Subthreshold opioid use disorder prevention (STOP) trial: a cluster randomized clinical trial: study design and methods.

BACKGROUND: Preventing progression to moderate or severe opioid use disorder (OUD) among people who exhibit risky opioid use behavior that does not meet criteria for treatment with opioid agonists or antagonists (subthreshold OUD) is poorly understood. The Subthreshold Opioid Use Disorder Prevention (STOP) Trial is designed to study the efficacy of a collaborative care intervention to reduce risky opioid use and to prevent progression to moderate or severe OUD in adult primary care patients with subthreshold OUD. METHODS: The STOP trial is a cluster randomized controlled trial, randomized at the PCP level, conducted in 5 distinct geographic sites. STOP tests the efficacy of the STOP intervention in comparison to enhanced usual care (EUC) in adult primary care patients with risky opioid use that does not meet criteria for moderate-severe OUD. The STOP intervention consists of (1) a practice-embedded nurse care manager (NCM) who provides patient participant education and supports primary care providers (PCPs) in engaging and monitoring patient-participants; (2) brief advice, delivered to patient participants by their PCP and/or prerecorded video message, about health risks of opioid misuse; and (3) up to 6 sessions of telephone health coaching to motivate and support behavior change. EUC consists of primary care treatment as usual, plus printed overdose prevention educational materials and an educational video on cancer screening. The primary outcome measure is self-reported number of days of risky (illicit or nonmedical) opioid use over 180 days, assessed monthly via text message using items from the Addiction Severity Index and the Current Opioid Misuse Measure. Secondary outcomes assess other substance use, mental health, quality of life, and healthcare utilization as well as PCP prescribing and monitoring behaviors. A mixed effects negative binomial model with a log link will be fit to estimate the difference in means between treatment and control groups using an intent-to-treat population. DISCUSSION: Given a growing interest in interventions for the management of patients with risky opioid use, and the need for primary care-based interventions, this study potentially offers a blueprint for a feasible and effective approach to improving outcomes in this population. TRIAL REGISTRATION: Clinicaltrials.gov, identifier NCT04218201, January 6, 2020.

Relative Effectiveness of Social Media, Dating Apps, and Information Search Sites in Promoting HIV Self-testing: Observational Cohort Study.

BACKGROUND: Social media sites, dating apps, and information search sites have been used to reach individuals at high risk for HIV infection. However, it is not clear which platform is the most efficient in promoting home HIV self-testing, given that the users of various platforms may have different characteristics that impact their readiness for HIV testing. OBJECTIVE: This study aimed to compare the relative effectiveness of social media sites, dating apps, and information search sites in promoting HIV self-testing among minority men who have sex with men (MSM) at an increased risk of HIV infection. Test kit order rates were used as a proxy to evaluate promotion effectiveness. In addition, we assessed differences in characteristics between participants who ordered and did not order an HIV test kit. METHODS: Culturally appropriate advertisements were placed on popular sites of three different platforms: social media sites (Facebook, Instagram), dating apps (Grindr, Jack'D), and information search sites (Google, Bing). Advertisements targeted young (18-30 years old) and minority (Black or Latinx) MSM at risk of HIV exposure. Recruitment occurred in 2 waves, with each wave running advertisements on 1 platform of each type over the same period. Participants completed a baseline survey assessing sexual or injection use behavior, substance use including alcohol, psychological readiness to test, attitudes toward HIV testing and treatment, and HIV-related stigma. Participants received an electronic code to order a free home-based HIV self-test kit. Follow-up assessments were conducted to assess HIV self-test kit use and uptake of pre-exposure prophylaxis (PrEP) at 14 and 60 days post enrollment. RESULTS: In total, 271 participants were enrolled, and 254 were included in the final analysis. Among these 254 participants, 177 (69.7%) ordered a home HIV self-test kit. Most of the self-test kits were ordered by participants enrolled from dating apps. Due to waves with low enrollment, between wave statistical comparisons were not feasible. Within wave comparison revealed that Jack'D showed higher order rates (3.29 kits/day) compared to Instagram (0.34 kits/day) and Bing (0 kits/day). There were no associations among self-test kit ordering and HIV-related stigma, perceptions about HIV testing and treatment, and mistrust of medical organizations. CONCLUSIONS: Our findings show that using popular dating apps might be an efficient way to promote HIV self-testing. Stigma, perceptions about HIV testing and treatment, or mistrust of medical organizations may not affect order rates of HIV test kits promoted on the internet. TRIAL REGISTRATION: ClinicalTrials.gov NCT04155502; https://clinicaltrials.gov/ct2/show/NCT04155502. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/20417.

Comparing Web-Based Platforms for Promoting HIV Self-Testing and Pre-Exposure Prophylaxis Uptake in High-Risk Men Who Have Sex With Men: Protocol for a Longitudinal Cohort Study.

BACKGROUND: The majority of those living with HIV in the United States are men who have sex with men (MSM), and young, minority MSM account for more new HIV infections than any other group. HIV transmission can be reduced through detection and early treatment initiation or by starting pre-exposure prophylaxis (PrEP), but rates of testing are lower than recommended among MSM, and PrEP uptake has been slow. Although promoting HIV testing and PrEP uptake by placing advertisements on web-based platforms - such as social media websites and dating apps - is a promising approach for promoting HIV testing and PrEP, the relative effectiveness of HIV prevention advertising on common web-based platforms is underexamined. OBJECTIVE: This study aims to evaluate the relative effectiveness of advertisements placed on 3 types of web-based platforms (social media websites, dating apps, and informational websites) for promoting HIV self-testing and PrEP uptake. METHODS: Advertisements will be placed on social media websites (Facebook, Instagram, and Twitter), dating apps (Grindr, Jack'd, and Hornet), and informational search websites (Google, Yahoo, and Bing) to recruit approximately 400 young (18-30 years old), minority (Black or Latino) MSM at elevated risk of HIV exposure. Recruitment will occur in 3 waves, with each wave running advertisements on 1 website from each type of platform. The number of participants per platform is not prespecified, and recruitment in each wave will occur until approximately 133 HIV self-tests are ordered. Participants will complete a baseline survey assessing risk behavior, substance use, psychological readiness to test, and attitudes and then receive an electronic code to order a free home-based HIV self-test kit. Two follow-ups are planned to assess HIV self-test results and PrEP uptake. RESULTS: Recruitment was completed in July 2020. CONCLUSIONS: Findings may improve our understanding of how the platform users' receptivity to test for HIV differs across web-based platforms and thus may assist in facilitating web-based HIV prevention campaigns. TRIAL REGISTRATION: ClinicalTrials.gov NCT04155502; https://clinicaltrials.gov/ct2/show/NCT04155502. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/20417.

Differential splicing of the lectin domain of an O-glycosyltransferase modulates both peptide and glycopeptide preferences.

Mucin-type O-glycosylation is an essential post-translational modification required for protein secretion, extracellular matrix formation, and organ growth. O-Glycosylation is initiated by a large family of enzymes (GALNTs in mammals and PGANTs in Drosophila) that catalyze the addition of GalNAc onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes contain two functional domains: a catalytic domain and a C-terminal ricin-like lectin domain comprised of three potential GalNAc recognition repeats termed α, ß, and γ. The catalytic domain is responsible for binding donor and acceptor substrates and catalyzing transfer of GalNAc, whereas the lectin domain recognizes more distant extant GalNAc on previously glycosylated substrates. We previously demonstrated a novel role for the α repeat of lectin domain in influencing charged peptide preferences. Here, we further interrogate how the differentially spliced α repeat of the PGANT9A and PGANT9B O-glycosyltransferases confers distinct preferences for a variety of endogenous substrates. Through biochemical analyses and in silico modeling using preferred substrates, we find that a combination of charged residues within the α repeat and charged residues in the flexible gating loop of the catalytic domain distinctively influence the peptide substrate preferences of each splice variant. Moreover, PGANT9A and PGANT9B also display unique glycopeptide preferences. These data illustrate how changes within the noncatalytic lectin domain can alter the recognition of both peptide and glycopeptide substrates. Overall, our results elucidate a novel mechanism for modulating substrate preferences of O-glycosyltransferases via alternative splicing within specific subregions of functional domains.

A molecular switch orchestrates enzyme specificity and secretory granule morphology.

Regulated secretion is an essential process where molecules destined for export are directed to membranous secretory granules, where they undergo packaging and maturation. Here, we identify a gene (pgant9) that influences the structure and shape of secretory granules within the Drosophila salivary gland. Loss of pgant9, which encodes an O-glycosyltransferase, results in secretory granules with an irregular, shard-like morphology, and altered glycosylation of cargo. Interestingly, pgant9 undergoes a splicing event that acts as a molecular switch to alter the charge of a loop controlling access to the active site of the enzyme. The splice variant with the negatively charged loop glycosylates the positively charged secretory cargo and rescues secretory granule morphology. Our study highlights a mechanism for dictating substrate specificity within the O-glycosyltransferase enzyme family. Moreover, our in vitro and in vivo studies suggest that the glycosylation status of secretory cargo influences the morphology of maturing secretory granules.

Evidence for GALNT12 as a moderate penetrance gene for colorectal cancer.

Characterizing moderate penetrance susceptibility genes is an emerging frontier in colorectal cancer (CRC) research. GALNT12 is a strong candidate CRC-susceptibility gene given previous linkage and association studies, and inactivating somatic and germline alleles in CRC patients. Previously, we found rare segregating germline GALNT12 variants in a clinic-based cohort (N = 118) with predisposition for CRC. Here, we screened a new population-based cohort of incident CRC cases (N = 479) for rare (MAF ≤1%) deleterious germline GALNT12 variants. GALNT12 screening revealed eight rare variants. Two variants were previously described (p.Asp303Asn, p.Arg297Trp), and additionally, we found six other rare variants: five missense (p.His101Gln, p.Ile142Thr, p.Glu239Gln, p.Thr286Met, p.Val290Phe) and one putative splice-altering variant (c.732-8 G>T). Sequencing of population-matched controls (N = 400) revealed higher burden of these variants in CRC cases compared with healthy controls (P = 0.0381). We then functionally characterized the impact of substitutions on GALNT12 enzyme activity using in vitro-derived peptide substrates. Three of the newly identified GALNT12 missense variants (p.His101Gln, p.Ile142Thr, p.Val290Phe) demonstrated a marked loss (>2-fold reduction) of enzymatic activity compared with wild-type (P ≤ 0.05), whereas p.Glu239Gln exhibited a ∼2-fold reduction in activity (P = 0.077). These findings provide strong, independent evidence for the association of GALNT12 defects with CRC-susceptibility; underscoring implications for glycosylation pathway defects in CRC.

Revisiting the human polypeptide GalNAc-T1 and T13 paralogs.

Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns of each GalNAc-T isoforms. Here, we present a comprehensive in vitro analysis of the peptide substrate specificity of GalNAc-T13, showing that it essentially overlaps with the ubiquitous expressed GalNAc-T1 isoform found in the same subfamily as T13. We have also identified and partially characterized nine splice variants of GalNAc-T13, which add further complexity to the GalNAc-T family. Two variants with changes in their lectin domains were characterized by in vitro glycosylation assays, and one (Δ39Ex9) was inactive while the second one (Ex10b) had essentially unaltered activity. We used reverse transcription-polymerase chain reaction analysis of human neuroblastoma cell lines, normal brain and a small panel of neuroblastoma tumors to demonstrate that several splice variants (Ex10b, ΔEx9, ΔEx2-7 and ΔEx6/8-39bpEx9) were highly expressed in tumor cell lines compared with normal brain, although the functional implications remain to be unveiled. In summary, the GalNAc-T13 isoform is predicted to function similarly to GalNAc-T1 against peptide substrates in vivo, in contrast to a prior report, but is unique by being selectively expressed in the brain.

Biochemical and functional characterization of glycosylation-associated mutational landscapes in colon cancer.

The molecular basis of aberrant protein glycosylation, a pathological alteration widespread in colorectal cancers (CRC), and the mechanisms by which it contributes to tumor progression remain largely unknown. We performed targeted re-sequencing of 430 glycosylation-associated genes in a series of patient-derived CRC cell lines (N = 31) and matched primary tumor tissues, identifying 12 new significantly mutated glycosylation-associated genes in colon cancer. In particular, we observed an enrichment of mutations in genes (B3GNT2, B4GALT2, ST6GALNAC2) involved in the biosynthesis of N- and Cores 1-3 O-linked glycans in the colon, accounting for ~16% of the CRCs tested. Analysis of independent large-scale tumor tissue datasets confirmed recurrent mutations within these genes in colon and other gastrointestinal cancers. Systematic biochemical and phenotypic characterization of the candidate wild-type and mutant glycosyltransferases demonstrated these mutations as either markedly altering protein localization, post-translational modification, encoded enzymatic activities and/or the migratory potential of colon carcinoma cells. These findings suggest that functionally deleterious mutations in glycosyltransferase genes in part underlie aberrant glycosylation, and contribute to the pathogenesis of molecular subsets of colon and other gastrointestinal malignancies.

Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family.

A large family of UDP-GalNAc:polypeptide GalNAc transferases (ppGalNAc-Ts) initiates and defines sites of mucin-type Ser/Thr-O-GalNAc glycosylation. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both families possess variable activities against glycopeptide substrates. All but one isoform contains a C-terminal carbohydrate-binding lectin domain whose roles in modulating glycopeptide specificity is just being understood. We have previously shown for several peptide-preferring isoforms that the presence of a remote Thr-O-GalNAc, 6-17 residues from a Ser/Thr acceptor site, may enhance overall catalytic activity in an N- or C-terminal direction. This enhancement varies with isoform and is attributed to Thr-O-GalNAc interactions at the lectin domain. We now report on the glycopeptide substrate utilization of a series of glycopeptide (human-ppGalNAc-T4, T7, T10, T12 and fly PGANT7) and peptide-preferring transferases (T2, T3 and T5) by exploiting a series of random glycopeptide substrates designed to probe the functions of their catalytic and lectin domains. Glycosylation was observed at the -3, -1 and +1 residues relative to a neighboring Thr-O-GalNAc, depending on isoform, which we attribute to specific Thr-O-GalNAc binding at the catalytic domain. Additionally, these glycopeptide-preferring isoforms show remote lectin domain-assisted Thr-O-GalNAc enhancements that vary from modest to none. We conclude that the glycopeptide specificity of the glycopeptide-preferring isoforms predominantly resides in their catalytic domain but may be further modulated by remote lectin domain interactions. These studies further demonstrate that both domains of the ppGalNAc-Ts have specialized and unique functions that work in concert to control and order mucin-type O-glycosylation.

The lectin domain of the polypeptide GalNAc transferase family of glycosyltransferases (ppGalNAc Ts) acts as a switch directing glycopeptide substrate glycosylation in an N- or C-terminal direction, further controlling mucin type O-glycosylation.

Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled.