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BackgroundThe 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. ObjectiveThis 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. MethodsAdvertisements 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. ResultsRecruitment was completed in July 2020. ConclusionsFindings 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 RegistrationClinicalTrials.gov NCT04155502; https://clinicaltrials.gov/ct2/show/NCT04155502 International Registered Report Identifier (IRRID)DERR1-10.2196/20417

Abstract: Platelet factor 4 (PF4) is an abundant chemokine that is released from platelet α-granules on activation. PF4 is central to the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT) in which antibodies to PF4 form immune complexes with PF4, which activate platelets and neutrophils through Fc receptors. In this study, we show that PF4 binds and activates the thrombopoietin receptor, cellular myeloproliferative leukemia protein (c-Mpl), on platelets. This leads to the activation of Janus kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5, leading to platelet aggregation. Inhibition of the c-Mpl-JAK2 pathway inhibits platelet aggregation to PF4, VITT sera, and the combination of PF4 and IgG isolated from VITT patient plasma. The results support a model in which PF4-based immune complexes activate platelets through binding of the Fc domain to FcγRIIA and PF4 to c-Mpl., (© 2024 by American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Platelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate-binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets., Objective: In this study, we investigated the role of galectin-9 (Gal-9) as a novel ligand for platelet glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2)., Methods: Platelet spreading, aggregation, and P-selectin expression in response to Gal-9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid-phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal-9 and GPVI, and immunoprecipitation for detecting CLEC-2 phosphorylation. Wild-type (WT), GPVI-knockout (Gp6 -/- ), and GPVI and CLEC-2-double knockout (Gp6 -/- /Gp1ba-Cre-Clec1b fl / fl ) mice were used., Results: We have shown that recombinant Gal-9 stimulates aggregation in human and mouse washed platelets dose-dependently. Platelets from both species adhere and spread on immobilized Gal-9 and express P-selectin. Gal-9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal-9 stimulated tyrosine phosphorylation of CLEC-2 and proteins known to lie downstream of GPVI and CLEC-2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI-deficient murine platelets exhibited significantly impaired aggregation in response to Gal-9, which was further abrogated in GPVI and CLEC-2-double-deficient platelets., Conclusions: We have identified Gal-9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC-2., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Objective: Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and Results: We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients., Conclusions: Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.

Objective: To develop and test a direct utility measure (McSad) for major, unipolar depression., Methods: A depression specific, multi-attribute health state classification system was created; clinical validity was evaluated by experts using specially designed structured exercises; a cross-sectional survey was conducted to obtain directly measured utilities for depression health states., Setting: Tertiary care, university medical centre., Participants: Three psychiatrists, 3 psychiatric nurses and 3 social workers assessed depression health state clinical validity. Survey participants were referred by psychiatrists and consisted of 105 outpatients, currently in remission with at least one episode of major, unipolar depression in the past two years., Survey Results: Respondent self-health state utility (mean and 95% confidence interval (CI)) was 0.79 (0.74-0.83). Utilities for hypothetical, untreated depression health states were: mild depression, 0.59 (0.55-0.62); moderate depression, 0.32 (0.29-0.34); severe depression, 0.04 (0.01-0.07). Fifty-six percent of respondents rated severe depression worse than being dead. Utilities for the hypothetical health states were not correlated with self-health utility. The intra-class correlation coefficient (ICC) was satisfactory for 13 of the 14 health states assessed., Conclusions: McSad was feasible and acceptable in patients with a history of major unipolar depression. The utilities for mild, moderate and severe untreated depression show the low health-related quality of life associated with depression. Initial assessments of test-retest reliability and validity yielded satisfactory results but further studies are needed to extend our knowledge of the measurement properties of McSad.

We have determined the nucleotide sequence of a stress-inducible mouse Hsp70-encoding gene named hsp70A1. The gene encodes a 641-amino-acid protein whose deduced sequence is similar to those of other members of the HSP70 family. The 5' end (tsp) of a heat-inducible mRNA is 225 bp upstream from the start codon, and several consensus recognition sequences for transcription factors lie upstream from this tsp. There are 17 putative binding sites for heat-shock transcription factor (HSF), including three clusters of multiple binding sites. We show that this upstream region is sufficient to direct heat-inducible expression of a hsp70A1::cat hybrid gene in mouse and human cells.

Immunoglobulin heavy chain binding protein (BiP) is present in the lumen of the mammalian endoplasmic reticulum, where it associates transiently with a variety of newly synthesized secretory and membrane proteins or permanently with mutant proteins that are incorrectly folded. We describe a unique member of the Saccharomyces cerevisiae 70-kDa heat shock protein gene family (HSP70) that encodes a protein homologous to mammalian BiP. The DNA sequence contains a 2046-nucleotide open reading frame devoid of introns, and examination of the predicted amino acid sequence reveals features not found in most other yeast HSP70 proteins but which are present in BiP. Most notable are a 42-residue sequence at the N terminus that exhibits characteristics of a cleavable signal sequence and a C-terminal sequence, -His-Asp-Glu-Leu, that is involved in determining endoplasmic reticulum localization in yeast. The 5' flanking region of this gene contains two overlapping sequences between nucleotides -146 and -169 that closely resemble consensus heat shock elements. The yeast BiP gene is strongly heat shock-inducible, whereas the BiP genes in various other species are either weakly or non-heat-inducible. We demonstrate that a functional BiP gene is essential for vegetative growth. An evolutionary comparison of amino acid sequences of 34 HSP70 proteins from 17 species suggests that BiP genes share a common ancestor, which diverged from other HSP70 genes near the time when eukaryotes first appeared.

We have used cloned fragments from a Drosophila melanogaster hsp70 gene and a mouse hsp68 cDNA in recombinational screens of mouse genomic libraries. Using the mouse probe we have isolated two overlapping recombinant lambda phages comprising 22 kb of cloned DNA. Southern analysis has localized the homology with the Drosophila hsp70 coding region to a 2.2-kb fragment containing the mouse heat-shock gene. Insertion accompanying recombinational screening can disrupt interesting sequences; we have overcome this inconvenience by developing a simple one-step genetic selection for phage which have precisely excised the microplasmid probe.

We used a cloned Drosophila melanogaster hsp70 gene to hybrid-select heat shock-induced mouse mRNA and showed that this mRNA encodes the major mouse heat shock protein. This result suggests that the sequence of the hsp70 gene(s) is highly conserved.

The T4 bacteriophage gene 41 protein is known from genetic analysis to be essential for phage DNA replication in vivo. It became possible to monitor the activity of this protein during purification after development of an "in vitro complementation assay," which measures its stimulation of DNA synthesis in a concentrated crude lysate prepared from Escherichia coli cells infected with a T4 bacteriophage mutant in gene 41 (L. Moran and B. Alberts, manuscript in preparation). In this report, a purification procedure involving three chromatographic steps is described which reproducibly yields a 90% homogeneous preparation of this rather unstable protein. The major polypeptide chain present (58,000 daltons) is shown to cosediment with a DNA-dependent GTPase (and ATPase) activity, and to induce extensive in vitro DNA synthesis on both single- and double-stranded DNA templates when incubated with our preparations of five other purified T4 DNA replication proteins (plus deoxyribonucleoside and ribonucleoside triphosphates).

Transgenic mice have been generated that express the E. coli beta-galactosidase gene under the control of the promoter from the mouse heat-shock gene, hsp68. Sequences from -664 to +113 relative to the start of transcription of the hsp68 gene were sufficient to direct stress-induced expression of the beta-galactosidase gene in adult tail tissue and various tissues of fetal stages of development. Expression was detected in situ by staining with the chromogenic substrate, X-gal. The hybrid gene was refractory to induction in preimplantation embryos until the blastocyst stage of development, as reported for the endogenous hsp68 gene. No constitutive expression was observed by in situ staining or Northern analysis at any stage of development, even in tissues that constitutively express the endogenous hsp68 gene. We conclude that the hsp68 promoter region included in the construct contains sufficient sequence information for heat and arsenite inducibility, but it does not contain sequences controlling tissue-specific expression during development. This tightly regulated inducible promoter may provide a useful tool for short-term inducible gene expression in transgenic mice.

Heat shock of mouse L cells induces the synthesis of two polypeptides of Mrs 68,000 and 89,000. Using a fragment of a cloned gene encoding the Drosophila melanogaster Mr 70,000 heat shock protein (hsp70), we have shown that this protein has been highly conserved during eukaryotic evolution. We extended this observation by probing at low stringency for the expression in mouse L cells of RNA homologous to the Drosophila hsp70 gene. In addition to the RNA encoding the inducible Mr 68,000 heat shock protein (hsp68), there are mouse mRNAs encoding proteins of Mrs 70,000 and 74,000 that are homologous to the Drosophila hsp70 gene. The Mrs 70,000 and 74,000 proteins and their mRNAs are abundant components of unstressed mouse L cells. These constituitively expressed proteins are unique polypeptides in contrast to the several isoelectric point variants of the inducible hsp68. We do not detect hsp68 or its mRNA in unstressed L cells. In addition to the mRNAs corresponding to hsp68 and the Mrs 74,000 and 70,000 proteins, we detect a fourth RNA homologous to the Drosophila hsp70 gene but whose protein product has not been identified. Our results suggest that the hsp68 gene of mouse L cells is a member of a multigene family and that the individual family members are distinguishable by their degree of similarity but show differences in the regulation of their expression.

The construction and isolation of three recombinant DNAs complementary to different mouse L-cell Mr = 68,000 heat shock protein (hsp68) mRNAs is described. cDNA libraries derived from heat-shocked mouse L-cell poly(A)+ RNA by the vector-linked primer strategy of cDNA synthesis and cloning of Okayama and Berg (Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170) were screened first with a Drosophila hsp70 heterologous probe and subsequently with a cDNA probe isolated from the first screening. Positive clones were assigned to one of three sets based on their restriction map, and the largest member of each group was chosen for further analysis. All three cDNAs hybrid-select mRNA for the mouse major heat shock protein (hsp68) as assayed by in vitro translation and hybridize preferentially to two heat shock-induced hsp68 mRNAs on Northern blots. The coding regions of the cDNAs are almost identical and closely resemble other HSP70 genes but the 3' untranslated regions diverge considerably. Differences in the lengths of the untranslated regions are responsible for the two different sized induced hsp68 mRNAs in mouse L-cells. The physical maps of these cDNA clones and the limited number of mouse genomic DNA fragments detected on Southern blots suggest that there are at least three closely related heat shock-inducible members of the mouse HSP70 gene family. None of the cloned cDNAs are derived from the two related cognate genes known to be present in the mouse genome.