Elevated levels of MMP12 sourced from macrophages are associated with poor prognosis in urothelial bladder cancer.

BACKGROUND: Urothelial bladder cancer is most frequently diagnosed at the non-muscle-invasive stage (NMIBC). However, recurrences and interventions for intermediate and high-risk NMIBC patients impact the quality of life. Biomarkers for patient stratification could help to avoid unnecessary interventions whilst indicating aggressive measures when required. METHODS: In this study, immuno-oncology focused, multiplexed proximity extension assays were utilised to analyse plasma (n = 90) and urine (n = 40) samples from 90 newly-diagnosed and treatment-naïve bladder cancer patients. Public single-cell RNA-sequencing and microarray data from patient tumour tissues and murine OH-BBN-induced urothelial carcinomas were also explored to further corroborate the proteomic findings. RESULTS: Plasma from muscle-invasive, urothelial bladder cancer patients displayed higher levels of MMP7 (p = 0.028) and CCL23 (p = 0.03) compared to NMIBC patients, whereas urine displayed higher levels of CD27 (p = 0.044) and CD40 (p = 0.04) in the NMIBC group by two-sided Wilcoxon rank-sum tests. Random forest survival and multivariable regression analyses identified increased MMP12 plasma levels as an independent marker (p < 0.001) associated with shorter overall survival (HR = 1.8, p < 0.001, 95% CI:1.3-2.5); this finding was validated in an independent patient OLINK cohort, but could not be established using a transcriptomic microarray dataset. Single-cell transcriptomics analyses indicated tumour-infiltrating macrophages as a putative source of MMP12. CONCLUSIONS: The measurable levels of tumour-localised, immune-cell-derived MMP12 in blood suggest MMP12 as an important biomarker that could complement histopathology-based risk stratification. As MMP12 stems from infiltrating immune cells rather than the tumor cells themselves, analyses performed on tissue biopsy material risk a biased selection of biomarkers produced by the tumour, while ignoring the surrounding microenvironment.

Switching to Bictegravir, Emtricitabine, and Tenofovir Alafenamide in Virologically Suppressed Adults With Human Immunodeficiency Virus.

BACKGROUND: Bictegravir (B)/emtricitabine (F)/tenofovir alafenamide (TAF) is guideline-recommended treatment for human immunodeficiency virus type 1 (HIV-1). We evaluated whether people receiving dolutegravir (DTG) plus F/TAF or F/TDF (tenofovir disoproxil fumarate) with viral suppression can switch to B/F/TAF without compromising safety or efficacy, regardless of preexisting nucleoside reverse transcriptase inhibitor (NRTI) resistance. METHODS: In this multicenter, randomized, double-blinded, active-controlled, noninferiority trial, we enrolled adults who were virologically suppressed for ≥6 months before screening (with documented/suspected NRTI resistance) or ≥3 months before screening (with no documented/suspected NRTI resistance) on DTG plus either F/TDF or F/TAF. We randomly assigned (1:1) participants to switch to B/F/TAF or DTG + F/TAF once daily for 48 weeks, each with matching placebo. The primary endpoint was proportion of participants with plasma HIV-1 RNA ≥50 copies/mL at week 48 (snapshot algorithm); the prespecified noninferiority margin was 4%. RESULTS: Five hundred sixty-seven adults were randomized; 565 were treated (284 B/F/TAF, 281 DTG + F/TAF). At week 48, B/F/TAF was noninferior to DTG + F/TAF, as 0.4% (1/284) vs 1.1% (3/281) had HIV-1 RNA ≥50 copies/mL (difference, -0.7% [95.001% confidence interval {CI}, -2.8% to 1.0%]). There were no significant differences in efficacy among participants with suspected or confirmed prior NRTI resistance (n = 138). No participant had treatment-emergent drug resistance. Median weight change from baseline at week 48 was +1.3 kg (B/F/TAF) vs +1.1 kg (DTG + F/TAF) (P = .46). Weight change differed by baseline NRTIs (+2.2 kg [F/TDF] and +0.6 kg [F/TAF], P < .001), with no differences between B/F/TAF and DTG + F/TAF. CONCLUSIONS: The single-tablet regimen B/F/TAF is a safe, effective option for people virologically suppressed on DTG plus either F/TDF or F/TAF, including in individuals with preexisting resistance to NRTIs. CLINICAL TRIALS REGISTRATION: NCT03110380.

The Sirenic Links between Diabetes, Obesity, and Bladder Cancer.

There is considerable evidence of a positive association between the incidence of type 2 diabetes mellitus (T2DM) and obesity with bladder cancer (BCa), with the link between T2DM and obesity having already been established. There also appear to be potential associations between Pleckstrin homology domain containing S1 (PLEKHS1) and the Insulin-like Growth Factor (IGF) axis. Seven literature searches were carried out to investigate the backgrounds of these potential links. PLEKHS1 is a candidate biomarker in BCa, with mutations that are easily detectable in urine and increased expression seemingly associated with worse disease states. PLEKHS1 has also been implicated as a potential mediator for the onset of T2DM in people with obesity. The substantial evidence of the involvement of IGF in BCa, the role of the IGF axis in obesity and T2DM, and the global prevalence of T2DM and obesity suggest there is scope for investigating the links between these components. Preliminary findings on the relationship between PLEKHS1 and the IGF axis signal possible associations with BCa progression. This indicates that PLEKHS1 plays a role in the pathogenesis of BCa that may be mediated by members of the IGF axis. Further detailed research is needed to establish the relationship between PLEKHS1 and the IGF axis in BCa and determine how these phenomena overlap with T2DM and obesity.

Chlamydia and Gonorrhea Incidence and Testing Among Patients in the Human Immunodeficiency Virus Outpatient Study (HOPS), 2007-2017.

BACKGROUND: Although chlamydia (CT) and gonorrhea (GC) infections are increasing in the United States, there are limited data on their incidence, testing rates, and associated risk factors among persons living with HIV (PLWH), including by anatomic site among men who have sex with men (MSM). METHODS: We analyzed 2007-2017 medical records data from Human Immunodeficiency Virus (HIV) Outpatient Study (HOPS) participants in care at 9 HIV clinics. We calculated CT (and GC) incidence and testing rates and assessed associations with sociodemographic and clinical factors using log-linear regression. RESULTS: Among 4727 PLWH, 397 had 881 CT infections and 331 had 861 GC infections, with an incidence of 2.95 and 2.88 per 100 person-years, respectively. From 2007 to 2017, incidence and testing rates increased by approximately 3.0- and 1.9-fold for CT and GC, respectively. Multivariable factors associated with incident CT (GC) included younger age, MSM, and prior diagnoses of sexually transmitted diseases (STDs). Among 1159 MSM, 583 (50.3%) had 844 CT and 843 GC tests during 2016-2017, and 26.6% of tests were 3-site (urethra, rectum, and pharynx), yielding the highest rates of CT (GC) detection. Multivariable factors associated with CT (GC) testing included younger age, non-Hispanic/Latino black race, and having prior STDs. CONCLUSIONS: Recent CT and GC incidence and testing increased among PLWH; however, only half of MSM were tested for CT or GC during 2016-2017 and less than a third of tests were 3-site. To promote sexual health and STD prevention among PLWH who are MSM, research regarding the added value of CT and GC testing across 3 anatomic sites is needed.

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23-gene panel with utility for non-invasive diagnosis and risk stratification.

OBJECTIVES: To develop a focused panel of somatic mutations (SMs) present in the majority of urothelial bladder cancers (UBCs), to investigate the diagnostic and prognostic utility of this panel, and to compare the identification of SMs in urinary cell-pellet (cp)DNA and cell-free (cf)DNA as part of the development of a non-invasive clinical assay. PATIENTS AND METHODS: A panel of SMs was validated by targeted deep-sequencing of tumour DNA from 956 patients with UBC. In addition, amplicon and capture-based targeted sequencing measured mutant allele frequencies (MAFs) of SMs in 314 urine cpDNAs and 153 urine cfDNAs. The association of SMs with grade, stage, and clinical outcomes was investigated by univariate and multivariate Cox models. Concordance between SMs detected in tumour tissue and cpDNA and cfDNA was assessed. RESULTS: The panel comprised SMs in 23 genes: TERT (promoter), FGFR3, PIK3CA, TP53, ERCC2, RHOB, ERBB2, HRAS, RXRA, ELF3, CDKN1A, KRAS, KDM6A, AKT1, FBXW7, ERBB3, SF3B1, CTNNB1, BRAF, C3orf70, CREBBP, CDKN2A, and NRAS; 93.5-98.3% of UBCs of all grades and stages harboured ≥1 SM (mean: 2.5 SMs/tumour). RAS mutations were associated with better overall survival (P = 0.04). Mutations in RXRA, RHOB and TERT (promoter) were associated with shorter time to recurrence (P < 0.05). MAFs in urinary cfDNA and cpDNA were highly correlated; using a capture-based approach, >94% of tumour SMs were detected in both cpDNA and cfDNA. CONCLUSIONS: SMs are reliably detected in urinary cpDNA and cfDNA. The technical capability to identify very low MAFs is essential to reliably detect UBC, regardless of the use of cpDNA or cfDNA. This 23-gene panel shows promise for the non-invasive diagnosis and risk stratification of UBC.

Tropomyosins: Potential Biomarkers for Urothelial Bladder Cancer.

Despite the incidence and prevalence of urothelial bladder cancer (UBC), few advances in treatment and diagnosis have been made in recent years. In this review, we discuss potential biomarker candidates: the tropomyosin family of genes, encoded by four loci in the human genome. The expression of these genes is tissue-specific. Tropomyosins are responsible for diverse cellular roles, most notably based upon their interplay with actin to maintain cellular processes, integrity and structure. Tropomyosins exhibit a large variety of splice forms, and altered isoform expression levels have been associated with cancer, including UBC. Notably, tropomyosin isoforms are detectable in urine, offering the potential for non-invasive diagnosis and risk-stratification. This review collates the basic knowledge on tropomyosin and its isoforms, and discusses their relationships with cancer-related phenomena, most specifically in UBC.

Risk Factors and Incidence of Syphilis in Human Immunodeficiency Virus (HIV)-Infected Persons: The HIV Outpatient Study, 1999-2015.

Background: Since 2000, the incidence of syphilis has been increasing, especially among gay, bisexual, and other men who have sex with men (MSM) in the United States. We assessed temporal trends and associated risk factors for newly diagnosed syphilis infections among human immunodeficiency virus (HIV)-infected patients during a 16-year period. Methods: We analyzed data from the HIV Outpatient Study (HOPS) cohort participants at 10 US HIV clinics during 1999-2015. New syphilis cases were defined based on laboratory parameters and clinical diagnoses. We performed Cox proportional hazards regression analyses of sociodemographic, clinical, and behavioral risk factors for new syphilis infections. Results: We studied 6888 HIV-infected participants; 641 had 1 or more new syphilis diagnoses during a median follow-up of 5.2 years. Most participants were male (78%), aged 31-50 years, and 57% were MSM. The overall incidence was 1.8 (95% confidence interval [CI], 1.6-1.9) per 100 person-years (PY) and it increased from 0.4 (95% CI, .2-.8) to 2.2 (95% CI, 1.4-3.5) per 100 PY during 1999-2015. In multivariable analyses adjusting for calendar year, risk factors for syphilis included age 18-30 years (hazard ratio [HR], 1.3 [95% CI, 1.1-1.6]) vs 31-40 years, being MSM (HR, 3.1 [95% CI, 2.4-4.1]) vs heterosexual male, and being non-Hispanic black (HR, 1.6 [95% CI, 1.4-1.9]) vs non-Hispanic white. Conclusions: The increases in the syphilis incidence rate through 2015 reflect ongoing sexual risk and highlight the need for enhanced prevention interventions among HIV-infected patients in care.

Globotriaosylsphingosine (Lyso-Gb3) as a biomarker for cardiac variant (N215S) Fabry disease.

Fabry disease (FD) is a multi-systemic X-linked lysosomal disorder caused by the deficient activity of α-galactosidase-A enzyme, which leads to accumulation of glycosphingolipids in various body tissues. The N215S mutation is a known variant of FD, with a late onset cardiac phenotype. Consensus guidelines acknowledged the use of globotriaosylsphingosine (Lyso-Gb3) as a diagnostic marker for classical FD but its utility for cardiac variant FD is not clear. We aim to characterize the clinical features and evaluate the diagnostic accuracy of plasma and urinary Lyso-Gb3 levels in N215S cardiac variant FD patients. Thirty-four FD patients with the late-onset N215S cardiac variant mutation were enrolled along with 62 classical FD patients and 109 healthy controls. Plasma and urinary Lyso-Gb3 and its analogues were analyzed by LC-MS/MS. Both FD males and females with N215S mutation showed Lyso-Gb3 levels of (mean ± SEM) 9.7 ± 1.0 and 5.4 ± 0.8 nM, respectively. These levels were significantly higher than healthy control and lower than classical FD patients (p < 0.0001). Plasma Lyso-Gb3 levels equal to or higher than 2.7 nM yielded a diagnostic sensitivity and specificity of 100% (AUC = 1, p < 0.0001). Cardiac involvement was frequent with 16/34 (47%) developing left ventricular hypertrophy. Three patients who underwent renal biopsy had the characteristic sphingolipid deposition in the podocytes while 6/19 (32%) had evidence of white matter changes or infarct on brain MRI. Taken together, cardiac variant N215S mutation is rather an attenuated form of classical FD. Plasma Lyso-Gb3 is a diagnostic hallmark to differentiate N215S variant phenotype from subjects with no FD.

Body size drives allochthony in food webs of tropical rivers.

Food web subsidies from external sources ("allochthony") can support rich biological diversity and high secondary and tertiary production in aquatic systems, even those with low rates of primary production. However, animals vary in their degree of dependence on these subsidies. We examined dietary sources for aquatic animals restricted to refugial habitats (waterholes) during the dry season in Australia's wet-dry tropics, and show that allochthony is strongly size dependent. While small-bodied fishes and invertebrates derived a large proportion of their diet from autochthonous sources within the waterhole (phytoplankton, periphyton, or macrophytes), larger animals, including predatory fishes and crocodiles, demonstrated allochthony from seasonally inundated floodplains, coastal zones or the surrounding savanna. Autochthony declined roughly 10% for each order of magnitude increase in body size. The largest animals in the food web, estuarine crocodiles (Crocodylus porosus), derived ~80% of their diet from allochthonous sources. Allochthony enables crocodiles and large predatory fish to achieve high biomass, countering empirically derived expectations for negative density vs. body size relationships. These results highlight the strong degree of connectivity that exists between rivers and their floodplains in systems largely unaffected by river regulation or dams and levees, and how large iconic predators could be disproportionately affected by these human activities.

Reduced Functional Connectivity Between the Hypothalamus and High-order Cortical Regions in Adolescent Patients With Irritable Bowel Syndrome.

The hypothalamus plays a critical role in maintaining visceral homeostasis. Altered hypothalamus activation has been implicated in functional gastrointestinal disorders, including irritable bowel syndrome (IBS). One important aspect of homeostatic regulation is the cortical modulation of limbic and paralimbic subsystems, including the hypothalamus, which in turn affects the descending regulatory processes mediating visceral homeostasis. Using neuroimaging, we evaluated hypothalamus functional connectivity in adolescent patients with IBS and age-matched healthy controls who received rectal distension stimulations. More extensive hypothalamus connectivity was observed in liminal than subliminal condition in controls, but not in patients with IBS. Compared with controls, patients with IBS showed significantly reduced hypothalamus connectivity in the bilateral prefrontal cortices, supplementary motor and premotor areas, bilateral sensorimotor cortex, and limbic subareas, which are specifically involved in homeostatic regulation. The findings support the generalized homeostatic regulation model that reduced cortical and limbic modulations of hypothalamus functioning underlies disrupted visceral homeostasis in patients with IBS.

Quantitative assessment of the cell surface proteome to identify novel therapeutic targets in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma has a high mortality and morbidity. Median survival is less than 6 months. Surgical resection is appropriate in certain circumstances. Because distal cholangiocarcinoma is difficult to distinguish from pancreatic cancers, patients might not receive optimum therapy. Proteomics is the study of complex cellular proteins using mass spectrometry. The aim of this study was to determine the constituent proteins on the cell surface of a model of cholangiocarcinoma. METHODS: A sample preparation technique to enrich for cell surface proteins of the intrahepatic cholangiocarcinoma cell line CC-SW-1 was developed by modifying a NeutrAvidin-biotin system. After isolation, trypsin digestion, and purification, peptides were fractionated for tandem mass spectrometry before being analysed with the NCBInr database and the Mascot search algorithm. Results were confirmed by immunohistochemistry using a peroxidase detection technique on paraffin-embedded sections from resected specimens. FINDINGS: Peptide enrichment was confirmed by electrophoresis. 862 proteins were consistently expressed between samples (n=3). 271 of these proteins were attributed only to the cell surface. They included proteins used clinically for staging disease (cytokeratin 19 [CK19]), identifying cancer stem cells (epithelial cell adhesion molecule [EpCAM], neural cell adhesion molecule [NCAM], epithelial growth factor receptor [EGFR]), and indicating potential for differentiation (Frozzled receptor, Notch pathway). Novel markers from the tumour necrosis factor (TNF) receptor superfamily were also identified. Immunohistochemistry confirmed these findings. INTERPRETATION: The results from this surface proteomic profiling could help to identify novel therapeutic targets in cholangiocarcinoma. Further development of this technique could be translated to distinguish between distal cholangiocarcinoma and pancreatic cancers. FUNDING: UK Medical Research Council.

Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca(2+)-sensitivity and suppress the modulation of Ca(2+)-sensitivity by troponin I phosphorylation.

We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca(2+)-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca(2+)-sensitivity when compared with donor heart troponin and the Ca(2+)-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca(2+)-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca(2+)-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca(2+)-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca(2+)-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca(2+)-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.

Does flood rhythm drive ecosystem responses in tropical riverscapes?

Biotic communities are shaped by adaptations from generations of exposure to selective pressures by recurrent and often infrequent events. In large rivers, floods can act as significant agents of change, causing considerable physical and biotic disturbance while often enhancing productivity and diversity. We show that the relative balance between these seemingly divergent outcomes can be explained by the rhythmicity, or predictability of the timing and magnitude, of flood events. By analyzing biological data for large rivers that span a gradient of rhythmicity in the Neotropics and tropical Australia, we find that systems with rhythmic annual floods have higher-fish species richness, more stable avian populations, and elevated rates of riparian forest production compared with those with arrhythmic flood pulses. Intensification of the hydrological cycle driven by climate change, coupled with reductions in runoff due to water extractions for human use and altered discharge from impoundments, is expected to alter the hydrologic rhythmicity of floodplain rivers with significant consequences for both biodiversity and productivity.

Proteomic profiling of N-linked glycoproteins identifies ConA-binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma.

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N-linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty-eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high-levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA-binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, "ConA-binding proCD" (ConA-pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5-5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA-pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConA-pCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.

Z-band alternatively spliced PDZ motif protein (ZASP) is the major O-linked ß-N-acetylglucosamine-substituted protein in human heart myofibrils.

We studied O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of contractile proteins in human heart using SDS-PAGE and three detection methods: specific enzymatic conjugation of O-GlcNAc with UDP-N-azidoacetylgalactosamine (UDP-GalNAz) that is then linked to a tetramethylrhodamine fluorescent tag and CTD110.6 and RL2 monoclonal antibodies to O-GlcNAc. All three methods showed that O-GlcNAc modification was predominantly in a group of bands ~90 kDa that did not correspond to any of the major myofibrillar proteins. MALDI-MS/MS identified the 90-kDa band as the protein ZASP (Z-band alternatively spliced PDZ motif protein), a minor component of the Z-disc (about 1 per 400 α-actinin) important for myofibrillar development and mechanotransduction. This was confirmed by the co-localization of O-GlcNAc and ZASP in Western blotting and by immunofluorescence microscopy. O-GlcNAcylation of ZASP increased in diseased heart, being 49 ± 5% of all O-GlcNAc in donor, 68 ± 9% in end-stage failing heart, and 76 ± 6% in myectomy muscle samples (donor versus myectomy p < 0.05). ZASP is only 22% of all O-GlcNAcylated proteins in mouse heart myofibrils.

Estimation of polyclonal IgG4 hybrids in normal human serum.

The in vivo or in vitro formation of IgG4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal IgG4 hybrids in normal human serum and comment on the existence of IgG4 molecules with different immunoglobulin light chains. Polyclonal IgG4 was purified from pooled or individual donor human sera and sequentially fractionated using light-chain affinity and size exclusion chromatography. Fractions were analysed by SDS-PAGE, immunoblotting, ELISA, immunodiffusion and matrix-assisted laser-desorption mass spectrometry. Polyclonal IgG4 purified from normal serum contained IgG4κ, IgG4λ and IgG4κ/λ molecules. Size exclusion chromatography showed that IgG4 was principally present in monomeric form (150 000 MW). SDS-PAGE, immunoblotting and ELISA showed the purity of the three IgG4 samples. Immunodiffusion, light-chain sandwich ELISA and mass spectrometry demonstrated that both κ and λ light chains were present on only the IgG4κ/λ molecules. The amounts of IgG4κ/λ hybrid molecules ranged from 21 to 33% from the five sera analysed. Based on the molecular weight these molecules were formed of two IgG4 heavy chains plus one κ and one λ light chain. Polyclonal IgG (IgG4-depleted) was similarly fractionated according to light-chain specificity. No evidence of hybrid IgG κ/λ antibodies was observed. These results indicate that hybrid IgG4κ/λ antibodies compose a substantial portion of IgG4 from normal human serum.

Two-Drug Regimens Dolutegravir/Lamivudine and Dolutegravir/Rilpivirine Are Effective with Few Discontinuations in US Real-World Settings: Results from the TANDEM Study.

INTRODUCTION: Dolutegravir/lamivudine (DTG/3TC) and dolutegravir/rilpivirine (DTG/RPV) are fixed-dose, complete, single-tablet, two-drug regimens (2DRs) indicated for HIV-1. DTG/3TC is approved for antiretroviral therapy (ART)-naive people with HIV-1 and virologically suppressed individuals to replace current ART; DTG/RPV is indicated for virologically suppressed individuals as a switch option. Virologic efficacy and effectiveness of these DTG-based 2DRs have been demonstrated in phase 3 clinical trials and real-world cohorts, primarily from Europe. This study characterized real-world use of DTG-based 2DRs for HIV-1 treatment in the USA. METHODS: TANDEM was a retrospective medical chart review across 24 US sites. Individuals aged ≥ 18 years who initiated DTG/3TC or DTG/RPV before September 30, 2020, with ≥ 6 months of follow-up were included. One cohort included ART-naive people who initiated DTG/3TC (n = 126), and two other cohorts included virologically suppressed (HIV-1 RNA < 50 copies/mL) people on stable ART regimens for ≥ 3 months before switch to either DTG/3TC (n = 192) or DTG/RPV (n = 151). Clinical characteristics, treatment history, and outcomes are described. RESULTS: Virologically suppressed individuals were older than those who were ART-naive, and the ART-naive cohort had higher proportions of individuals assigned male at birth and of Hispanic ethnicity. The most common healthcare provider-reported reason for choosing a DTG-based 2DR was avoidance of long-term toxicities (25-33% across cohorts), followed by simplification/streamlining of treatment. Among ART-naive people on DTG/3TC, 94% achieved virologic suppression after initiation, and 83% maintained suppression at last follow-up; discontinuation rate was < 1%. Among cohorts who switched to DTG-based 2DRs, 96% maintained virologic suppression on DTG/3TC and 93% on DTG/RPV; 2% on DTG/3TC and 3% on DTG/RPV discontinued. CONCLUSION: Motivation for selecting DTG-based 2DRs was primarily driven by a desire to avoid or manage toxicities and simplify treatment. Results demonstrate that DTG/3TC and DTG/RPV are effective in real-world settings, with few discontinuations, reflecting data from clinical trials.

Structural basis of ligand interactions of the large extracellular domain of tetraspanin CD81.

Hepatitis C virus (HCV) causes chronic liver disease, cirrhosis, and primary liver cancer. Despite 130 million people being at risk worldwide, no vaccine exists, and effective therapy is limited by drug resistance, toxicity, and high costs. The tetraspanin CD81 is an essential entry-level receptor required for HCV infection of hepatocytes and represents a critical target for intervention. In this study, we report the first structural characterization of the large extracellular loop of CD81, expressed in mammalian cells and studied in physiological solutions. The HCV E2 glycoprotein recognizes CD81 through a dynamic loop on the helical bundle, which was shown by nuclear magnetic resonance (NMR) spectroscopy to adopt a conformation distinct from that seen in crystals. A novel membrane binding interface was revealed adjacent to the exposed HCV interaction site in the extracellular loop of CD81. The binding pockets for two proposed inhibitors of the CD81-HCV interaction, namely, benzyl salicylate and fexofenadine, were shown to overlap the HCV and membrane interaction sites. Although the dynamic loop region targeted by these compounds presents challenges for structure-based design, the NMR assignments enable realistic screening and validation of ligands. Together, these data provide an improved avenue for developing potent agents that specifically block CD81-HCV interaction and also pave a way for elucidating the recognition mechanisms of diverse tetraspanins.

Tropomyosin isoform expression and phosphorylation in the human heart in health and disease.

We determined the isoforms of tropomyosin expressed and the level of tropomyosin phosphorylation in donor, end-stage failing and hypertrophic obstructive cardiomyopathy samples of human heart muscle. Western blots and isoform-specific antibodies showed that α-tropomyosin was the only significant isoform expressed and that tropomyosin was 25-30% phosphorylated at serine 283. Mass spectrometry confirmed directly that α-tropomyosin made up over 95% of tropomyosin but also indicated the presence of up to 4% κ-tropomyosin and much smaller amounts of ß-, γ- and smooth ß-tropomyosin and about 26% phosphorylation. Neither the isoform distribution nor the level of phosphorylation changed significantly in the pathological heart muscle samples.