Safety of carrageenan-based gels as preventive microbicides: a narrative review.

BACKGROUND: Carrageenan-containing gels researched for the prevention of sexually transmitted infections (STIs) have shown promising results for human papillomavirus prevention in women, but not in men. We conducted a narrative review to assess the safety of these gels for genital use. METHODS: We searched PubMed using MeSH terms and keywords on 5 November 2023. Title/abstract of articles were screened to identify relevant ones. Full-text screening determined eligibility: empirical study evaluating safety of carrageenan-containing gel(s) for genital use. RESULTS: Of the 125 identified records, 15 were eligible, comprising 14 (10 randomised controlled trials and 4 cohorts) unique study populations. Studies included women only (n=11), men only (n=1) or both (n=3); number of participants ranged from 4 to 6202. Safety was assessed for vaginal (n=13), penile (n=3) and anal use (n=2). Most studies assessed safety of Carraguard (53%), followed by Divine9 (14%), and one each of iota-carrageenan gel, lambda-carrageenan gel, Carvir, PC-6500 (griffithsin and carrageenan) and PC-1005 (MIV-150/zinc acetate/carrageenan). Safety assessment relied on self-report (80.0%), testing for STIs (53.3%), investigator-identified genital findings (93.3%) and/or testing for changes in genital flora (60.0%). Adverse events (AEs) were described by investigators as mostly mild, (mostly) comparable between groups, not observed and/or not significant for vaginal and penile use. Only one study, assessing anal use of carrageenan, reported a significantly higher proportion of AEs in the carrageenan compared with placebo group. CONCLUSIONS: Carrageenan-based gels are generally well tolerated for vaginal and penile, but not anal use. Studies on carrageenan gel's safety for anal use are scarce.

Impact of a carrageenan gel on viral load of genital human papillomavirus infections in sexually active women: Findings from the Carrageenan-gel Against Transmission of Cervical Human papillomavirus (CATCH) trial.

Previous research has shown that women's use of a carrageenan gel reduces the risk of acquiring genital human papillomavirus (HPV) infections but does not help to clear existing ones. Although gel use may not result in complete clearance, it may decrease the viral load of HPV infections. We tested this hypothesis in the Carrageenan-gel Against Transmission of Cervical Human papillomavirus (CATCH) randomized controlled trial. Participants of the CATCH study were selected for viral load testing if they had completed the first four study visits and tested positive for HPV42 or HPV51 in at least one of these visits. HPV42 and HPV51 were chosen as they were among the most abundant low- and high-risk types, respectively, in the study sample. We measured viral load with a type-specific real-time polymerase chain reaction. Results were displayed using summary statistics. Of 461 enrolled participants, 39 were included in the HPV42 analysis set and 56 in the HPV51 analysis set. The median time between visits 1 and 4 was 3.7 months. The viral load (copies/cell) of HPV42 ranged from <0.001 to 13 434.1, and that of HPV51 from <0.001 to 967.1. The net median change in HPV42 viral load over all four visits was -1.04 copies/cell in the carrageenan and -147 copies/cell in the placebo arm (Wilcoxon rank sum test, p = 0.26). There was no net median change in HPV51 viral load over all four visits in either arm (p = 0.45). The use of a carrageenan-based gel is unlikely to reduce the viral load of HPVs 42 or 51.

Use of a carrageenan-based gel had no impact on anal HPVs 16 and 18 viral loads in gay, bisexual, and other men who have sex with men.

The Lubricant Investigation in Men to Inhibit Transmission of human papillomavirus (HPV) Infection randomized control trial in gay, bisexual, and other men who have sex with men (gbMSM) found that carrageenan use neither reduced acquisition of anal HPV infections nor influenced infection clearance. To investigate carrageenan's lack of protective effect, we compared the change in anal HPV16 and HPV18 viral loads following carrageenan use against placebo. We restricted our analysis to participants who completed the first four study visits and had a valid baseline sample (n = 161, 54 HIV-positive). Samples were tested for HPV detection using the linear array PCR assay. HPV16- and/or HPV18-positive samples were tested for viral load using real-time PCR. For participants who tested HPV16- (n = 29) or HPV18-positive (n = 10) at least once across visits 1-4, we compared the change in type-specific viral load between study arms using the Mann-Whitney U test. Although the median net change in HPV16 and HPV18 viral loads across visits 1-4 was higher in the treatment than placebo arm (HPV16: 0.68 vs. 0.18 copies/cell, p = 0.60; HPV18: 18.32 vs. 10.12 copies/cell, p = 0.52), these differences were not statistically significant. Results were similar by HIV status. Carrageenan use did not impact anal HPV16 or HPV18 viral loads, which may further explain its lack of protective effect in gbMSM.

Human Papillomavirus Detectability and Cervical Cancer Prognosis: A Systematic Review and Meta-analysis.

OBJECTIVE: To evaluate whether testing positive for human papillomavirus (HPV) before treatment is associated with cervical cancer recurrence and disease-free, cancer-specific, and overall survival and to report the relationship of HPV to cervical cancer histology, stage, grade, tumor size, lymph node involvement, and treatment response. DATA SOURCES: EMBASE and MEDLINE were searched from inception to January 27, 2022, with the use of MeSH terms and keywords relating to cervical cancer, HPV, and prognosis. ClinicalTrials.gov was not searched because of the nature of our review question. METHODS OF STUDY SELECTION: Studies must have assessed HPV DNA or RNA in cervical pretreatment biopsies or cells from 20 or more patients with invasive cervical cancer followed up for any length of time and reported the effect of testing positive or negative for HPV on cervical cancer recurrence, disease-free survival, cancer-specific survival, or overall survival. We extracted data on HPV-detection methods, patient and tumor characteristics, and clinical outcomes. TABULATION, INTEGRATION, AND RESULTS: Hazard ratios (HRs) and 95% CIs were pooled with a random-effects model. Meta-regression was performed to explore heterogeneity. Of 11,179 titles or abstracts and 474 full-text articles reviewed, 77 studies were included in the systematic review. Among these 77 studies, 30 reported on the relationship of HPV status to histology, 39 to cancer stage, 13 to tumor grade, 17 to tumor size, 23 to lymph node involvement, and four to treatment response. Testing positive for HPV was associated with better disease-free survival (HR 0.38, 95% CI 0.25-0.57; 15 studies with 2,564 cases), cancer-specific survival (HR 0.56, 95% CI 0.44-0.71; nine studies with 1,398 cases), and overall survival (HR 0.59, 95% CI 0.47-0.74; 36 studies with 9,169 cases), but not recurrence (HR 0.59, 95% CI 0.33-1.07; eight studies with 1,313 cases). Meta-regression revealed that the number of cases, tumor grade, specimen type, gene target, and HPV prevalence together explained 73.8% of the between-study heterogeneity. CONCLUSION: This review indicates that HPV detectability in cervical cancer is associated with a better clinical prognosis. SYSTEMATIC REVIEW REGISTRATION: https://osf.io/dtyeb .

Efficacy and safety of a self-applied carrageenan-based gel to prevent human papillomavirus infection in sexually active young women (CATCH study): an exploratory phase IIB randomised, placebo-controlled trial.

Background: Carrageenan demonstrated potent anti-HPV (human papillomavirus) activity in vitro and in animal models. The Carrageenan-gel Against Transmission of Cervical Human papillomavirus trial's interim analysis (n = 277) demonstrated a 36% protective effect of carrageenan against incident HPV infections. Herein, we report the trial's final results. Methods: In this exploratory phase IIB randomised, placebo-controlled trial, we recruited healthy women aged ≥18 years primarily from health service clinics at two Canadian Universities in Montreal. Participants were randomised (1:1) by the study coordinator (using computer-assisted block randomisation with randomly variable block sizes up to a block size of eight) to a carrageenan-based or placebo gel to be self-applied every other day for the first month and before/after intercourse. Participants, study nurses, and laboratory technicians (HPV testing and genotyping) were blinded to group assignment. At each visit (months 0, 0.5, 1, 3, 6, 9, 12), participants provided questionnaire data and a self-collected vaginal sample (tested for 36 HPV types, Linear Array). The primary outcome was type-specific HPV incidence (occurring at any follow-up visit). Intention-to-treat analyses for incidence were conducted using Cox proportional hazards regression models, including participants with ≥2 visits. Safety analyses included all participants randomised. This trial is registered with the ISRCTN registry, ISRCTN96104919. Findings: Between Jan 16, 2013 and Sept 30, 2020, 461 participants (enrolled) were randomly assigned to the carrageenan (n = 227) or placebo (n = 234) groups. Incidence and safety analyses included 429 and 461 participants, respectively. We found 51.9% (108/208) of participants in carrageenan and 66.5% (147/221) in placebo arm acquired ≥1 HPV type (hazard ratio 0.63 [95% CI: 0.49-0.81], p = 0.0003). Adverse events were reported by 34.8% (79/227) and 39.7% (93/234) of participants in carrageenan and placebo arm (p = 0.27), respectively. Interpretation: Consistent with the interim analysis, use of a carrageenan-based gel compared to placebo resulted in a 37% reduction in risk of incident genital HPV infections in women with no increase in adverse events. A carrageenan-based gel may complement HPV vaccination. Funding: Canadian Institutes of Health Research, CarraShield Labs Inc.

Association between male circumcision and human papillomavirus infection in males and females: a systematic review, meta-analysis, and meta-regression.

BACKGROUND: Previous studies have suggested a protective effect of male circumcision on human papillomavirus (HPV) infections in males, and that this protection may be conferred to their female sexual partners. OBJECTIVES: To synthesize the available evidence on the association between male circumcision and HPV infections in males and females. DATA SOURCES: We searched MEDLINE, Embase, Scopus, Cochrane, LILACS, and ProQuest Dissertations & Theses Global for records published up to 22 June 2022. STUDY ELIGIBILITY: We considered observational and experimental studies that assessed male circumcision status and HPV prevalence, incidence, or clearance in males or females for inclusion. PARTICIPANTS: Males and their female sexual partners who were tested for genital HPV infection. INTERVENTIONS: Male circumcision compared with no circumcision. THE RISK-OF-BIAS ASSESSMENT: The Newcastle-Ottawa scale was used for observational studies, and the Cochrane risk-of-bias tool was used for randomized trials. DATA SYNTHESIS: We estimated summary measures of effect and 95% CIs for the prevalence, incidence, and clearance of HPV infections in males and females using random-effects meta-analysis. We assessed the effect modification of circumcision on HPV prevalence by the penile site in males using random-effects meta-regression. RESULTS: Across 32 studies, male circumcision was associated with decreased odds of prevalent HPV infections (odds ratio, 0.45; 95% CI, 0.34-0.61), a reduced incidence rate of HPV infections (incidence rate ratio, 0.69; 95% CI, 0.57-0.83), and an increased risk of clearing HPV infections (risk ratio, 1.44; 95% CI, 1.28-1.61) at the glans penis among male subjects. Circumcision conferred greater protection against infection at the glans than the shaft (odds ratio, 0.68; 95% CI, 0.48-0.98). Females with circumcised partners were protected from all outcomes. CONCLUSIONS: Male circumcision may protect against various HPV infection outcomes, suggesting its prophylactic potential. Understanding the site-specific effects of circumcision on HPV infection prevalence has important implications for studies of HPV transmission.

Efficacy of a Carrageenan Gel in Increasing Clearance of Anal Human Papillomavirus Infections in Men: Interim Analysis of a Double-Blind, Randomized Controlled Trial.

Preclinical studies have demonstrated carrageenan's anti-human papillomavirus (HPV) activity. We assessed efficacy of a carrageenan-based gel compared to a placebo gel in increasing the clearance of anal HPV infections among gay, bisexual, and other men who have sex with men (gbMSM). Of 255 enrolled gbMSM, 134 were HPV positive at baseline and had valid HPV results for ≥2 visits. Carrageenan did not differ from placebo in clearing all baseline infections (hazard ratio, 0.84 [95% confidence interval, .31-2.27]), based on having 2 consecutive HPV-negative visits following at least 1 HPV-positive visit. There were no remarkable differences for analyses at the HPV type level or by human immunodeficiency virus status. CLINICAL TRIALS REGISTRATION: NCT02354144.

A Review of Ethical and Legal Aspects of Gender-Neutral Human Papillomavirus Vaccination.

While launching a campaign to eliminate cervical cancer, the World Health Organization called to halt human papillomavirus (HPV) gender-neutral vaccination (GNV) because of limited vaccine supply, raising ethical and legal questions about female-only vaccination versus GNV. We identified ethical and legal aspects of HPV GNV by searching MEDLINE for records up to February 19, 2021. We also provided an overview of HPV vaccines, the evolution of HPV vaccine recommendations in North America, and a timeline of male HPV vaccination introduction by searching PubMed, Google, and government websites. Four HPV vaccines are available: Cervarix, Gardasil, Gardasil9, and Cecolin. Vaccine recommendations in North America evolved from female only to eventually include males. Following the FDA's approval of the first HPV vaccine for males (2009), 35 countries began vaccinating males (2011-2020). On the basis of 59 eligible records out of 652, we identified the following constructs: lower male awareness of HPV and vaccination (n = 13), limited economic resources (n = 5), shared social responsibility (n = 18), unprotected groups from female-only HPV vaccination (n = 10), limited screening for HPV-associated noncervical cancers (n = 6), consideration of ethical principles (n = 17), and HPV vaccine mandates (n = 5). Ethical and legal aspects must be considered when recommending vaccination for females only or GNV.

Efficacy of a carrageenan gel in preventing anal human papillomavirus (HPV) infection: interim analysis of the Lubricant Investigation in Men to Inhibit Transmission of HPV Infection (LIMIT-HPV) randomised controlled trial.

BACKGROUND: Carrageenan, a non-toxic gelling agent derived from red algae, has potent anti-human papillomavirus (HPV) activity in in vitro and animal studies. We assessed, in an interim analysis, the efficacy of a carrageenan-based gel in reducing the risk of new detections of anal HPV among gay, bisexual and other men who have sex with men (gbMSM). METHODS: The LIMIT-HPV study (Lubricant Investigation in Men to Inhibit Transmission of HPV Infection) is a phase IIb, double-blind, placebo-controlled randomised controlled trial conducted in Montreal, Canada. gbMSM were randomly assigned (1:1) to receive a carrageenan-based or placebo gel. Participants were instructed to apply the gel to the anus, condom and/or partners' penis before and-as required-during receptive anal intercourse. Questionnaire data and anal samples were collected at 0, 1, 2, 3, 6, 9 and 12 months. We estimated new detections of anal HPV infection(s) detected via Linear Array using Cox proportional hazards models. RESULTS: Participants recruited from February 2016 to December 2019 were randomly assigned to the carrageenan (n=127) or placebo (n=128) arm. The efficacy and safety analyses included 201 and 210 participants. The median follow-up time was 7.6 months (range: 0-28.5) in the carrageenan group and 9.3 months (range: 0-40.7) in the placebo group. The HR for new detections was 1.21 (95% CI 0.86 to 1.70): 69.4% and 65.1% new detections of HPV in the carrageenan and placebo arms, respectively. More adverse events were reported in the carrageenan (59.8%) compared with the placebo (39.8%) arm. CONCLUSIONS: The interim analysis did not demonstrate a protective effect of carrageenan on the risk of new detections of anal HPV infection among gbMSM. Carrageenan gel use was associated with a higher proportion of adverse events. Given these findings and the (assumed) low probability that a beneficial effect would be found by the study's end, the trial was terminated as recommended by the Data Safety and Monitoring Board. TRIAL REGISTRATION NUMBER: NCT02354144.

Design and methods for the Carrageenan-gel Against Transmission of Cervical Human papillomavirus (CATCH) study: A randomized controlled trial.

Human papillomavirus (HPV) infection, a common sexually transmitted infection, is causally associated with cervical cancer. Vaccination against HPV provides protection; however, HPV vaccines are exclusively prophylactic. Carrageenan, an extract from red algae, demonstrated potent anti-HPV activity in in vitro and animal studies. We describe the protocol for the Carrageenan-gel Against Transmission of Cervical Human papillomavirus (CATCH) study, an ongoing randomized controlled trial among sexually active young females, aimed at evaluating the efficacy of a carrageenan-based gel in reducing type-specific incidence (i.e. new detections of HPV) and prevalence (i.e. absence of a previously detected HPV) of genital HPV infections as well as participant adherence to the intervention. The CATCH study is a phase IIB double-blind randomized placebo-controlled trial. Eligible women 18 years and older are randomized 1:1 to the carrageenan-containing gel or placebo gel arm. For the first month, participants use the study gel intra-vaginally every other day, and over the 12-month study period, prior to and after each act of vaginal intercourse. At each study visit (months 0, 0.5, 1, 3, 6, 9, 12), participants provide a self-collected vaginal sample and record information on sexual activities, adherence, and adverse events using a computerized questionnaire. The primary outcomes are incident and prevalent type-specific cervicovaginal HPV infection. The primary analyses are based on intention-to-treat whereas per-protocol analyses are conducted based on measures of adherence. Trial registration: ISRCTN96104919.

Assessment of the possible inhibitory effect of carrageenan in human papillomavirus DNA testing by polymerase chain reaction amplification.

We assessed carrageenan's potential to inhibit human papillomavirus (HPV) DNA extraction and amplification in vaginal swab samples collected in a trial, assessing the efficacy of a carrageenan-based gel against HPV infections. Experiment #1 consisted of adding gel (carrageenan-containing or placebo) to swabs and comparing HPV DNA detection by polymerase chain reaction (PCR) to unmanipulated samples collected from the same participants. For Experiments #2 and #3, we tested vaginal samples for inhibition by addition of an internal control and amplification by real-time PCR. Experiment #4 investigated carrageenan's interference with the extraction process by assessing HPV45 detectability in undiluted and diluted HPV45 positive samples (n = 3) with carrageenan versus no gel. In Experiment #1, there was a loss of HPV positivity with the addition of carrageenan (n = 9), but none with placebo gel (n = 5). In Experiments #2 and #3, the absence of the amplified product was observed in samples from the carrageenan arm: 3.3% (1/30) and 0.5% (1/199) of samples. In Experiment #4, HPV45 was not detected in undiluted carrageenan-containing samples, but after 1/50 dilution, the same HPV45 copy number was detected. Carrageenan does not affect the DNA extraction process, and inhibition of HPV DNA amplification by carrageenan occurs infrequently.

Carrageenan as a Preventive Agent Against Human Papillomavirus Infection: A Narrative Review.

ABSTRACT: Carrageenan, an extract from red algae, was identified over a decade ago as a potent inhibitor of human papillomavirus (HPV) infection in vitro. After this discovery, several studies evaluated carrageenan's anti-HPV activity in cells, experimental animals, and humans. We reviewed the evidence for carrageenan's anti-HPV activity. Studies had to be in vitro, in vivo, or in humans and report on carrageenan's anti-HPV activity. Of the 39 records identified in PubMed and 29 records in Clinicaltrials.gov, 22 records were included after screening: 8 in vitro (including 2 ex vivo), 3 in vivo, 5 in vitro and in vivo, 3 clinical studies, and 3 trial protocols. A total of 12 studies evaluated carrageenan exclusively, whereas 7 considered carrageenan combined with additional antiviral or other agents. One study protocol will evaluate carrageenan exclusively, and 2 others will evaluate carrageenan-combination products. Most clinical studies evaluated carrageenan's ability to prevent HPV acquisition (n = 4), whereas one study explored its ability to promote clearance of existing infection (defined as the absence of HPV DNA detection). Carrageenan's anti-HPV activity was observed consistently across study designs, except in 2 studies: 1 in vitro study where 2 of the HPV types tested were not significantly inhibited by carrageenan and 1 phase IIB trial in gay, bisexual, and other men who have sex with men. This review supports the premise that carrageenan, alone or in combination with other antiviral agents, might be a potential prevention strategy complementary to HPV vaccination for women.

Clinical studies assessing the efficacy, effectiveness and safety of remdesivir in management of COVID-19: A scoping review.

AIMS: Remdesivir is 1 of the repurposed drugs under investigation to treat patients with COVID-19. Clinicians and decision-makers need a summary of the most recent evidence. This scoping review maps the evidence on the efficacy, effectiveness and safety of remdesivir for patients with COVID-19, up to 14 September 2020. METHODS: Our scoping review searched Pubmed, Embase (Ovid), Scopus and 17 primary trial registries for empirical publications or active registered clinical trials for data on the efficacy, effectiveness, or safety of remdesivir for COVID-19 or SARS-CoV-2. We conducted a narrative synthesis of the included publications. RESULTS: Seventeen empirical studies and 23 clinical trial registrations (n = 40) accumulated 46 508 participants. We found 4 published randomized-controlled trials accumulating 2293 patients. Two trials reported shorter median recovery time and better clinical status among patients who received remdesivir compared with the control groups. Observational studies report an association between remdesivir treatment and decreased mortality, as well as increased survival. The most common adverse reaction was hepatic impairment, although the trials reported a similar proportion of adverse events in the intervention and control groups. CONCLUSION: Remdesivir might shorten the time to clinical improvement among hospitalized adults with severe COVID-19. Trial data report a similar proportion of adverse events in treated and control groups. The results of the 23 registered active trials, including more than 30 000 participants, will shed light on the efficacy and safety of the antiviral. The findings of the remaining clinical trials expected to report results in 2020 will allow a quantitative synthesis of available evidence.

Lubricant Investigation in Men to Inhibit Transmission of HPV Infection (LIMIT-HPV): design and methods for a randomised controlled trial.

INTRODUCTION: Gay, bisexual and other men who have sex with men (gbMSM) have an increased risk of human papillomavirus (HPV) infection and HPV-associated diseases, such as anal cancer and anogenital warts. A carrageenan-based lubricant could prevent HPV infection, thereby reducing the disease burden in this population. This paper describes the protocol for the Lubricant Investigation in Men to Inhibit Transmission of HPV Infection (LIMIT-HPV) study, an ongoing randomised controlled trial (RCT), evaluating efficacy of a carrageenan-based personal lubricant in reducing type-specific anal HPV incidence and prevalence among sexually active gbMSM, efficacy by HIV status, safety and tolerability of the gel and participant adherence to the intervention. METHODS AND ANALYSIS: The study is a double-blinded, placebo-controlled RCT. Volunteer gbMSM 18 years and older are randomly assigned 1:1 to receive the treatment (a self-applied anal microbicide gel with carrageenan) or placebo (a self-applied placebo gel). At each visit, computerised questionnaires are used to collect data on sociodemographic and clinical variables, lifestyle, sexual behaviour and the gels' safety and tolerability. At baseline and each follow-up visit (months 1, 2, 3, 6, 9 and 12), nurses collect anal specimens tested for 36 HPV types (linear array assay). HIV status is determined at baseline and 12 months. The primary outcome is incidence of type-specific anal HPV infection(s) undetected at baseline. Secondary outcomes are prevalence of type-specific anal HPV infection, safety, tolerability and adherence. We aim to recruit 380 participants to attain the study's objectives. Data will be analysed using intention-to-treat and per-protocol approaches with subgroup analyses by HIV status. ETHICS AND DISSEMINATION: Ethics approval was obtained by the Research Ethics Boards of McGill University, the McGill University Health Centre, Concordia University and Centre Hospitalier de l'Université de Montréal. Trial results will be disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: NCT02354144.

Human papillomavirus vaccination and the role of herd effects in future cancer control planning: a review.

INTRODUCTION: Vaccine herd effects are the indirect protection that vaccinated persons provide to those who remain susceptible to infection, due to the reduced transmission of infections. Herd effects have been an important part of the discourse on how to best implement human papillomavirus (HPV) vaccines and prevent HPV-related diseases. AREAS COVERED: In this paper, we review the theory of HPV vaccine herd effects derived from mathematical models, give an account of observed HPV vaccine herd effects worldwide, and examine the implications of vaccine herd effects for future cervical cancer screening efforts. EXPERT COMMENTARY: HPV vaccine herd effects improve the cost-effectiveness of vaccinating preadolescent girls, but contribute to making gender-neutral vaccination less economically efficient. Vaccination coverage and sexual mixing patterns by age are strong determinants of herd effects. Many countries worldwide are starting to observe reductions in HPV-related outcomes likely attributable to herd effects, most notably declining anogenital warts in young men, and declining HPV-16/18 infection prevalence in young unvaccinated women. Policy makers making recommendations for cervical cancer screening will have to consider HPV vaccination coverage and herd effects, as these will affect the positive predictive value of screening and the risk of cervical cancer in unvaccinated women.

The Details of Glycolipid Glycan Hydrolysis by the Structural Analysis of a Family 123 Glycoside Hydrolase from Clostridium perfringens.

Clostridium perfringens is an opportunistic pathogen of humans and animals whose genome encodes a wide variety of putative carbohydrate-hydrolyzing enzymes that are increasingly being shown to be directed toward the cleavage of host glycans. Among these putative enzymes is a member of glycoside hydrolase family 123. Here we show that the recombinant enzyme (referred to as CpNga123) encoded by the gene cloned from C. perfringens strain ATCC 13124 (locus tag CPF_1473) is a ß-N-acetylgalactosaminidase, similar to NgaP from Paenibacillus sp. TS12. Like NgaP, CpNga123 was able to cleave the terminal ß-D-GalNAc-(1→4)-D-Gal and ß-D-GalNAc-(1→3)-D-Gal motifs that would be found in glycosphigolipids. The X-ray crystal structure of CpNga123 revealed it to have an N-terminal ß-sandwich domain and a (ß/α)8-barrel catalytic domain with a C-terminal α-helical elaboration. The structures determined in complex with reaction products provide details of the -1 subsite architecture, catalytic residues, and a structural change in the active site that is likely required to enable hydrolysis of the glycosidic bond by promoting engagement of the substrate by the catalytic residues. The features of the active site support the likelihood of a substrate-assisted catalytic mechanism for this enzyme. The structures of an inactive mutant of CpNga123 in complex with intact GA2 and Gb4 glycosphingolipid motifs reveal insight into aglycon recognition and suggest that the kinked or pleated conformation of GA2 caused by the ß-1,4-linkage between N-acetylgalactosamine and galactose, and the accommodation of this conformation by the enzyme active site, may be responsible for greater activity on GA2.

A PCR assay detects a male-specific duplicated copy of Anti-Müllerian hormone (amh) in the lingcod (Ophiodon elongatus).

BACKGROUND: Anti-Müllerian hormone (amh) or Müllerian-inhibiting substance (mis) is a member of the transforming growth factor-ß family of hormones. This gene plays a key role in vertebrate male sex-determination by inhibiting the development of the Müllerian ducts, and has been shown to be the master sex-determinant in the Patagonian pejerrey. RESULTS: In the lingcod, Ophiodon elongatus, both males and females share one copy of amh, however we have identified a second duplicate copy that appears solely in the male individuals. We have developed a PCR-based assay targeting the TGF-ß domain of amh that provides a simple method with which to sex lingcod from a small amount of tissue. An analysis across 57 individuals gave a 100% success rate in identifying the phenotypic sex. CONCLUSIONS: We present a simple method to sex lingcod through non-lethal tissue sampling. A third, independent, male-specific duplication of amh in a teleost fish has been identified in the lingcod.