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Background: The role of barbed sutures in preventing myometrial defects and enhancing postpartum outcomes after cesarean section (C-section) is uncertain., Objective: This study compared clinical and ultrasonographic outcomes of uterine scar defects after C-section with barbed and conventional smooth thread sutures., Study Design: This was a multicenter, parallel-group, randomized, controlled clinical trial. Four obstetrics and gynecology departments across three Japanese healthcare regions were included. The participants were women requiring their first cesarean delivery between May 2020 and March 2023. Of the 1211 participants enrolled, 298 underwent C-section and 253 were followed up until July 2023. Participants with singleton pregnancies were randomly assigned (1:1 ratio) to receive either conventional or spiral thread sutures with a double-layer continuous suture. The study period comprised the time of consent to the 6- to 7-month examination. The primary endpoint was the rate of scar niches >2 mm evaluated using transvaginal ultrasonography at 6 to 7 months after surgery. Additional metrics included the total operative time, suture application time, operative blood loss, number of additional sutures required for hemostasis, maternal surgical complications, postoperative infections, surgeon's years of experience, and individual subscale scores., Results: All data of the 220 participants (barbed suture group: 110; conventional suture group: 110) were available, thus enabling a full analysis set. A comparison of the barbed and conventional suture groups, respectively, revealed the following: niche length, 2.45±1.65 mm (range: 1.0-6.7) vs 3.79±1.84 mm (range: 1.0-11.0) (P<.001); niche depth, 1.78±1.07 mm (range: 1.0-5.7) vs 2.70±1.34 mm (range: 1.0-7.3) (P<.001); residual myometrial thickness (RMT), 8.46±1.74 mm (range: 4.8-13.0) vs 7.07±2.186 mm (range: 2.2-16.2) (P<.001); and niche width, 1.58±2.73 mm (range: 0.0-14.0) vs 2.88±2.36 mm (range: 0.0-11.0) (P<.001), respectively. The barbed suture group exhibited no defects and an RMT <3 mm. Furthermore, the barbed suture group had a lower rate of uterine niches (29.1%; n=32/110) than the conventional suture group (68.2%; n=75/110). Secondary outcomes showed no significant differences in operative times, maternal surgical complications, or postoperative complications., Conclusion: Double-layer barbed sutures during cesarean delivery may prevent C-section scar defects and postoperative complications. El resumen está disponible en Español al final del artículo., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

During the coronavirus pandemic, face-to-face simulation education became impossible. Therefore, we aimed to develop remote-access simulation education with a sense of realism through Information and Communication Technology (ICT) using a perinatal whole-body management and delivery simulator. In September 2021, we administered a multi-center simultaneous remote simulation based on our developed model. Ten universities in the Chugoku-Shikoku region were connected via a web-conferencing system to a live broadcast of a virtual vaginal birth in which a fictional hospitalized pregnant woman experienced accelerated labor and gave birth through vacuum delivery for fetal distress. A Video on Demand (VOD) was made beforehand using a new simulator that allowed for a visual understanding of the process of the inter-vaginal examination. We provided a participatory program that enhanced the sense of realism by combining VOD and real-time lectures on each scenario, with two-way communication between participants and trainee doctors using a chat function. Most participants answered "satisfied" or "very satisfied" with the content, level of difficulty, and level of understanding. From November 2021, we have used the videos of all processes in face-to-face classes. Our construction of a high-flexibility education system using remote simulation in the field of obstetrics and gynecology, especially in the vaginal delivery module, is unique, creative, and sustainable., Competing Interests: No potential conflict of interest relevant to this article was reported.

Immune checkpoint inhibitors (ICIs) have been widely used as standard therapies for various cancers. However, in 20-30% of cases, ICIs can lead to immune-related adverse events (irAEs), which sometimes require discontinuation of treatment. Due to the increased risk of irAEs, patients with pre-existing autoimmune diseases (AI) are often advised against receiving ICIs. However, there has not been sufficient objective risk assessment for AI. In our study, we conducted logistic regression analysis to assess the risk of irAEs by analyzing 478 cases that received anti-PD-(L)1 Ab and/or anti-CTLA4 Ab at our hospital between April 3, 2017, and May 24, 2022. Among these cases, 28 (5.9%) had pre-existing AI. We selected several independent factors for analysis: gender, age, performance status (PS), cancer type, type of ICI, type of combined anti-cancer agents, best overall response, and pre-existing AI. The adjusted odds ratio (OR) of AI for irAE occurrence was 2.52 [95% CI: 1.08-5.86] (p = 0.033), and the adjusted OR of AI for ICI discontinuation due to irAE was 3.32 [1.41-7.78] (p = 0.006). Patients with pre-existing AI experienced a significantly shorter irAE-free survival time compared to those without AI (median irAE-free survival: 5.7 months [95% CI: 3.5-7.8] vs 10.4 months [95% CI: 7.9-12.9], respectively, p = 0.035). Frequently observed irAEs in full ICI cohort, such as dermatologic issues (7.5%), pneumonitis (7.1%), hepatitis (4.6%), and hypothyroidism (4.2%), were often accompanied by pre-existing AI. Furthermore, pre-existing AI flared up in 6 cases (37.5% in AI-positive irAE-positive cases). The activity of AI was not related to the occurrence of irAEs. Grade 3 or higher irAEs were observed in 6 out of 20 (30.0%) cases in AI-accompanied patients complicated with irAEs. Although having a complicated AI increases the risk of irAEs, it may not necessarily be a contraindication for ICI treatment if closely monitored. (292<300 characters)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sumimoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We investigated the effectiveness of simulation-based education in Focused Assessment with Sonography for Trauma (FAST) to increase the number of Emergency Medical Technicians (EMTs) capable of performing ultrasound examinations in vehicles under the guidance of a physician. Twenty-eight paramedics watched a 14-min video on the features of the ultrasound system, its use, and the scanning method for each part of the body. Each participant performed four FAST examinations using a portable ultrasound device, and the task performance was rated using the Task Specific Checklist (TSC) and Global Rating Scale (GRS). The time required for visualizing each examination site and each FAST was assessed. The mean time required for the first and fourth FAST was 144.6 ± 52.4 s and 90.5 ± 31.0 s, respectively. The time required for each test significantly decreased with repeated testing (p < 0.001). The time to complete FAST was significantly shortened for the pericardial cavity (33.4 ± 23.1/15.3 ± 10.6 s, p < 0.01), right thoracic cavity (25.2 ± 11.8/12.1 ± 8.3 s, p < 0.01), Morrison fossa (19.1 ± 10.8/10.8 ± 6.3 s, p < 0.05), and left thoracic cavity (19.0 ± 8.3/15.6 ± 8.3 s, p < 0.05). TSC and GRS scores were elevated, and all EMTs could obtain valid images. The combination of a brief video lecture and hands-on training significantly reduced the time required for FAST performance. Moreover, repeated practice enabled the EMTs to efficiently obtain accurate and clinically useful images., (© 2024. The Author(s).)

Heparan sulfate proteoglycans (HSPGs) serve as co-receptors for growth factor signaling during development. It is well known that the level and patterns of sulfate groups of heparan sulfate (HS) chains, or HS fine structures, have a major impact on HSPG function. On the other hand, the physiological significance of other structural features of HS, including NS/NA domain organization, remains to be elucidated. A blueprint of the HS domain structures is mainly controlled by HS N -deacetylase/ N -sulfotransferases (NDSTs). To analyze in vivo activities of differentially modified HS, we established two knock-in (KI) Drosophila strains with the insertion of mouse Ndst1 ( mNdst1 ) or Ndst2 ( mNdst2 ) in the locus of sulfateless ( sfl ), the only Drosophila NDST. In these KI lines, mNDSTs are expressed from the sfl locus, in the level and patterns identical to the endogenous sfl gene. Thus, phenotypes of Ndst1 KI and Ndst2 KI animals reflect the ability of HS structures made by these enzymes to rescue sfl mutation. Remarkably, we found that mNdst1 completely rescued the loss of sfl. mNdst2 showed a limited rescue ability, despite a higher level of HS sulfation compared to HS in mNdst1 KI. Our study suggests that independent of sulfation levels, additional HS structural features controlled by NDSTs play key roles during tissue patterning., Competing Interests: CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest.

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dally ΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dally ΔGAG -specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dally ΔGAG , dlp ΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Persons with HIV (PWH) with high mobility face obstacles to HIV care engagement and viral suppression. We sought to understand whether a patient-centered intervention for mobile PWH would improve viral suppression and retention in care, and if so, which subgroups would benefit most., Methods: In a randomized trial, we evaluated the effect of an intervention designed to address barriers to care among mobile (≥2 weeks out of community in previous year) PWH with viral nonsuppression or recent missed visits in Kenya and Uganda (NCT04810650). The intervention included dynamic choice of a "travel pack" (emergency antiretroviral therapy [ART] supply, discrete ART packaging, and travel checklist), multimonth and offsite refills, facilitated transfer to out-of-community clinics, and hotline access to a mobility coordinator. The primary outcome was viral suppression (<400 copies/mL) at 48 weeks. Secondary outcomes included retention in care and ART possession., Results: From April 2021 to July 2022, 201 participants were enrolled and randomized (102 intervention, 99 control): 109 (54%) were female participants and 101 (50%) from Kenya; median age was 37 years (interquartile range: 29-43). At 48 weeks, there was no significant difference in viral suppression in intervention (85%) vs. control (86%). The intervention improved retention in care (risk ratio: 1.06[1.02-1.1]; P < 0.001) and ART possession (risk ratio: 1.07[1.03-1.11]; P < 0.001), with larger effect sizes among persons with baseline nonsuppression and high mobility (≥2 weeks out of community in previous 3 months)., Conclusions: Mobile PWH-centered care should be considered for high-risk mobile populations, including nonsuppressed and highly mobile PWH, to improve retention in care and sustain viral suppression over time., Trial Registration: NCT04810650., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Heparan sulfate (HS) and chondroitin sulfate (CS) are evolutionarily conserved glycosaminoglycans that are found in most animal species, including the genetically tractable model organism Drosophila. In contrast to extensive in vivo studies elucidating co-receptor functions of Drosophila HS proteoglycans (PGs), only a limited number of studies have been conducted for those of CSPGs. To investigate the global function of CS in development, we generated mutants for Chondroitin sulfate synthase (Chsy), which encodes the Drosophila homolog of mammalian chondroitin synthase 1, a crucial CS biosynthetic enzyme. Our characterizations of the Chsy mutants indicated that a fraction survive to adult stage, which allowed us to analyze the morphology of the adult organs. In the ovary, Chsy mutants exhibited altered stiffness of the basement membrane and muscle dysfunction, leading to a gradual degradation of the gross organ structure as mutant animals aged. Our observations show that normal CS function is required for the maintenance of the structural integrity of the ECM and gross organ architecture., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Introduction: Health workers' failure to adhere to guidelines for screening, diagnosis and management of HIV-associated cryptococcal meningitis (CM) remains a significant public health concern. We aimed to assess adherence to the standards of care and management of HIV patients at risk of CM per the MoH guidelines and assess stock management of CM supplies in the period of January to June 2021 at selected public health facilities (HFs) in Uganda., Methods: The study employed an observational cross-sectional design to assess the level of adherence of health workers to standards of clinical care and management of HIV positive patients at risk of CM as per the clinical guidelines for Uganda, and stock management of CM supplies in the period of January to June 2021in selected public health facilities. The study team used a survey guide designed by MoH to assess and score the screening, diagnosis and management practices of Health Facilities towards CM. Scoring was categorized as red (< 80%), light green (80%-95%), and dark green (˃95%) in the order from worst to best adherence. The data was transcribed into a spread sheet and analysed using STATA-v15., Results: The study team visited a total of 15 public health facilities including 5 general hospitals, 9 regional referral hospitals (RRHs) and 1 National Referral hospital (NRH). The mean score for adherence to screening and management of CM for all the combined facilities was 15 (64.7%) classified as red. 10 (66.7%) HFs had not performed a baseline CD4 test for eligible patients within 2 weeks of ART initiation. With regards to treatment, 9 (60%) of the HFs were scored as light green on knowledge of the procedure for reconstituting intravenous Liposomal Amphotericin B. None of the HFs visited had potassium chloride tablets in stock., Conclusion: Major MoH guidelines are generally not being adhered to by health workers while managing cryptococcal meningitis. It is vital that government and implementing partners regularly support HFs with training, mentorship, and support supervision on CM management to improve adherence to CM screening and treatment guidelines., Competing Interests: The Authors have declared that no competing interests exists., (Copyright: © 2023 Namuju et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The uptake of HIV prevention services is lower among youth than adults in sub-Saharan Africa. Existing youth livelihood trainings offer a potential entry point to HIV prevention services. We determined feasibility and preliminary effectiveness of integrating HIV prevention into youth clubs implementing an empowerment and livelihood for adolescents (ELA) intervention in rural Uganda. Staff conducted community mobilization for youth (15-24 years) over one month. Clubs met (3×/week) over six months, with local peer mentors trained to teach life-skills and sexual/reproductive health education. We integrated mentor-led education on HIV prevention, including pre- and post-exposure prophylaxis (PrEP/PEP). Clubs offered on-site HIV testing, a field trip to a local clinic and PrEP referrals after one month and six months. Surveys were conducted at baseline and six months. Forty-two participants (24 adolescent girls/young women (AGYW) and 18 adolescent boys/young men (ABYM)) joined the clubs. At baseline, no participants accepted referral for PrEP, whereas 5/18 (28%) sexually active, HIV-negative AGYW requested PrEP referral at follow-up. One ABYM requested PEP referral. Integration of HIV prevention services into an established ELA curriculum at mentor-led youth clubs in rural Uganda was feasible. PrEP uptake increased among sexually active AGYW. Evaluation of this approach for HIV prevention among youth merits further study.

Background: Mycobacterium tuberculosis presents several lineages each with distinct characteristics of evolutionary status, transmissibility, drug resistance, host interaction, latency, and vaccine efficacy. Whole genome sequencing (WGS) has emerged as a new diagnostic tool to reliably inform the occurrence of phylogenetic lineages of Mycobacterium tuberculosis and examine their relationship with patient demographic characteristics and multidrug-resistance development., Methods: 191 Mycobacterium tuberculosis isolates obtained from a 2017/2018 Tanzanian drug resistance survey were sequenced on the Illumina Miseq platform at Supranational Tuberculosis Reference Laboratory in Uganda. Obtained fast-q files were imported into tools for resistance profiling and lineage inference (Kvarq v0.12.2, Mykrobe v0.8.1 and TBprofiler v3.0.5). Additionally for phylogenetic tree construction, RaxML-NG v1.0.3(25) was used to generate a maximum likelihood phylogeny with 800 bootstrap replicates. The resulting trees were plotted, annotated and visualized using ggtree v2.0.4 RESULTS: Most [172(90.0%)] of the isolates were from newly treated Pulmonary TB patients. Coinfection with HIV was observed in 33(17.3%) TB patients. Of the 191 isolates, 22(11.5%) were resistant to one or more commonly used first line anti-TB drugs (FLD), 9(4.7%) isolates were MDR-TB while 3(1.6%) were resistant to all the drugs. Of the 24 isolates with any resistance conferring mutations, 13(54.2%) and 10(41.6%) had mutations in genes associated with resistance to INH and RIF respectively. The findings also show four major lineages i.e. Lineage 3[81 (42.4%)], followed by Lineage 4 [74 (38.7%)], the Lineage 1 [23 (12.0%)] and Lineages 2 [13 (6.8%)] circulaing in Tanzania., Conclusion: The findings in this study show that Lineage 3 is the most prevalent lineage in Tanzania whereas drug resistant mutations were more frequent among isolates that belonged to Lineage 4., (© 2022. The Author(s).)

Background: Uganda remains one of the countries with the highest burden of TB/HIV. Drug-resistant TB remains a substantial challenge to TB control globally and requires new strategic effective control approaches. Drug resistance usually develops due to inadequate management of TB patients including improper treatment regimens and failure to complete the treatment course which may be due to an unstable supply or a lack of access to treatment, as well as patient noncompliance., Methods: Two sputa samples were collected from Xpert MTB/RIF® assay-diagnosed multi-drug resistant tuberculosis (MDR-TB) patient at Lira regional referral hospital in northern Uganda between 2020 and 2021 for comprehensive routine mycobacterial species identification and drug susceptibility testing using culture-based methods. Detection of drug resistance-conferring genes was subsequently performed using whole-genome sequencing with Illumina MiSeq platform at the TB Supranational Reference Laboratory in Uganda., Results: In both isolates, extensively drug-resistant TB (XDR-TB) was identified including resistance to Isoniazid (katG p.Ser315Thr), Rifampicin (rpoB p.Ser450Leu), Moxifloxacin (gyrA p.Asp94Gly), Bedaquiline (Rv0678 Glu49fs), Clofazimine (Rv0678 Glu49fs), Linezolid (rplC Cys154Arg), and Ethionamide (ethA c.477del). Further analysis of these two high quality genomes revealed that this 32 years-old patient was infected with the Latin American Mediterranean TB strain (LAM)., Conclusions: This is the first identification of extensively drug-resistant Mycobacterium tuberculosis clinical isolates with bedaquiline, linezolid and clofazimine resistance from Uganda. These acquired resistances were because of non-adherence as seen in the patient's clinical history. Our study also strongly highlights the importance of combating DR-TB in Africa through implementing next generation sequencing that can test resistance to all drugs while providing a faster turnaround time. This can facilitate timely clinical decisions in managing MDR-TB patients with non-adherence or lost to follow-up., (© 2022. The Author(s).)

Several classes of heparan sulfate proteoglycan (HSPG) core proteins and all HS biosynthetic/modifying enzymes are evolutionarily conserved from human to Drosophila melanogaster. This genetically tractable model offers highly sophisticated techniques to manipulate gene function in a spatially and temporally controlled manner. Thus, Drosophila genetics has been a powerful system to explore functions of HSPGs in vivo. In this chapter, we will introduce three genetic techniques available in Drosophila: TARGET (temporal and regional gene expression targeting), MARCM (mosaic analysis with a repressible cell marker), and FLP-Out., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Genetic studies using a model organism, Drosophila melanogaster, have been contributing to elucidating the in vivo functions of heparan sulfate proteoglycans (HSPGs). On the other hand, biochemical analysis of Drosophila glycosaminoglycans (GAGs) has been limited, mainly due to the insufficient amount of the material obtained from the animal. Recently, a novel in vitro system has been developed by establishing mutant cell lines for heparan sulfate (HS)-modifying enzyme genes. Metabolic radiolabeling of GAGs allows us to assess uncharacterized features of Drosophila GAGs and the effects of the mutations on HS structures and function. The novel in vitro system will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Unpaired1 (Upd1) is a ligand of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway in Drosophila . In this study, using the CRISPR/Cas9 technique, we generate a transgenic fly strain in which a hemagglutinin (HA) epitope tag sequence is inserted into the endogenous locus of the upd1 gene. Anti-HA antibody staining confirms that the distribution of the epitope-tagged Upd1::HA in various tissues is consistent with upd1 expression patterns revealed by previous studies. This transgenic fly strain will be useful in studying the expression, localization, and association partners of Upd1, and thus will contribute to understanding how activation of the JAK/STAT pathway is regulated., (Copyright: © 2021 by the authors.)

The molecular mechanisms by which stem cell proliferation is precisely controlled during the course of regeneration are poorly understood. Namely, how a damaged tissue senses when to terminate the regeneration process, inactivates stem cell mitotic activity, and organizes ECM integrity remain fundamental unanswered questions. The Drosophila midgut intestinal stem cell (ISC) offers an excellent model system to study the molecular basis for stem cell inactivation. Here, we show that a novel gene, CG6967 or dMOV10, is induced at the termination stage of midgut regeneration, and shows an inhibitory effect on ISC proliferation. dMOV10 encodes a putative component of the microRNA (miRNA) gene silencing complex (miRISC). Our data, along with previous studies on the mammalian MOV10, suggest that dMOV10 is not a core member of miRISC, but modulates miRISC activity as an additional component. Further analyses identified direct target mRNAs of dMOV10-containing miRISC, including Daughter against Dpp (Dad), a known inhibitor of BMP/TGF-β signaling. We show that RNAi knockdown of Dad significantly impaired ISC division during regeneration. We also identified six miRNAs that are induced at the termination stage and their potential target transcripts. One of these miRNAs, mir-1, is required for proper termination of ISC division at the end of regeneration. We propose that miRNA-mediated gene regulation contributes to the precise control of Drosophila midgut regeneration., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Competing Interests: Competing interests: None declared.

Proteoglycans, a class of carbohydrate-modified proteins, often modulate growth factor signaling on the cell surface. However, the molecular mechanism by which proteoglycans regulate signal transduction is largely unknown. In this study, using a recently developed glycoproteomic method, we found that Windpipe (Wdp) is a novel chondroitin sulfate proteoglycan (CSPG) in Drosophila . Wdp is a single-pass transmembrane protein with leucin-rich repeat (LRR) motifs and bears three CS sugar chain attachment sites in the extracellular domain. Here we show that Wdp modulates the Hedgehog (Hh) pathway. In the wing disc, overexpression of wdp inhibits Hh signaling, which is dependent on its CS chains and the LRR motifs. The wdp null mutant flies show a specific defect (supernumerary scutellar bristles) known to be caused by Hh overexpression. RNA interference knockdown and mutant clone analyses showed that loss of wdp leads to the up-regulation of Hh signaling. Altogether, our study demonstrates a novel role of CSPGs in regulating Hh signaling.

Background: Universal antiretroviral therapy (ART) with annual population testing and a multidisease, patient-centered strategy could reduce new human immunodeficiency virus (HIV) infections and improve community health., Methods: We randomly assigned 32 rural communities in Uganda and Kenya to baseline HIV and multidisease testing and national guideline-restricted ART (control group) or to baseline testing plus annual testing, eligibility for universal ART, and patient-centered care (intervention group). The primary end point was the cumulative incidence of HIV infection at 3 years. Secondary end points included viral suppression, death, tuberculosis, hypertension control, and the change in the annual incidence of HIV infection (which was evaluated in the intervention group only)., Results: A total of 150,395 persons were included in the analyses. Population-level viral suppression among 15,399 HIV-infected persons was 42% at baseline and was higher in the intervention group than in the control group at 3 years (79% vs. 68%; relative prevalence, 1.15; 95% confidence interval [CI], 1.11 to 1.20). The annual incidence of HIV infection in the intervention group decreased by 32% over 3 years (from 0.43 to 0.31 cases per 100 person-years; relative rate, 0.68; 95% CI, 0.56 to 0.84). However, the 3-year cumulative incidence (704 incident HIV infections) did not differ significantly between the intervention group and the control group (0.77% and 0.81%, respectively; relative risk, 0.95; 95% CI, 0.77 to 1.17). Among HIV-infected persons, the risk of death by year 3 was 3% in the intervention group and 4% in the control group (0.99 vs. 1.29 deaths per 100 person-years; relative risk, 0.77; 95% CI, 0.64 to 0.93). The risk of HIV-associated tuberculosis or death by year 3 among HIV-infected persons was 4% in the intervention group and 5% in the control group (1.19 vs. 1.50 events per 100 person-years; relative risk, 0.79; 95% CI, 0.67 to 0.94). At 3 years, 47% of adults with hypertension in the intervention group and 37% in the control group had hypertension control (relative prevalence, 1.26; 95% CI, 1.15 to 1.39)., Conclusions: Universal HIV treatment did not result in a significantly lower incidence of HIV infection than standard care, probably owing to the availability of comprehensive baseline HIV testing and the rapid expansion of ART eligibility in the control group. (Funded by the National Institutes of Health and others; SEARCH ClinicalTrials.gov number, NCT01864603.)., (Copyright © 2019 Massachusetts Medical Society.)

A class of carbohydrate-modified proteins, heparan sulfate proteoglycans (HSPGs), play critical roles both in normal development and during disease. Genetic studies using a model organism, Drosophila, have been contributing to understanding the in vivo functions of HSPGs. Despite the many strengths of the Drosophila model for in vivo studies, biochemical analysis of Drosophila HS is somewhat limited, mainly due to the insufficient amount of the material obtained from the animal. To overcome this obstacle, we generated mutant cell lines for four HS modifying enzymes that are critical for the formation of ligand binding sites on HS, Hsepi, Hs2st, Hs6st and Sulf1, using a recently established method. Morphological and immunological analyses of the established lines suggest that they are spindle-shaped cells of mesodermal origin. The disaccharide profiles of HS from these cell lines showed characteristics of lack of each enzyme as well as compensatory modifications by other enzymes. Metabolic radiolabeling of HS allowed us to assess chain length and net charge of the total population of HS in wild-type and Hsepi mutant cell lines. We found that Drosophila HS chains are significantly shorter than those from mammalian cells. BMP signaling assay using Hs6st cells indicates that molecular phenotypes of these cell lines are consistent with previously known in vivo phenomena. The established cell lines will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The stem cell niche normally prevents aberrant stem cell behaviors that lead to cancer formation. Recent studies suggest that some cancers are derived from endogenous populations of adult stem cells that have somehow escaped from normal control by the niche. However, the molecular mechanisms by which the niche retains stem cells locally and tightly controls their divisions are poorly understood. Here, we demonstrate that the presence of heparan sulfate (HS), a class glygosaminoglycan chains, in the Drosophila germline stem cell niche prevents tumor formation in the testis. Loss of HS in the niche, called the hub, led to gross changes in the morphology of testes as well as the formation of both somatic and germline tumors. This loss of hub HS resulted in ectopic signaling events in the Jak/Stat pathway outside the niche. This ectopic Jak/Stat signaling disrupted normal somatic cell differentiation, leading to the formation of tumors. Our finding indicates a novel non-autonomous role for niche HS in ensuring the integrity of the niche and preventing tumor formation., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Adult stem cells reside in specialized microenvironments called niches, which provide signals for stem cells to maintain their undifferentiated and self-renewing state. To maintain stem cell quality, several types of stem cells are known to be regularly replaced by progenitor cells through niche competition. However, the cellular and molecular bases for stem cell competition for niche occupancy are largely unknown. Here, we show that two Drosophila members of the glypican family of heparan sulfate proteoglycans (HSPGs), Dally and Dally-like (Dlp), differentially regulate follicle stem cell (FSC) maintenance and competitiveness for niche occupancy. Lineage analyses of glypican mutant FSC clones showed that dally is essential for normal FSC maintenance. In contrast, dlp is a hypercompetitive mutation: dlp mutant FSC progenitors often eventually occupy the entire epithelial sheet. RNA interference knockdown experiments showed that Dally and Dlp play both partially redundant and distinct roles in regulating Jak/Stat, Wg, and Hh signaling in FSCs. The Drosophila FSC system offers a powerful genetic model to study the mechanisms by which HSPGs exert specific functions in stem cell replacement and competition., (Copyright © 2018 by the Genetics Society of America.)

Despite the importance of precisely regulating stem cell division, the molecular basis for this control is still elusive. Here, we show that surface glia in the developing Drosophila brain play essential roles in regulating the proliferation of neural stem cells, neuroblasts (NBs). We found that two classes of extracellular factors, Dally-like (Dlp), a heparan sulfate proteoglycan, and Glass bottom boat (Gbb), a BMP homologue, are required for proper NB proliferation. Interestingly, Dlp expressed in perineural glia (PG), the most outer layer of the surface glia, is responsible for NB proliferation. Consistent with this finding, functional ablation of PG using a dominant-negative form of dynamin showed that PG has an instructive role in regulating NB proliferation. Gbb acts not only as an autocrine proliferation factor in NBs but also as a paracrine survival signal in the PG. We propose that bidirectional communication between NBs and glia through TGF-β signaling influences mutual development of these two cell types. We also discuss the possibility that PG and NBs communicate via direct membrane contact or transcytotic transport of membrane components. Thus, our study shows that the surface glia acts not only as a simple structural insulator but also a dynamic regulator of brain development.

Stem cell division is activated to trigger regeneration in response to tissue damage. The molecular mechanisms by which this stem cell mitotic activity is properly repressed at the end of regeneration are poorly understood. Here, we show that a specific modification of heparan sulfate is crucial for regulating Drosophila intestinal stem cell (ISC) division during normal midgut homeostasis and regeneration. Loss of the extracellular heparan sulfate endosulfatase Sulf1 resulted in increased ISC division during normal homeostasis, which was caused by upregulation of mitogenic signaling including the JAK-STAT, EGFR and Hedgehog pathways. Using a regeneration model, we found that ISCs failed to properly halt division at the termination stage in Sulf1 mutants, showing that Sulf1 is required for terminating ISC division at the end of regeneration. We propose that post-transcriptional regulation of mitogen signaling by heparan sulfate structural modifications provides a new regulatory step for precise temporal control of stem cell activity during regeneration., Competing Interests: The authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. Here we show that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. We found that GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the niche, termed the hub, led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome-anchoring machinery, at the hub-GSC interface. This perturbation of the centrosome-anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control., (© 2016 Levings et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Several classes of heparan sulfate proteoglycan (HSPG) core proteins and all HS biosynthetic/modifying enzymes are evolutionarily conserved from human to Drosophila melanogaster. This genetically tractable model offers highly sophisticated techniques to manipulate gene function in a spatially and temporally controlled manner. Thus, Drosophila has been a powerful system to explore the functions of HSPGs in vivo. In this chapter, we will introduce two genetic techniques available in Drosophila: TARGET (temporal and regional gene expression targeting) and MARCM (mosaic analysis with a repressible cell marker).

During the biosynthesis of heparan sulfate (HS), glucuronyl C5-epimerase (Hsepi) catalyzes C5-epimerization of glucuronic acid (GlcA), converting it to iduronic acid (IdoA). Because HS 2-O-sulfotransferase (Hs2st) shows a strong substrate preference for IdoA over GlcA, C5-epimerization is required for normal HS sulfation. However, the physiological significance of C5-epimerization remains elusive. To understand the role of Hsepi in development, we isolated Drosophila Hsepi mutants. Homozygous mutants are viable and fertile with only minor morphological defects, including the formation of an ectopic crossvein in the wing, but they have a short lifespan. We propose that two mechanisms contribute to the mild phenotypes of Hsepi mutants: HS sulfation compensation and possible developmental roles of 2-O-sulfated GlcA (GlcA2S). HS disaccharide analysis showed that loss of Hsepi resulted in a significant impairment of 2-O-sulfation and induced compensatory increases in N- and 6-O-sulfation. Simultaneous block of Hsepi and HS 6-O-sulfotransferase (Hs6st) activity disrupted tracheoblast formation, a well established FGF-dependent process. This result suggests that the increase in 6-O-sulfation in Hsepi mutants is critical for the rescue of FGF signaling. We also found that the ectopic crossvein phenotype can be induced by expression of a mutant form of Hs2st with a strong substrate preference for GlcA-containing units, suggesting that this phenotype is associated with abnormal GlcA 2-O-sulfation. Finally, we show that Hsepi formed a complex with Hs2st and Hs6st in S2 cells, raising the possibility that this complex formation contributes to the close functional relationships between these enzymes.

The biosynthesis of heparan sulfate proteoglycans is tightly regulated by multiple feedback mechanisms, which support robust developmental systems. One of the regulatory network systems controlling heparan sulfate (HS) biosynthesis is sulfation compensation. A previous study using Drosophila HS 2-O- and 6-O-sulfotransferase (Hs2st and Hs6st) mutants showed that loss of sulfation at one position is compensated by increased sulfation at other positions, supporting normal FGF signaling. Here, we show that HS sulfation compensation rescues both Decapentaplegic and Wingless signaling, suggesting a universal role of this regulatory system in multiple pathways in Drosophila. Furthermore, we identified Sulf1, extracellular HS 6-O-endosulfatase, as a novel component of HS sulfation compensation. Simultaneous loss of Hs2st and Sulf1 led to 6-O-oversulfation, leading to patterning defects, overgrowth, and lethality. These phenotypes are caused at least partly by abnormal up-regulation of Hedgehog signaling. Thus, sulfation compensation depends on the coordinated activities of Hs2st, Hs6st, and Sulf1.

Heparan sulfate proteoglycans regulate various physiological and developmental processes through interactions with a number of protein ligands. Heparan sulfate (HS)-ligand binding depends on the amount and patterns of sulfate groups on HS, which are controlled by various HS sulfotransferases in the Golgi apparatus as well as extracellular 6-O-endosulfatases called "Sulfs." Sulfs are a family of secreted molecules that specifically remove 6-O-sulfate groups within the highly sulfated regions on HS. Vertebrate Sulfs promote Wnt signaling, whereas the only Drosophila homologue of Sulfs, Sulf1, negatively regulates Wingless (Wg) signaling. To understand the molecular mechanism for the negative regulation of Wg signaling by Sulf1, we studied the effects of Sulf1 on HS-Wg interaction and Wg stability. Sulf1 overexpression strongly inhibited the binding of Wg to Dally, a potential target heparan sulfate proteoglycan of Sulf1. This effect of Drosophila Sulf1 on the HS-Wg interaction is similar to that of vertebrate Sulfs. Using in vitro, in vivo, and ex vivo systems, we show that Sulf1 reduces extracellular Wg protein levels, at least partly by facilitating Wg degradation. In addition, expression of human Sulf1 in the Drosophila wing disc lowers the levels of extracellular Wg protein, as observed for Drosophila Sulf1. Our study demonstrates that vertebrate and Drosophila Sulfs have an intrinsically similar activity and that the function of Sulfs in the fate of Wnt/Wg ligands is context-dependent.

In Drosophila, ligands of the Unpaired (Upd) family activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. The JAK/STAT pathway controls many developmental events, including multiple functions in the ovary. These include an early role in the germarium for specification of stalk cells and a later role in the vitellarium to pattern the follicular epithelium surrounding each cyst. In this latter role, graded JAK/STAT activation specifies three distinct anterior follicular cell fates, suggesting that Upd is a morphogen in this system. Consistent with the JAK/STAT activation pattern in the vitellarium, Upd forms a concentration gradient on the apical surface of the follicular epithelium with a peak at its source, the polar cells. Like many morphogens, signaling and distribution of Upd are regulated by the heparan sulfate proteoglycans (HSPGs) Dally and Dally-like. Mutations in these glypican genes and in heparan sulfate biosynthetic genes result in disruption of JAK/STAT signaling, loss or abnormal formation of the stalk and significant reduction in the accumulation of extracellular Upd. Conversely, forced expression of Dally causes ectopic accumulation of Upd in follicular cells. Furthermore, biochemical studies reveal that Upd and Dally bind each other on the surface of the cell membrane. Our findings demonstrate that Drosophila glypicans regulate formation of the follicular gradient of the Upd morphogen, Upd. Furthermore, we establish the follicular epithelium as a new model for morphogen signaling in complex organ development.

Sulfs are secreted sulfatases that catalyse removal of sulfate from Heparan Sulfate Proteoglycans (HSPGs) in the extracellular space. These enzymes are well known to regulate a number of crucial signalling pathways during development. In this study, we report that DSulfatase-1 (DSulf1), the unique Drosophila Sulf protein, is a regulator of Hedgehog (Hh) signalling during wing development. DSulf1 activity is required in both Hh source and Hh receiving cells for proper positioning of Hh target gene expression boundaries. As assessed by loss- and gain-of-function experiments in specific compartments, DSulf1 displays dual functions with respect to Hh signalling, acting as a positive regulator in Hh producing cells and a negative regulator in Hh receiving cells. In either domain, DSulf1 modulates Hh distribution by locally lowering the concentration of the morphogen at the apical pole of wing disc cells. Thus, we propose that DSulf1, by its desulfation catalytic activity, lowers Hh/HSPG interaction in both Hh source and target fields, thereby enhancing Hh release from its source of production and reducing Hh signalling activity in responding cells. Finally, we show that Dsulf1 pattern of expression is temporally regulated and depends on EGFR signalling, a Hh-dependent secondary signal in this tissue. Our data reveal a novel Hh regulatory feedback loop, involving DSulf1, which contributes to maintain and stabilise expression domains of Hh target genes during wing disc development., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We previously proposed a model that DALLY, a Drosophila glypican, acts as a trans co-receptor to regulate BMP signaling in the germ line stem cell niche. To investigate the molecular mechanisms of contact-dependent BMP signaling, we developed novel in vitro assay systems to monitor trans signaling using Drosophila S2 cells. Using immunoblot-based as well as single-cell assay systems, we present evidence that Drosophila glypicans indeed enhance BMP signaling in trans in a contact-dependent manner in vitro. Our analysis showed that heparan sulfate modification is required for the trans co-receptor activity of DALLY. Two BMP-like molecules, Decapentaplegic (DPP) and Glass bottom boat, can mediate trans signaling through a heparan sulfate proteoglycan co-receptor in S2 cells. The in vitro systems reflect the molecular characteristics of heparan sulfate proteoglycan functions observed previously in vivo, such as ligand specificity and biphasic activity dependent on the ligand dosage. In addition, experiments using a DALLY-coated surface suggested that DALLY regulates DPP signaling in trans by its effect on the stability of DPP protein on the surface of the contacting cells. Our findings provide the molecular foundation for novel contact-dependent signaling, which defines the physical space of the stem cell niche in vivo.

Heparan sulfate proteoglycans (HSPGs) participate in a wide range of biological processes through interactions with a number of ligand proteins. The nature of these interactions largely depends on the heparan sulfate (HS) moiety of HSPGs, which undergoes a series of modifications by various HS-modifying enzymes (HSMEs). Although the effects of alterations in a single HSME on physiological processes have started to be studied, it remains elusive how a combination of these molecules control the structure and function of HS. Here we systematically manipulated the HS structures and analyzed their effect on morphogenesis and signaling, using the genetically tractable model organism, Drosophila. We generated transgenic fly strains overexpressing HSMEs alone or in combination. Unsaturated disaccharide analyses of HS showed that expression of various HSMEs generates distinct HS structures, and the enzymatic activities of HSMEs are influenced by coexpression of other HSMEs. Furthermore, these transgenic HSME animals showed a different extent of lethality, and a subset of HSMEs caused specific morphological defects due to defective activities of Wnt and bone morphogenetic protein signaling. There is no obvious relationship between HS unsaturated disaccharide composition and developmental defects in HSME animals, suggesting that other structural factors, such as domain organization or sulfation sequence, might regulate the function of HS.

Heparan sulfate proteoglycans (HSPGs) play critical roles in the distribution and signaling of growth factors, but the molecular mechanisms regulating HSPG function are poorly understood. Here, we characterized Sulf1, which is a Drosophila member of the HS 6-O endosulfatase class of HS modifying enzymes. Our genetic and biochemical analyses show that Sulf1 acts as a novel regulator of the Wg morphogen gradient by modulating the sulfation status of HS on the cell surface in the developing wing. Sulf1 affects gradient formation by influencing the stability and distribution of Wg. We also demonstrate that expression of Sulf1 is induced by Wg signaling itself. Thus, Sulf1 participates in a feedback loop, potentially stabilizing the shape of the Wg gradient. Our study shows that the modification of HS fine structure provides a novel mechanism for the regulation of morphogen gradients., ((c) 2010 Elsevier Inc. All rights reserved.)

Stem cells are maintained in vivo by short-range signaling systems in specialized microenvironments called niches, but the molecular mechanisms controlling the physical space of the stem cell niche are poorly understood. In this study, we report that heparan sulfate (HS) proteoglycans (HSPGs) are essential regulators of the germline stem cell (GSC) niches in the Drosophila melanogaster gonads. GSCs were lost in both male and female gonads of mutants deficient for HS biosynthesis. dally, a Drosophila glypican, is expressed in the female GSC niche cells and is responsible for maintaining the GSC niche. Ectopic expression of dally in the ovary expanded the niche area, showing that dally is required for restriction of the GSC niche space. Interestingly, the other glypican, dally-like, plays a major role in regulating male GSC niche maintenance. We propose that HSPGs define the physical space of the niche by serving as trans coreceptors, mediating short-range signaling by secreted factors.

Decapentaplegic (Dpp), a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. Here, we characterized activity, stability, and gradient formation of a truncated form of Dpp (Dpp(Delta N)), which lacks a short domain at the N-terminus essential for its interaction with Dally. Dpp(Delta N) shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, we propose a model in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex.

Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, we characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. We found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts.