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Background: Vaccine development against Chlamydia, a prevalent sexually transmitted infection (STI), is imperative due to its global public health impact. However, significant challenges arise in the production of effective subunit vaccines based on recombinant protein antigens, particularly with membrane proteins like the Major Outer Membrane Protein (MOMP)., Methods: Cell-free protein synthesis (CFPS) technology is an attractive approach to address these challenges as a method of high-throughput membrane protein and protein complex production coupled with nanolipoprotein particles (NLPs). NLPs provide a supporting scaffold while allowing easy adjuvant addition during formulation. Over the last decade, we have been working toward the production and characterization of MOMP-NLP complexes for vaccine testing., Results: The work presented here highlights the expression and biophysical analyses, including transmission electron microscopy (TEM) and dynamic light scattering (DLS), which confirm the formation and functionality of MOMP-NLP complexes for use in animal studies. Moreover, immunization studies in preclinical models compare the past and present protective efficacy of MOMP-NLP formulations, particularly when co-adjuvanted with CpG and FSL1., Conclusion: Ex vivo assessments further highlight the immunomodulatory effects of MOMP-NLP vaccinations, emphasizing their potential to elicit robust immune responses. However, further research is warranted to optimize vaccine formulations further, validate efficacy against Chlamydia trachomatis , and better understand the underlying mechanisms of immune response.

Background: Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen in humans worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and decentralized production of recombinant protein vaccine antigens. Methods: Here, we use CFPS to produce the full-length putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for evaluation as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) (RIBM, Tsukuba, Japan) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four weeks later by an i.m. boost before respiratory challenge with 10 4 inclusion forming units (IFU) of Chlamydia muridarum . Results: Immunization with CT584 generated robust antibody responses but weak cell-mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lung weights, and the presence of high numbers of IFUs in the lungs. Conclusion: While CT584 was not a protective vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens make it an attractive technique for antigen production.

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and de-centralized production of recombinant protein vaccine antigens. Here, we use CFPS to produce the putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for use as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four-weeks later by an i.m. boost before respiratory challenge with 10 4 inclusion forming units (IFU) of Chlamydia muridarum . Immunization with CT584 generated robust antibody responses but weak cell mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lungs' weights and the presence of high numbers of IFUs in the lungs. While CT584 alone may not be the ideal vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens makes it an attractive technique for antigen production., Competing Interests: Conflicts of Interest The authors declare no conflict of interest.

Healthcare workers (HCWs) are a key population at high risk for hepatitis B (HBV) and hepatitis C (HCV) infections. We aim to study HBV vaccination coverage, seroprevalence, knowledge, attitudes, and practices towards HBV and HCV infections among HCWs in public sector in Cambodia. A nationally representative cross-sectional study was implemented in 2019, among Cambodian HCWs. A standardized questionnaire was administered to randomly selected HCWs whose blood was then sampled. We used univariate and multivariate regression to determine predictors of outcomes. Among 755 participants, we found 4.9% positive HBsAg and 2.3% positive anti-HCV Ab. HBV vaccination coverage was 59.3%. Lack of knowledge was found on the route of transmission, HBV vaccination, diagnosis and treatment of HBV and HCV. 67% of HCWs thought that all patients should be screened for HBV and HCV and about 30% of them would refuse to take care of infected patients. 58% of HCWs always recapped the needle after use. In univariate analysis, older age-group (> 50 years) is more likely to have positive anti-HCV (OR: 9.48; 95% CI: 2.36-38.18). HCWs who were younger, female or having higher education or having ever been tested, were more likely to have gotten HBV vaccinated. Multivariate analysis reconfirmed these predictors of getting vaccinated. Study findings indicated an urgent need of a national policy for Cambodian HCWs given the high prevalence of hepatitis among this group. Policy should include an effective in-service training program to improve knowledge and practices, a testing and vaccination program for HCWs and it should emphasize stigma intervention towards people living with HBV/HCV., Competing Interests: The authors have no conflicts of interest to disclose., (2024, National Center for Global Health and Medicine.)

Somatic cells of human males and females have 45 chromosomes in common, including the "active" X chromosome. In males the 46 th chromosome is a Y; in females it is an "inactive" X (Xi). Through linear modeling of autosomal gene expression in cells from individuals with zero to three Xi and zero to four Y chromosomes, we found that Xi and Y impact autosomal expression broadly and with remarkably similar effects. Studying sex chromosome structural anomalies, promoters of Xi- and Y-responsive genes, and CRISPR inhibition, we traced part of this shared effect to homologous transcription factors-ZFX and ZFY-encoded by Chr X and Y. This demonstrates sex-shared mechanisms by which Xi and Y modulate autosomal expression. Combined with earlier analyses of sex-linked gene expression, our studies show that 21% of all genes expressed in lymphoblastoid cells or fibroblasts change expression significantly in response to Xi or Y chromosomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Somatic cells of human males and females have 45 chromosomes in common, including the "active" X chromosome. In males the 46 th chromosome is a Y; in females it is an "inactive" X (Xi). Through linear modeling of autosomal gene expression in cells from individuals with zero to three Xi and zero to four Y chromosomes, we found that Xi and Y impact autosomal expression broadly and with remarkably similar effects. Studying sex-chromosome structural anomalies, promoters of Xi- and Y-responsive genes, and CRISPR inhibition, we traced part of this shared effect to homologous transcription factors - ZFX and ZFY - encoded by Chr X and Y. This demonstrates sex-shared mechanisms by which Xi and Y modulate autosomal expression. Combined with earlier analyses of sex-linked gene expression, our studies show that 21% of all genes expressed in lymphoblastoid cells or fibroblasts change expression significantly in response to Xi or Y chromosomes., Competing Interests: Declaration of interests: The authors declare no competing interests.

Background: Bacterial contamination of hematopoietic stem cell (HSC) products is most commonly due to normal skin flora. Salmonella in HSC products is rare, and to our knowledge safe administration of an autologous HSC product containing Salmonella has not been reported., Study Design and Methods: We describe two patients undergoing autologous HSC transplant: peripheral blood HSC collection was performed by leukapheresis, and samples were cultured according to standard institutional protocol. Subsequent microorganism identification was performed using MALDI-TOF (Bruker Biotyper). Strain-relatedness was investigated by infrared spectroscopy using the IR Biotyper (Bruker)., Results: The patients were asymptomatic throughout the collection process; however, HSC products collected on two consecutive days from each patient were positive for Salmonella. Isolates from both cultures were further characterized as Salmonella enterica serovar Dublin by the local public health department. Antibiotic susceptibility testing revealed different sensitivity patterns for the two strains. IR Biotyper demonstrated significant discriminatory power among the clinically significant Salmonella enterica subspecies, serogroups B, C1, and D. The patient strains were similar as both belonged to Group D Salmonella enterica serovar Dublin but were not identical. The Salmonella positive autologous HSC products were infused to both patients following administration of empiric antibiotic therapy. Both patients successfully engrafted and did well., Conclusion: Salmonella is rarely seen in cellular therapy products and positivity may be the result of asymptomatic bacteremia at the time of collection. We present two instances of autologous HSC products containing Salmonella that were infused, along with prophylactic antimicrobial therapy without significant adverse clinical effects., (© 2023 AABB.)

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen. The number of chlamydial infections continuous to increase and there is an urgent need for a safe and efficacious vaccine. To assess the ability of the Chlamydia muridarum polymorphic membrane protein G (PmpG) and the plasmid glycoprotein 3 (Pgp3) as single antigens, and in combination with the major outer-membrane protein (MOMP) to induce protection, BALB/c mice were immunized utilizing CpG-1826 and Montanide ISA 720 VG as adjuvants. Following vaccination with MOMP, significant humoral and cell-mediated immune responses were observed, while immunization with PmpG, or Pgp3, elicited weaker immune responses. Weaker immune responses were induced with MOMP+Pgp3 compared with MOMP alone. Following the intranasal challenge with C. muridarum, mice vaccinated with MOMP showed robust protection against body-weight loss, inflammatory responses in the lungs and number of Chlamydia recovered from the lungs. PmpG and Pgp3 elicited weaker protective responses. Mice immunized with MOMP+PmpG, were no better protected than animals vaccinated with MOMP only, while Pgp3 antagonized the protection elicited by MOMP. In conclusion, PmpG and Pgp3 elicited limited protective immune responses in mice against a respiratory challenge with C. muridarum and failed to enhance the protection induced by MOMP alone. The virulence of Pgp3 may result from its antagonistic effect on the immune protection induced by MOMP.

The "inactive" X chromosome (Xi) has been assumed to have little impact, in trans , on the "active" X (Xa). To test this, we quantified Xi and Xa gene expression in individuals with one Xa and zero to three Xis. Our linear modeling revealed modular Xi and Xa transcriptomes and significant Xi-driven expression changes for 38% (162/423) of expressed X chromosome genes. By integrating allele-specific analyses, we found that modulation of Xa transcript levels by Xi contributes to many of these Xi-driven changes (≥121 genes). By incorporating metrics of evolutionary constraint, we identified 10 X chromosome genes most likely to drive sex differences in common disease and sex chromosome aneuploidy syndromes. We conclude that human X chromosomes are regulated both in cis , through Xi-wide transcriptional attenuation, and in trans , through positive or negative modulation of individual Xa genes by Xi. The sum of these cis and trans effects differs widely among genes., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats., (© 2023. The Author(s).)

Background: There has been insufficient attention to a fundamental force shaping healthcare policies-conflicts of interest (COI). We investigated COI, which results in the professional judgement of a policymaker or healthcare provider being compromised by a secondary interest, in relation to antimicrobial use, thereby illuminating challenges to the regulation of medicines use more broadly. Our objectives were to characterise connections between three groups-policymakers, healthcare providers and pharmaceutical companies-that can create COI, and elucidate the impacts of COI on stages of the policy process., Methods: Using an interpretive approach, we systematically analysed qualitative data from 136 in-depth interviews and five focus group discussions in three Asian countries with dominant private healthcare sectors: Cambodia, Indonesia and Pakistan., Findings: We characterised four types of connections that were pervasive between the three groups: financial, political, social and familial. These connections created strong COI that could impact all stages of the policy process by: preventing issues related to medicines sales from featuring prominently on the agenda; influencing policy formulation towards softer regulatory measures; determining resource availability for, and opposition to, policy implementation; and shaping how accurately the success of contested policies is reported., Interpretation: Our multicountry study fills a gap in empirical evidence on how COI can impede effective policies to improve the quality of healthcare. It shows that COI can be pervasive, rather than sporadic, in influencing regulation of medicine use, and highlights that, in addition to financial connections, other types of connections should be examined as important drivers of COI., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Daratumumab, a human anti-CD38 monoclonal antibody used to treat multiple myeloma, interferes with pretransfusion testing and can mask alloantibodies. Incidence of alloimmunization in patients on daratumumab has not been well characterized, and optimal transfusion guidelines regarding prophylactic antigen matching, accounting for both patient safety and efficiency, have not been well established for these patients., Methods: Records of patients who received daratumumab between January 1, 2014 and July 2, 2019 were reviewed. Daratumumab interference with pretransfusion testing was managed by testing with reagent red blood cells (RBCs) treated with 0.2 M dithiothreitol. When daratumumab was present during antibody testing, patients were transfused with RBC units prophylactically matched for D, C, c, E, e, and K antigens per hospital policy., Results: Out of 90 patients identified, 52 received a total of 638 RBC transfusions (average of 12.3 units per patient, SD 17.2, range 1-105, median 5 among those transfused). Alloantibodies existing before daratumumab initiation were identified in seven patients. No new alloantibodies were detected in any patients after starting daratumumab treatment., Conclusions: The incidence of alloimmunization in patients receiving daratumumab is low. Whether this is due to the effect of daratumumab, underlying pathophysiology, or other factors, is unknown. Because these patients require a large number of RBC transfusions overall and have little observed alloimmunization, phenotype matching (beyond RhD) may be unnecessary. Since the use of dithiothreitol cannot rule out the presence of anti-K, we recommend transfusion of ABO-compatible units, prophylactically matched for the D and K antigens only., (© 2021 AABB.)

Purpose: Uveal melanomas are associated with characteristic genetic changes. Germline mutations in mismatch repair (MMR) genes and microsatellite instability have been implicated in the development of numerous malignant neoplasms such as colon and ovarian cancers. The frequency of MMR defects in uveal melanomas has yet to be determined., Methods: Here, we analyzed the frequency of MMR gene mutations in uveal melanoma specimens from the University of California, San Diego (UCSD), The Cancer Genome Atlas (TGCA), and the Catalogue of Somatic Mutations in Cancer (COSMIC)., Results: We identified only two mutations in a MMR gene: one premature stop codon in the PMS gene within the UCSD cohort (0.5% frequency) and one in-frame deletion in MSH3 within the COSMIC database (0.8% frequency). We report copy number variation of MLH1 in monosomy 3 and show decreased mRNA expression of MLH1 in uveal melanoma specimens with monosomy 3. Expression levels of MLH1 were not found to correlate with the observed number of total mutations., Conclusion: Overall, we show that mutations in MMR genes in uveal melanoma specimens are exceedingly rare, and although one copy of MLH1 is lost in monosomy 3, it does not seem to have pathologic consequences in uveal melanoma pathogenesis.

Background: Healthcare providers' (HCPs) professionalism refers to their commitment and ability to respond to the health needs of the communities they serve and to act in the best interest of patients. Despite attention to increasing the number of HCPs in low- and middle-income countries (LMIC), the quality of professional education delivered to HCPs and their resulting professionalism has been neglected. The Global Action Plan on Antimicrobial Resistance (AMR) seeks to reduce inappropriate use of antibiotics by urging patients to access antibiotics only through qualified HCPs, on the premise that qualified HCPs will act as more responsible and competent gatekeepers of access to antibiotics than unqualified HCPs., Methods: We investigate whether weaknesses in HCP professionalism result in boundaries between qualified HCPs and unqualified providers being blurred, and how these weaknesses impact inappropriate provision of antibiotics by HCPs in two LMIC with increasing AMR-Pakistan and Cambodia. We conducted 85 in-depth interviews with HCPs, policymakers, and pharmaceutical industry representatives. Our thematic analysis was based on a conceptual framework of four components of professionalism and focused on identifying recurring findings in both countries., Results: Despite many cultural and sociodemographic differences between Cambodia and Pakistan, there was a consistent finding that the behaviour of many qualified HCPs did not reflect their professional education. Our analysis identified five areas in which strengthening HCP education could enhance professionalism and reduce the inappropriate use of antibiotics: updating curricula to better cover the need for appropriate use of antibiotics; imparting stronger communication skills to manage patient demand for medications; inculcating essential professional ethics; building skills required for effective collaboration between doctors, pharmacists, and lay HCPs; and ensuring access to (unbiased) continuing medical education., Conclusions: In light of the weaknesses in HCP professionalism identified, we conclude that global guidelines urging patients to only seek care at qualified HCPs should consider whether HCP professional education is equipping them to act in the best interest of the patient and society. Our findings suggest that improvements to HCP professional education are needed urgently and that these should focus not only on the curriculum content and learning methods, but also on the social purpose of graduates.

Background: Global attention to antimicrobial resistance has increased interest in tackling the widespread inappropriate dispensing of antibiotics by informal, for-profit healthcare providers (HCPs). We provide new evidence on an understudied group of informal HCPs: invisible medicine sellers (IMS) who operate without any marked facility. We investigated factors that influence community decisions on which HCPs to purchase medicines from, focusing on reasons for using IMS, and compared different HCPs' knowledge of antibiotic use., Methods: We conducted community focus group discussions (FGDs) in seven purposively selected villages representing high and low informal HCPs use in two peri-urban districts in Phnom Penh, Cambodia. Using information from the FGDs to identify HCPs that sell medicines, we interviewed 35 participants: 21 HCPs (including five IMS) and 14 key informants, including government HCPs and village leaders. We adopted an interpretative approach and conducted a thematic analysis., Results: Community members typically knew of several formal and informal HCPs selling medicines nearby, and IMS were common, as were doctors that sell medicines covertly. Two factors were most salient in influencing the choice of HCP for medicine purchasing. The first was trust in the effectiveness of medicines provided, judged by the speed of symptomatic relief. This pushed HCPs to provide several medicines, including antibiotics, at the first consultation. The second was the convenience offered by IMS and other informal HCPs: supplying medicines when other facilities are closed, accepting delayed payments, providing incomplete courses of medication and selling human antibiotics for animal use., Conclusion: This first study focusing on IMS indicates that it is important, but challenging, for public health agencies to engage with them to reduce inappropriate use of antibiotics. Although public health facilities must fill some gaps that informal HCPs are currently addressing, such as access to medicines at night, reducing demand for unnecessary antibiotics is also critical., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.)

Basal tumor propagating cells (TPCs) control squamous cell carcinoma (SCC) growth by self-renewing and differentiating into supra-basal SCC cells, which lack proliferative potential. While transcription factors such as SOX2 and KLF4 can drive these behaviors, their molecular roles and regulatory interactions with each other have remained elusive. Here, we show that PITX1 is specifically expressed in TPCs, where it co-localizes with SOX2 and TRP63 and determines cell fate in mouse and human SCC. Combining gene targeting with chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic analyses reveals that PITX1 cooperates with SOX2 and TRP63 to sustain an SCC-specific transcriptional feed-forward circuit that maintains TPC-renewal, while inhibiting KLF4 expression and preventing KLF4-dependent differentiation. Conversely, KLF4 represses PITX1, SOX2, and TRP63 expression to prevent TPC expansion. This bi-stable, multi-input network reveals a molecular framework that explains self-renewal, aberrant differentiation, and SCC growth in mice and humans, providing clues for developing differentiation-inducing therapeutic strategies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Receptor tyrosine kinases (RTKs) play critical roles in physiological and pathological processes, and are important anticancer drug targets. In vitro mechanistic and drug discovery studies of full-length RTKs require protein that is both fully functional and free from contaminating proteins. Here we describe a rapid cell-free and detergent-free co-translation method for producing full-length and functional ERBB2 and EGFR receptor tyrosine kinases supported by water-soluble apolipoprotein A-I based nanolipoprotein particles.