the women who were interviewed and from whom samples were collected, some were either divorced, widowed or separated at the time of the interview  and information on their husband’s residence or condom use was not appropriate to collect and therefore, these women were excluded from the analysis.While the majority of women resided with their husbands in both areas , a higher proportion of rural women had a husband living overseas (18%) compared to urban women (6%). About half of the women had three or more children with no difference in the mean number of children between urban and rural women. Most women in both areas reported one lifetime sex partner. While the majority of women reported their husband to have one lifetime sex partner, one in five women reported their husband as having more than one lifetime sex partner, with no difference between urban and rural women. Ever use of oral contraceptives was slightly higher among rural women compared to urban women ; whereas ever use of condoms was significantly higher among urban women compared to rural women .Overall, the prevalence of any HPV infection was 7.7% with no significant difference between urban and rural women . A total of 93 women had single-type HPV infection  and 52 women had multiple HPV infection . HR-HPV infections, single and co-infection combined, were more common (4.2%) than LR-HPV infections (3.4%) with no significant variation between urban and rural women. The most commonly found HR-HPV types in either single or co-infection were HPV16, HPV66, HPV18, HPV45, HPV31 and HPV53. Other HR-HPV types included HPV33, HPV35, HPV52 and HPV58 .Age-specific prevalence of any HPV infection and infection by HR and LR-HPV types are shown in A slightly different HPV infection pattern was observed for rural women. Age-specific prevalence decreased from 9.3% at 24 years or below to 8.3% and 4.4% respectively for the next two age groups of 25–34 and 35–44 years; thereafter, it increased to 7.5% for women aged 45 years and older . When stOccupation had the strongest effect on HPV infection for urban women. Urban women who were working as housemaids or garment workers were at higher risk of any HPV infection  compared to those who reported being housewives. The risk doubled for infection with HR-HPV subtypes  for this group of women.Parity was found to be associated with any HPV infection for rural women only. In particular, having three or more children lowered the risk of any HPV infection for rural women . Similarly, husband’s residency status was found to be associated with any HPV infection for rural women. Rural women whose husband lived overseas were almost twice as likely to have any HPV infection  compared to those women whose husbands lived with them.The number of lifetime sex partners of urban women was significantly associated with detection of any HPV infection; women reporting two or more partners were almost four times more likely to have any HPV infection compared to those reporting only one sex partner . We did not find any association between the numbers of lifetime sex partners of women with HR-HPV infection. We also did not find any association between the number of the husband’s sex partners with the risk of any HPV infection or HR-HPV infection. No effect of contraceptives or tobacco use was found in our analysis. We did not have enough rural samples to investigate the effect of occupation or number of sexual partners on HPV status.To the best of our knowledge, this is the first study to investigate the prevalence of HPV infection and its type-specific distribution in population-based samples of urban and rural women in Bangladesh. The overall prevalence of any HPV infection was found to be 7.7% in this study. This is similar to the prevalence reported in other Asian countries –9% in Nepal HR-HPV was more common than LR-HPV infection in almost all age groups, except among rural women older than 44 years. HPV16 was the most frequently detected type, as found in other parts of the world, including Asia Similar to other countries, we found HPV infection to be most common in younger women with the peak prevalence occurring in women younger than 25 years of age; prevalence started to decline after 30 years of age. We found a secondary increase after age 44 for rural women and ages 35–44 for urban women. In Bangladesh girls marry at an early age and the study was conducted among married women. This may explain high HPV infection in the younger age group. However, the secondary peak seen during 35–44 years in urban women and 45–64 years for rural women requires further explanation.Bangladesh has made remarkable progress in terms of population, economic and health indicators in recent years Occupation was also a significant predictor for HPV infection in the rural study site. In this case however, it was the risk posed by the occupation of women’s husbands. Economic opportunities outside of Bangladesh draw rural men to leave their families for long periods of time for work overseas. This may increase their opportunity for engaging in risky sexual behaviors, thus increasing their wives vulnerability to HPV infection. All these factors may help to explain the secondary peak of HPV infection in our study in women in their late 30′s and 40′s.While we found a similar prevalence of any HPV infection and HR-HPV infections among rural and urban women, we found some important differences in risk factors for infection between rural and urban women. Education had a protective effect for rural women, but not for urban women. Urban women working as garment workers or housemaids were at an increased risk of HPV infection. For urban women, the number of sexual partners was highly associated with HPV infection, a finding consistent with other settings Another common economic opportunity for women in urban areas is to work as domestic servants or housemaids. This occupation may also expose women to increased risk of sexual exploitation as supported by other studies We did not find any association between oral contraceptive use with HPV infection as found in other studies Our study used population-based socio-economically diverse samples from two existing surveillance systems. However, limited participation  may raise questions about the representativeness of the study findings. To address this, we examined the basic socio-demographic characteristics of those participating in the study and those who did not, and we found the two groups to be similar, suggesting limited plausibility for selection bias.Our study adds immense value to the existing knowledge of the burden of HPV infection among the selected urban and rural female population in Bangladesh. The identification of genotypic distribution will help further understanding of the risk of developing cervical cancer in the study population. While we found HPV16 to be the predominant type, HPV66 was the second major HR-HPV type identified in our study population, followed by HPV18. Epidemiological evidence of the carcinogenicity of HPV66 was previously judged sufficient; however, HPV66 has rarely been identified in populations Given that HPV infection is a necessary cause of cervical cancer and preventing it is the most effective way to prevent cervical cancer. HPV vaccines have been proven to be an effective and safe measure to prevent HPV infection and disease related to the vaccine-specific genotypes in women with no evidence of past or current HPV infection This study provides the first population-based HPV prevalence data in Bangladesh. While the findings are similar to studies from neighboring countries, the type-specific distribution, as well as identification of risk factors will provide additional evidence to guide Bangladesh’s response through a cervical cancer prevention programme. The findings are especially timely as the Government of Bangladesh is planning to apply to the Global Alliance for Vaccines and Immunization (GAVI) for funding for pilot testing of the HPV vaccine in a peri-urban setting. The evidence from this study will provide necessary country-specific evidence for this application.


Drosophila larvae and its neural basis. Drosophila larvae display defecation cycles of stereotypic frequency, involving sequential contraction of hindgut and anal sphincter. The defecation behavior requires two groups of motor neurons that innervate hindgut and anal sphincter, respectively, and can excite gut muscles directly. These two groups of motor neurons fire sequentially with the same periodicity as the defecation behavior, as revealed by in vivo Ca2+ imaging. Moreover, we identified a single mechanosensitive sensory neuron that innervates the anal slit and senses the opening of the intestine terminus. This anus sensory neuron relies on the TRP channel NOMPC but not on INACTIVE, NANCHUNG, or PIEZO for mechanotransduction.Defecation allows the body to eliminate waste, an essential step in food processing for animal survival. In contrast to the extensive studies of feeding, its obligate counterpart, defecation, has received much less attention until recently. In this study, we report our characterizations of the defecation behavior of DOI:http://dx.doi.org/10.7554/eLife.03293.001 In animals, the final stage in the digestion of food is the removal of waste from the body. Until recently, however, defecation has received less attention than other aspects of digestion such as feeding behavior and nutrition.Drosophila, are commonly used in research as a model of animal biology. Food is moved through the digestive tract of fruit fly larvae when the muscles that circle the wall of the intestine contract. This process continues until the waste reaches the anus and is expelled from the body.Fruit flies, also known as Now Zhang et al. have found that when fruit fly larvae defecate, the muscles at the end of the intestine contract just before the muscles in the anus contract. The nervous system controls these muscles via sequential firing of two sets of nerve cells that connect to the intestine and anus muscles, respectively.Zhang et al. also identified a nerve cell that can sense when the anus is opened and relay this information back to the nervous system. The nerve cell is activated when stretched by the opening of the anus in a process that requires a protein called NOMPC.Problems with defecation can lead to constipation and other diseases. For example, Hirschsprung's disease—a birth defect that affects one in 4000—is caused by abnormal development of the nerve cells that control muscles in the gut. Experiments on fruit flies could help us to understand how defecation works in humans and to develop new treatments for disease.DOI:http://dx.doi.org/10.7554/eLife.03293.002 Defecation is important for food processing that provides nourishment to the animal. It eliminates waste (feces) from the digestive tract via the anus , an unglDrosophila larvae provide a useful model system for the studies of feeding behavior and nutrition intake   . Little Drosophila. Studies in adult flies have demonstrated that internal sensory neurons are important in regulating behaviors such as feeding, defecation, and egg laying . First, we show that Drosophila larvae exhibit rhythmic cycles of sequential contractions of the hindgut and the anal sphincter to expel feces. Second, we identify the motor neurons that innervate the hindgut and anal sphincter and show that these two groups of neurons fire sequentially with the same periodicity as the defecation cycle. Unexpectedly, we found that a single sensory neuron innervates the anal slit to sense its opening. Finally, we show that the TRP channel NOMPC but not other known mechanosensitive channels in Drosophila is required for the mechanosensation of this anus sensory neuron.In this study we establish Drosophila larval hindgut is the last part of the intestine, posterior to the Malpighian tubule, on the dorsal side under larval cuticle. At the posterior end of the hindgut is anal sphincter, which has a layer of thick sphincter muscles and a much narrower canal , the cell bodies of the neurons that innervate the hindgut and anal sphincter are most likely in the terminal segments of the VNC. We identified two groups of neurons, labeled by PDF-Gal4 and HGN1-Gal4 , which ila vGlut  , a light activated cation channel, in the HGN1 neurons and recorded the excitatory junction potentials (EJPs) in the gut muscles before and after activating ChR2 by light. The gut muscles received tonic excitatory inputs . Due to To test whether direct activation of PDF or HGN1 neurons could trigger gut muscle contraction, we first expressed ChR2 in the PDF neurons and use a Minos insertion line to label the hindgut. Because the blue light used to excite GFP in the hindgut can also activate ChR2, we could activate the neurons expressing ChR2 and monitor the gut movements at the same time. Both the hindgut and anal sphincter contracted strongly  upon stiTo test whether PDF and HGN1 neurons are important for the normal defecation behaviors, we expressed Kir2.1 in these neurons to inhibit their activities. Silencing PDF neurons caused the interval of the defecation to increase from 38 s to 94 s . SilenciDrosophila larvae is associated with rhythmic firing of the PDF and HGN1 neurons innervating the hindgut and anal sphincter, we performed in vivo whole-mount Ca2+ imaging by monitoring the neuronal activity with a genetically encoded Ca2+ indicator GCaMP5  neurons are primary sensory neurons, which cover the entire body wall of a ulations . In the ulations . The majulations . There aulations . The axo2+ level with GCaMP5. We found that opening of the anal slit is accompanied with Ca2+ elevation in both dendrites and soma of this neuron (2+ response is similar to that of the movements of the anus and the oscillations of PDF neurons and HGN1 neurons in the VNC (This single anus sensory neuron (ASN) has its cell body stochastically located on one side of the midline but its dendrites are highly symmetric. When the anal slit opens, the dendrites are dramatically stretched . To inves neuron . The per the VNC . This re2+ elevation of the ASN axon terminals in the VNC . The ASN was completely abolished 48 hr after laser ablation, while the other PPK neurons remained intact . We thenThese results suggest that the central neurons in the VNC receive excitatory feedback from ASN to increase motor neuron firing. Together with the GRASP analysis revealing the physical proximity of the processes of ASN and PDF neurons, our results indicate that the sensory neuron in the anus might sense the stretch when the anal slit is open and respond by provide feedback modulation of the PDF neurons and HGN1 neurons in the central nervous system.iav, nan, and piezo mutants. However, the defecation interval in the nompC mutants was significantly increased . Flies are kept in 12 hr/12 hr dark/light circle at 25°C. PDF-Gal4, HGN1-Gal4, and UAS-ChR2 are from Bloomington stock center. GRASP was done using lines: PDF-loxA > loxAop-mCherry and HGN1-Gal4 > UAS-GFP or PPK-Gal4 > UAS-GFP. w[1118]; Gr28b[MB03888] is a Minos insertion which is from stock center (#24190), UAS-GCaMP5 fly line is from Loren L Looger lab in Janelia Farm. GRASP between PDF neurons and HGN1 neurons: w; PDF-Lexa/UAS-CD4-sp1-10; HGN1-Gal4/LexAOp-CD4-sp11. GRASP between PPK neurons and HGN1 neurons: w; +/UAS-CD4-sp1-10; HGN1-Gal4/PPK-tdTomato-sp11. UAS-CD4-sp1-10; LexAOp-CD4-sp11 are from K Scott lab (UC Berkeley).For the whole-mounting imaging, a freely moving larva was picked up and rinsed with distilled water. Then the larva was transferred into 4% PFA overnight at 4°C. The larva was put between cover glass and images were taken by Zeiss confocal microscopy. In some cases, the whole VNC or different part of the gut was dissected out and mounted on a cover glass in PBS for imaging.Drosophila larvae, third instar larvae were dissected in PBS. The whole hindgut and anus were isolated from their bodies. The tissues were then fixed in 4% PFA solution for 20 min at room temperature and treated with the primary antibody  overnight at 4°C and secondary antibody for 2 hr at room temperature. Images were acquired with Leica SP5 confocal microscope.For immunostaining of Third instar larvae were picked up, rinsed, and transferred on to agar plate with yeast paste supplied with food dye . The larvae were fed with food dye for 2 hr and mounted between cover glasses for experiment.A hindgut-specific byn-Gal4 was crossed with UAS-GFP to visualize the hindgut. A third instar larva was gently picked up from the food surface and rinsed with distilled water briefly. The larva was then transferred into a drop of PBS on the slide. A cover glass was put on the larva and pressed slightly to reduce larval movement. The larva was mounted ventral side up under a Leica stereoscope and video-taped for later analysis.Mercury light filtered with a GFP filter was applied to the larva preparation with a certain duration of time. The movements of the hindgut and anus sphincter were video-taped for further analysis. The delay of contraction was calculated between the light onset and the anus sphincter contraction. Both hindgut and anus sphincter movements could be easily visualized with auto-fluorescence of food debris in the intestines.3, 1 NaH2PO4, and 4 MgCl2, adjusted to pH 7.25 and 310 mOsm. 2 mM Ca2+ was added to the saline fresh before use. Fat bodies were gently removed from the gut surface. Additional pins were used to immobilize the gut. The preparation was visualized by Zeiss axioscope microscopy with 40× water lens. Sharp electrode with resistance around 80 MΩ was filled with 3 M KCl. The electrode tip was approached to the gut surface under the control of the MP-285 manipulator . The signal was acquired by the Axon 200B amplifier and filter at 2 kHz. The electrode was moved forward until the voltage suddenly dropped to around −40 mV.Free moving third instar larva was pinned onto Sylgard coated chamber dorsal side up and filleted along the dorsal body wall. The larva was dissected in a saline containing: (in mM): 103 NaCl, 3 KCl, 5 TES, 10 trehalose, 10 glucose, 7 sucrose, 26 NaHCOBlue light was generated by mercury lamp with multiple filters. For ChR2 activation, a GFP filter was used to give out blue light with wavelength around 488 nm. The light application was controlled by a shutter equipped on the microscope. For pulse light activation, 2 s light pulse was repeated for three times. Recording data were analyzed with Clampfit and Matlab. The frequency before and during light application were calculated and compared as the index of activation.3, 1 mM NaH2PO4, 1.5 mM CaCl2, and 4 mM MgCl2 . The dissected VNC were transferred to a glass-bottom recording chamber containing recording saline and immobilized with a platinum frame. The HGN1 neurons were identified by their GFP signals under a 40× water objective. Current-clamp and voltage-clamp recordings were performed using patch-clamp electrodes (9–10 MΩ) filled with internal solution . Cells were used for recording if the Rm value was greater than 500 MΩ and the membrane potential value was lower than −50 mV. A small constant hyperpolarizing current was injected during recording, immediately after break-in, to bring the membrane potential of neurons to approximately −60 mV. Cells were held at −60 mV in voltage-clamp mode for EPSC recordings. Signals were acquired with an Axon-700B multiclamp amplifier and were digitized at 10 kHz and filtered at 2 kHz using a 1322A D-A converter. Data were analyzed using Clampex 9.0 software (Molecular Devices).The recordings were performed following the protocol described by A larva was mounted between two cover glasses with ventral side up. Its tail end was exposed for access of probe stimulation. A glass pipette was pulled and polished to form a taper-shaped probe with a diameter around 20 µm. The probe was spread on with grease to reduce friction. A piezo-controller was used to control the movement of the probe with fixed angle and increment. For imaging of ASN axons and PDF/HGN1 cell bodies, the probe was advanced 10 µm and the stimulation lasted for 1 s.ASN ablation was carried out as previously described . BrieflyA third instar larva was gently picked up from the food surface and rinsed with distilled water several times. The larva was transferred into a drop of PBS on the slide. A cover glass was put on the larva and pressed slightly to reduce larval movement. The preparation was then put under the Zeiss Pascal 510 confocal microscopy equipped with a 20× air objective. Time series images were acquired and used for analysis.For HGN1/PDF dual imaging, the larva was mounted with the lateral side up, so that both groups of neurons could be visualized simultaneously.For PPK neurons imaging, larvae were transferred to a piece of filter paper saturated with 100 mM sucrose for 4–6 hr to remove the food debris in the gut which could have potentially covered the neuron's images during experiments.An automatic alignment was made to most image series since the larvae tend to move slightly during image acquisition. An ImageJ plugin ‘registration ROI’ was utilized to correct the movements of the images during recording. review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 2 reviewers, one of whom, Howard Baylis, has agreed to reveal his identity.Thank you for sending your work entitled “Identification of motor neurons and a mechanosensitive sensory neuron in the defecation circuitry of Drosophila larvae” for consideration at The Senior editor and the two reviewers discussed their comments before we reached the decision that the manuscript needs revisions, and the Senior editor has assembled the following comments to help you prepare a revised submission.Drosophila larvae and to identify neuronal and molecular substrates for this behaviour. The paper has three main topics in the results; characterisation of the defecation motor programme, characterisation of three neuronal populations which control hind-gut muscle contractions and identification and characterisation of a neuronal TRP channel involved in mechanosensory feedback. Some of the data is of high quality  and the potential finding of a mechanosensitive neuron involved in the process is novel and potentially interesting. The work could have wider implications for understanding defecation and the generation of rhythmic behaviour, but there are substantial concerns with the paper as it stands.This is an interesting paper that seeks to define the defecation programme in Contrary to what the manuscript seems to imply, defecation behaviour has been previously studied in flies . In particular, Cognigni et al developed a method that allows quantification of diuresis, enteric function, and food intake, and showed complex dietary and reproductive modulation of some of these features. The characterisation of the neuronal circuitry underlying defecation per se is useful but linking to the above features is of broader interest and should be attempted. Furthermore, contrary to what the authors state, only one of the three neuronal populations that they “identify” is novel in this context, given that: 1) The PDF neurons have been previously shown not only to innervate the hindgut . The experiments suggested below are achievable in a reasonable timeframe of about three months if the authors have the tools to manipulate those neurons with cellular resolution . Substantial points are elaborated and listed below.Characterization of “defecation behaviour” and its rhythmicity. The authors infer, but never show, effects of these neurons on defecation based on their effects on hindgut muscle contractions. Defecation rate could be subject to additional layers of neuronal control involving both visceral and somatic muscles, so the authors would need to show that these neurons do regulate defecation by, for example, using Fluoropoo  if it works in larvae or by loading larval guts with dye-supplemented yeast and then recording defecation rate on unlabelled yeast. Related to this, the authors often make statements about defecation “rhythms/cycles” akin to those reported in C. elegans. These are based, as far as one can tell, on the rhythmic Ca2+ oscillations observed in resting PDF and RN2 neurons. However, the muscle recordings are not particularly rhythmic in resting conditions, and no evidence for rhythmic defecation events has been provided. For example, the pattern of EJPs shown in Gut peristalsis and defecation rate may vary depending on the larval stage. The authors seem to do all their experiments in L3 larva. Were these early, post-feeding, post critical weight or wandering L3s? Quantifications would need to be confined to one of these substages or data should be provided showing no differences between substages. Again there is a need for precise details of the behavioural assay and data.Loss and gain-of-function phenotypes of PDF and RN2 neurons. Loss-of-function experiments were carried out constitutively throughout embryonic and larval development using Pdf-Gal4 and RN2-Gal4. Both drivers are expressed in other neurons that, when inactivated, could affect behaviours  that could indirectly impact gut movements . Importantly, PDF has been shown to regulate tracheal branching in the hindgut, which could also affect muscle contractions . In the GoF muscle physiology experiments, no “raw” data has been provided for PDF neurons, and different experiments seem to have been conducted for the two neuronal populations. Only one control seems to have been used for the ChR2 experiments – several are normally required . More details would also have to be provided about quantifications .The authors state that both PDF and RN2 neurons are motor neurons, based on comparisons with the gut innervation seen using VGLUT-Gal4. This is unconvincing. The authors should show the positions of the PDF, RN2 and OK371 axons on the hindgut muscles side by side, and use a broad neuronal marker like 22c10 to co-label the major nerves. What functional data shows a motor rather than neuromodulatory role for these neurons, especially for PDF?The role of RN2 (see above too) is unclear. Activation of RN2 by ChR2 causes EJPs in the anal sphincter. This is the only direct evidence for the proposed role of RN2 as exciting the anal sphincter muscles. Other evidence suggests a more complex situation, ablation of RN2 does not prevent sphincter opening but does act additively with ablation of PDF on hindgut contraction period  below. SThe sequential firing is very neat. There are several experimental predictions that come from this. One is that activation of PDF by ChR2 should induce anal muscle contraction (via RN2), data should therefore be included in The authors seem to have used HGN1-Gal4 to label the RN2 neurons. As described in Cognigni et al, HGN1-Gal4 is a particular line (the D insertion) resulting from mobilizing the initial RN2-Gal4 . This is an important distinction because other RN2-Gal4 lines are either expressed more broadly, or are not expressed in those hindgut-innervating neurons and HGN1-Gal4 should be used in the text as this will allow other workers to connect the two studies.The data showing that PDF neurons are presynaptic to RN2 is not very convincing  - both neurons could be putting out dendrites in the same VNC region in order to respond to the same presynaptic partner.The sensory role of the ASN is potentially interesting, but there is no data linking that particular ppk neuron (as opposed to other ppk+ neurons) with the hindgut muscle contraction phenotypes and/or defecation control. The authors could use the elegant approach previously used by this lab  in turn activates ASN. The feedback experiments use a very indirect test of feedback onto PDF and/or RN2, EJPs in the anal sphincter muscles. This may be acting through ASN-PDF-RN2-anal sphincter muscle series. However there are other explanations, in particular it does not rule out other routes of ASN feedback onto the anal sphincter muscles. It would be much better to test the effect of ASN activity more directly on PDF. For example do NOMPC mutants have changes in PDF Ca2+ signals, which are alleviated by NOMPC rescue in ASN? Does ablation of ASN change PDF calcium signals? Even better would be activation of ASN and direct measurement of signals in PDF. This would add significant weight to the argument that ASN is a functional modify of PDF properties. Major additions and changes made in the revised paper:) We have extensively cited the work on adult fly defecation  and clarified the correspondence of our RN2-Gal4 with HGN1-Gal4 by using the latter terminology in the revised manuscript. We have also compared our study of larval defecation with previous studies of defecation of the adult fly in Discussion.1Drosophila; 2) PDF neurons could be double labeled with vGlut-Gal4 (shown in the new Drosophila motor neurons.2) We have conducted additional experiments to test whether PDF and HGN1 neurons display certain features characteristic of motor neurons. 1) These neurons express vGlut, which is a motor neuron marker in 3) We have performed additional experiments to show ASN is necessary to modulate motor neuron activities. We used laser ablation to specifically kill ASN without affecting other neurons and observed a reduction of HGN1 rhythmic activity (shown in the new 4) We have shown ASN activation is sufficient to induce activity of PDF and HGN1 neurons by mechanically stimulating this neuron with a probe without affecting adjacent neurons (shown in the new 5) We have included additional control experiments and detailed methods in the revised manuscript.This is an interesting paper that seeks to define the defecation programme in Drosophila larvae and to identify neuronal and molecular substrates for this behaviour. The paper has three main topics in the results; characterisation of the defecation motor programme, characterisation of three neuronal populations which control hind-gut muscle contractions and identification and characterisation of a neuronal TRP channel involved in mechanosensory feedback. Some of the data is of high quality  and the potential finding of a mechanosensitive neuron involved in the process is novel and potentially interesting. The work could have wider implications for understanding defecation and the generation of rhythmic behaviour, but there are substantial concerns with the paper as it stands.Contrary to what the manuscript seems to imply, defecation behaviour has been previously studied in flies . In particular, Cognigni et al developed a method that allows quantification of diuresis, enteric function, and food intake, and showed complex dietary and reproductive modulation of some of these features. The characterisation of the neuronal circuitry underlying defecation per se is useful but linking to the above features is of broader interest and should be attempted. Furthermore, contrary to what the authors state, only one of the three neuronal populations that they “identify” is novel in this context, given that: 1) The PDF neurons have been previously shown not only to innervate the hindgut , and also to promote visceral muscle contractions , and 2) The RN2 neurons have also been shown to innervate the hindgut and to regulate faecal output . [The authors should refer to the HGN1-Gal4 as this will allow other workers to connect the two studies.]We agree that the Cognigni et al Cell Metab paper has pioneered the study of defecation behaviors of fly, and we should have included more extensive discussion and reference to this study. In this revised manuscript, we have cited this study extensively and attempted to make connection between our study and previous studies. We have also cited the other relevant papers as the reviewers have suggested and made modification in the text accordingly.Both the Abstract and the body of the manuscript contain over-interpretations of the data and important omissions of previously published data. These need to be addressed in a revised manuscript. A revised manuscript should also show that what is being examined is indeed a circuit and that it controls actual defecation (as opposed to subtly modulating muscle contractions of the hind-gut). The experiments suggested below are achievable in a reasonable timeframe of about three months if the authors have the tools to manipulate those neurons with cellular resolution . Substantial points are elaborated and listed below.Characterization of “defecation behaviour” and its rhythmicity. The authors infer, but never show, effects of these neurons on defecation based on their effects on hindgut muscle contractions. Defecation rate could be subject to additional layers of neuronal control involving both visceral and somatic muscles, so the authors would need to show that these neurons do regulate defecation by, for example, using Fluoropoo  if it works in larvae or by loading larval guts with dye-supplemented yeast and then recording defecation rate on unlabelled yeast.The methods used in this paper  are very elegant. We have tried to use a similar approach to study larval defecation as the referees suggested, but failed for the following two reasons: 1) the feces of fly larvae are very tiny, making it hard to quantify; 2) the larval feces are actually thick liquid rather than solid. They got smeared and wiped away when larvae crawled over them.For an alternative approach, we took advantage of the transparency of the larval body wall and visualized defecation process by labeling the fly food with a blue food dye following the referees’ suggestion. The defecation cycle revealed by the expulsion of blue feces is similar to what we have seen in our previous experiments. These data are included in the new Related to this, the authors often make statements about defecation “rhythms/cycles” akin to those reported in C. elegans. These are based, as far as one can tell, on the rhythmic Ca2+ oscillations observed in resting PDF and RN2 neurons. However, the muscle recordings are not particularly rhythmic in resting conditions, and no evidence for rhythmic defecation events has been provided. For example, the pattern of EJPs shown inis described as rhythmic, however whilst it looks sporadic it does not look overtly rhythmic, analysis should be performed to support this statement and in support of a correlation between hind-gut contraction and defecation itself.We agree with the referees that the gut muscle activity as revealed by EJP recordings should have rhythmicity similar to that of the firing of PDF and HGN1 neurons and defecation. However, our recording from dissected larvae may not capture accurately the features of motor activities in intact animals. The purpose of showing these data is to demonstrate that gut muscles receive excitatory input from motor neurons. We have clarified this point by rephrasing the descriptions of the EJP recordings and making note of the likely difference in firing patterns observed in fillet preparations compared to intact animals in the revised manuscript.Previously published data  argues against a robust rhythm at least in adults, where defecation rate is regulated by both environmental (e.g. dietary content) and internal (e.g. reproductive) state. Thus, the conditions in which rhythmic defecation is observed should be precisely described.We appreciate the suggestion of the referees. We think the defecation behaviors in larvae could exhibit a more robust rhythm, which is different from that in adult flies, possibly because larvae spend the majority of their time consuming food to sustain their rapid growth whereas adult flies may need to command greater control of their feeding and defecation behaviors to cope with a more complex environment. It is very possible that larval defecation behavior is also modulated by other cues; however, exploration of such modulation is beyond the scope of this study which focuses on neuronal control of rhythmic defecation cycles. We have also discussed this point and compared the larval and adult fly behaviors in the revised text.which describes defecation data is not clear. What is being measured? The data inis described as hind-gut contraction, but 3A is simply described as “defecation” in the text. “Defecation” is too vague in this context, the exact step that is being measured needs to be defined. If the two measurements are the same thing, which one would hope, then the data inshould be presented using the same measure, i.e. either both should be period in seconds or both should be defecation per period. This reflects a need as mentioned above to give precise details of the behavioural assays and data.We thank the referees for pointing out this issue. We have clarified this in the revised text and added more details on methods of behavioral test and analysis. Also, The reason we used gut contraction time as a measurement in Gut peristalsis and defecation rate may vary depending on the larval stage. The authors seem to do all their experiments in L3 larva. Were these early, post-feeding, post critical weight or wandering L3s? Quantifications would need to be confined to one of these substages or data should be provided showing no differences between substages. Again there is a need for precise details of the behavioural assay and data.Throughout our experiment, we used larvae 96 hrs AEL. They are at active feeding stage. We have spelled it out in our revised text.Loss and gain-of-function phenotypes of PDF and RN2 neurons. Loss-of-function experiments were carried out constitutively throughout embryonic and larval development using Pdf-Gal4 and RN2-Gal4. Both drivers are expressed in other neurons that, when inactivated, could affect behaviours  that could indirectly impact gut movements . Importantly, PDF has been shown to regulate tracheal branching in the hindgut, which could also affect muscle contractions . In the GoF muscle physiology experiments, no “raw” data has been provided for PDF neurons, and different experiments seem to have been conducted for the two neuronal populations. Only one control seems to have been used for the ChR2 experiments - several are normally required . More details would also have to be provided about quantifications .Loss-of-function experiments were carried out with reagents that are currently available. PDF-Gal4 was the Gal4 line with the most restricted expression pattern for those neurons. Although we weren’t able to reach single cell resolution, our data support the notion that PDF and HGN1 neurons in the VNC are important for larval defecation. We have spelled out the caveats concerning the broader expression patterns of Gal4 lines in Discussion.We have tried to study the connectivity specificity between PDF neurons and hindgut muscles but failed to do so due to the special structure of hindguts. They only have a very thin layer of muscles, which tend to be more vigorously contracting even in a fillet preparation. We were unable to achieve stable intracellular recording of hindguts as we have done for anus sphincter muscles. However, we managed to activate PDF via ChR2 and induce anal muscle contraction, similar to what was done in HGN1>ChR2 larvae . RecordiWe have added the data of control experiments  to the figures.We have also provided more details about the quantification of this behavior.The authors state that both PDF and RN2 neurons are motor neurons, based on comparisons with the gut innervation seen using VGLUT-Gal4. This is unconvincing. The authors should show the positions of the PDF, RN2 and OK371 axons on the hindgut muscles side by side, and use a broad neuronal marker like 22c10 to co-label the major nerves. What functional data shows a motor rather than neuromodulatory role for these neurons, especially for PDF?We have performed additional experiments to address this question:For PDF neurons: We carried out anti-vGlut staining on hindgut and found the PDF axons are vGlut positive in new . We alsoFor HGN1 neurons: We have found the HGN1 axons over anal sphincter are vGlut positive in new . TogetheAdditionally, we have performed imaging and whole-cell recording of HGN1 neurons in dissected VNC, which were devoid of sensory inputs. We observed a robust rhythmic activity for HGN1 neurons. HGN1 neurons also exhibited burst firing patterns, which is a feature for fly motor neurons in new .These new data have been integrated into our revised manuscript.The role of RN2 (see above too) is unclear. Activation of RN2 by ChR2 causes EJPs in the anal sphincter. This is the only direct evidence for the proposed role of RN2 as exciting the anal sphincter muscles. Other evidence suggests a more complex situation, ablation of RN2 does not prevent sphincter opening but does act additively with ablation of PDF on hindgut contraction period . So does RN2 activation, by ChR2, alone have an effect on hind gut peristalsis? This data should be added toWe have monitored the contraction of hindgut while stimulating HGN1 neurons using a setup similar to the one we used to observe the contraction of anus sphincter. We found activation of HGN1 neurons had no effect on hindgut movement in new .Regarding the question why “ablation of HGN1 does not prevent sphincter opening but does act additively with ablation of PDF on hindgut contraction period”, one speculation is that the remaining PDF neurons can still trigger hindgut contractions, which generate pressure and push the feces along the intestine to induce passive sphincter opening. We have included possible explanation in the revised text.The sequential firing is very neat. There are several experimental predictions that come from this. One is that activation of PDF by ChR2 should induce anal muscle contraction (via RN2), data should therefore be included inon the anal sphincter muscle contraction when PDF is activated by ChR2. Similarly activation of PDF by ChR2 would be expected to give rise to anal sphincter EJPs. Another expectation is that inactivation of PDF may result in no Ca2+ signals in RN2, this could be tested by combining available reagents. Finally would it also be possible to test whether activation of PDF using ChR2 led to calcium signals in RN2?We used PDF-Gal4 to drive ChR2 in PDF neurons and stimulated these neurons with blue light. We found that activation of PDF via ChR2 induced anal muscle contraction, similar to what was observed in HGN1>ChR2 larvae new . RecordiThe authors seem to have used HGN1-Gal4 to label the RN2 neurons. As described in Cognigni et al, HGN1-Gal4 is a particular line (the D insertion) resulting from mobilizing the initial RN2-Gal4 . This is an important distinction because other RN2-Gal4 lines are either expressed more broadly, or are not expressed in those hindgut-innervating neurons and HGN1-Gal4 should be used in the text as this will allow other workers to connect the two studies.We have replaced RN2 with HGN1 throughout the manuscripts. We agree with the referees that the latter terminology will help the readers to connect our study with the previous work in the adult system.The data showing that PDF neurons are presynaptic to RN2 is not very convincing  - both neurons could be putting out dendrites in the same VNC region in order to respond to the same presynaptic partner.We have modified the text in the revised manuscript accordingly. For now we don’t have enough evidence to show PDF neurons are direct pre-synaptic partner of HGN1 neurons even though their aborizations overlap intensely. Based on our functional study , they are likely two components within the defecation motor circuitry where PDF neurons fire shortly before the firing of HGN1 neurons.The sensory role of the ASN is potentially interesting, but there is no data linking that particular ppk neuron (as opposed to other ppk+ neurons) with the hindgut muscle contraction phenotypes and/or defecation control. The authors could use the elegant approach previously used by this lab  to strengthen this link.The method suggested by the referees would provide one test as to whether ASN itself is important for the defecation. With the reagents currently available, the experiment we could do is to use 19-12-Gal4/UAS-Kir to silence all class III da neurons and PPK-Gal80 to suppress the silencing effect in ASN . However, this would generate indirect evidence.We used an alternative and more direct approach instead. We labeled all PPK neurons with PPK-Gal4 and used 2-photon laser to specifically ablate the ASN soma near the anal slit. Then we imaged the HGN1 neurons to see if ablation of ASN altered the activity of HGN1 neurons. Indeed, we found alteration of the periodicity of rhythmic firing of HGN1 neurons new .The result supports the conclusion that ASN (a particular PPK neuron) but not other PPK neurons modulates motor control of gut movements.ASN is identified as sensory neuron providing mechanosensory feedback from the anus. ASN shows periodic Ca2+ oscillations. These correlate to the opening of the anus and activity in PDF and RN2, however no functional test of the hypothesis that ASN activation lies downstream of PDF, RN2 and anal opening is shown. One experiment would be to show that inactivation of PDF results in a loss of Ca2+ signals in ASN. A more positive experiment would be to show that activation of RN2 (using ChR2 or dTRPA1) in turn activates ASN. The feedback experiments use a very indirect test of feedback onto PDF and/or RN2, EJPs in the anal sphincter muscles. This may be acting through ASN-PDF-RN2-anal sphincter muscle series. However there are other explanations, in particular it does not rule out other routes of ASN feedback onto the anal sphincter muscles. It would be much better to test the effect of ASN activity more directly on PDF. For example do NOMPC mutants have changes in PDF Ca2+ signals, which are alleviated by NOMPC rescue in ASN? Does ablation of ASN change PDF calcium signals? Even better would be activation of ASN and direct measurement of signals in PDF. This would add significant weight to the argument that ASN is a functional modify of PDF properties.2+ increase in the VNC (new We agree that recording from gut muscles while stimulating ASN didn’t provide direct evidence that ASN feedback to PDF and HGN1 neurons. Hence we carried out experiments using new strategies in order to show that ASN does send feedback to PDF and HGN1 when it receives mechanical stimulus. We imaged PDF neurons and HGN1 neurons with GCaMP5 while inserting a tapered glass probe to the anus slit and advanced it to split the anus sphincter, which mimicked anus opening during defecation. We found that PDF and HGN1 neurons responded to this stimulation new . As a co VNC new , which iThis result, together with the previous GRASP experiments between ASN and PDF neurons, support our model that ASN provide feedback to motor neurons.