Viral protein Nef is detected in plasma of half of HIV-infected adults with undetectable plasma HIV RNA

ObjectiveTo address the role of translationally active HIV reservoir in chronic inflammation and non-AIDS related disorders, we first need a simple and accurate assay to evaluate viral protein expression in virally suppressed subjects.DesignWe optimized an HIV Nef enzyme-linked immunosorbent assay (ELISA) and used it to quantify plasma Nef levels as an indicator of the leaky HIV reservoir in an HIV-infected cohort.MethodsThis study accessed 134 plasma samples from a well-characterized cohort study of HIV-infected and uninfected adults in San Francisco (the SCOPE cohort). We optimized an ELISA for detection of plasma Nef in HIV-negative subjects and HIV-infected non-controllers, and evaluated its utility to quantify plasma Nef levels in a cross-sectional study of ART-suppressed and elite controller HIV-infected subjects.ResultsHere, we describe the performance of an optimized HIV Nef ELISA. When we applied this assay to the study cohort we found that plasma Nef levels were correlated with plasma HIV RNA levels in untreated disease. However, we were able to detect Nef in plasma of approximately half of subjects on ART or with elite control, despite the lack of detectable plasma HIV RNA levels using standard assays. Plasma Nef levels were not consistently associated with CD4+ T-cell count, CD8+ T-cell count, self-reported nadir CD4+ T-cell count or the CD4+/CD8+ T-cell ratio in HIV-infected subjects.ConclusionSince plasma HIV RNA levels are undetectable in virally suppressed subjects, it is reasonable to assume that viral protein expression in leaky reservoir, and not plasma virions, is the source of Nef accumulating in plasma. To examine this further, improvements of the assay sensitivity, by lowering the background through improvements in the quality of Nef antibodies, and detailed characterization of the HIV reservoirs are needed

Enhanced antimicrobial efficacy by co-delivery of PGA capped silver nanoparticles and ascorbic acid with poly(lactide-co-glycolide)

Silver nanoparticles (AgNps) were prepared by modified chemical reduction with poly (Lglutamic acid) (PGA) as capping agent. These Ag/PGA nanoparticles (AgNpPGAs) were highly stable over the long periods of time without signs of precipitation. Ascorbic acid, a water soluble antioxidant, was encapsulated together with these stable AgNpPGAs within poly(DL-lactide-coglycolide) polymeric matrix and their synergistic antimicrobial effect was studied. The antimicrobial activity of the samples was investigated towards six laboratory control strains from the American Type Culture Collection (ATCC) and one clinical isolate methicillin-resistant Staphylococcus aureus strain by the broth microdilution method. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated good biocompatibility of the samples. To establish the influence of PLGA/AgNpPGA/ascorbic acid nanoparticles on intracellular ROS formation, we measured the kinetics of their formation in HepG2 cells by DCFH-DA assay. The samples were characterized by UV-VIS spectrometry, field-emission scanning electron microscopy, and transmission electron microscopy

Updating MISEV: evolving the minimal requirements for studies of extracellular vesicles

The minimal information for studies of extracellular vesicles (EVs, MISEV) is a field-consensus rigour initiative of the International Society for Extracellular Vesicles (ISEV). The last update to MISEV, MISEV2018, was informed by input from more than 400 scientists and made recommendations in the six broad topics of EV nomenclature, sample collection and pre-processing, EV separation and concentration, characterization, functional studies, and reporting requirements/exceptions. To gather opinions on MISEV and ideas for new updates, the ISEV Board of Directors canvassed previous MISEV authors and society members. Here, we share conclusions that are relevant to the ongoing evolution of the MISEV initiative and other ISEV rigour and standardization efforts

LC–DAD–MS phenolic characterisation of six invasive plant species in Croatia and determination of their antimicrobial and cytotoxic activity

Invasive plants’ phytochemicals are important for their invasiveness, enabling them to spread in new environments. However, these chemicals could offer many pharmaceutical compounds or active ingredients for herbal preparations. This study provides the first LC–MS phytochemical screening of six invasive alien plant species (IAPS) in the Istria region (Croatia): Ailanthus altissima, Ambrosia artemisiifolia, Conyza canadensis, Dittrichia viscosa, Erigeron annuus, and Xanthium strumarium. The study aims to identify and quantify the phenolic content of their leaf extracts and assess their antimicrobial and cytotoxic potential. A total of 32 species-specific compounds were recorded. Neochlorogenic, chlorogenic, and 5-p-coumaroylquinic acids, quercetin-3-glucoside, and kaempferol hexoside were detected in all the tested IAPS. Hydroxycinnamic acid derivatives were the main components in all the tested IAPS, except in E. annuus, where flavanones dominated with a share of 70%. X. strumarium extract had the best activity against the tested bacteria, with an average MIC value of 0.11 mg/mL, while A. altissima and X. strumarium extracts had the best activity against the tested fungi, with an average MIC value of 0.21 mg/mL in both cases. All the plant extracts studied, except X. strumarium, were less cytotoxic than the positive control. The results provided additional information on the phytochemical properties of IAPS and their potential for use as antimicrobial agents.SUPPLEMENTARY MATERIALS : Figure S1: Heat map presenting the representations of phenolic groups in different invasive plants; Figure S2: Chromatogram of the acetone extracts of the plant species leaves developed in ethyl acetate/methanol/water (EMW) solvent system sprayed with vanillin– sulphuric acid and TLC bioautograms; Table S1: Spectrum, mass-to-charge ratio (m/z) values of the molecular masses, and main fragments (MS2—second-generation product ion, MS3—thirdgeneration product ion) in negative ion mode ((M-H)−) identified with ESI–MS and the distribution of individual compounds in different invasive plants.The Croatian Science Foundation, the Slovenian Research Agency (ARRS) and the University of Pretoria, South Africa.https://www.mdpi.com/journal/plantsdm2022Paraclinical Science

Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids (?liquid biopsies?). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.Non peer reviewe

Presence of activating KRAS mutations correlates significantly with expression of tumour suppressor genes DCN and TPM1 in colorectal cancer

<p>Abstract</p> <p>Background</p> <p>Despite identification of the major genes and pathways involved in the development of colorectal cancer (CRC), it has become obvious that several steps in these pathways might be bypassed by other as yet unknown genetic events that lead towards CRC. Therefore we wanted to improve our understanding of the genetic mechanisms of CRC development.</p> <p>Methods</p> <p>We used microarrays to identify novel genes involved in the development of CRC. Real time PCR was used for mRNA expression as well as to search for chromosomal abnormalities within candidate genes. The correlation between the expression obtained by real time PCR and the presence of the <it>KRAS </it>mutation was investigated.</p> <p>Results</p> <p>We detected significant previously undescribed underexpression in CRC for genes <it>SLC26A3</it>, <it>TPM1 </it>and <it>DCN</it>, with a suggested tumour suppressor role. We also describe the correlation between <it>TPM1 </it>and <it>DCN </it>expression and the presence of <it>KRAS </it>mutations in CRC. When searching for chromosomal abnormalities, we found deletion of the <it>TPM1 </it>gene in one case of CRC, but no deletions of <it>DCN </it>and <it>SLC26A3 </it>were found.</p> <p>Conclusion</p> <p>Our study provides further evidence of decreased mRNA expression of three important tumour suppressor genes in cases of CRC, thus implicating them in the development of this type of cancer. Moreover, we found underexpression of the <it>TPM1 </it>gene in a case of CRCs without <it>KRAS </it>mutations, showing that <it>TPM1 </it>might serve as an alternative path of development of CRC. This downregulation could in some cases be mediated by deletion of the <it>TPM1 </it>gene. On the other hand, the correlation of <it>DCN </it>underexpression with the presence of <it>KRAS </it>mutations suggests that <it>DCN </it>expression is affected by the presence of activating <it>KRAS </it>mutations, lowering the amount of the important tumour suppressor protein decorin.</p

Roadblocks of Urinary EV Biomarkers:Moving Toward the Clinic

Despite remarkable interest in the biomarker potential of urinary extracellular vesicles (uEVs) and the identification of numerous promising candidates, their clinical translation still presents multiple challenges. The opportunities for successful translation are obvious, yet the main roadblocks on the way have hardly been systematically considered and more coordinated approaches are needed to overcome them. In the present review article, we have identified the most relevant roadblocks of clinical translation of urinary EV-based biomarkers and discuss possible solutions to overcome them. These roadblocks are categorized as fundamental and technical but also related to development of novel biomarker assays and clinical acceptance. In addition, hurdles within the regulatory approval process are discussed. It is clear that various roadblocks to clinical translation of urinary EV biomarkers exist; however, they are addressable by promoting rigor and reproducibility as well as collaboration between basic and clinical scientists, clinicians, industry and regulatory bodies. Moreover, knowledge of obstacles for assay development and regulatory requirements should already be considered when developing a new biomarker to maximize the chance of successful translation. This review presents not only a status quo, but also a roadmap for the further development of the field.</p

The association of breast mitogens with mammographic densities

Radiologically dense breast tissue (mammographic density) is strongly associated with risk of breast cancer, but the biological basis for this association is unknown. In this study we have examined the association of circulating levels of hormones and growth factors with mammographic density. A total of 382 subjects, 193 premenopausal and 189 postmenopausal, without previous breast cancer or current hormone use, were selected in each of five categories of breast density from mammography units. Risk factor information, anthropometric measures, and blood samples were obtained, and oestradiol, progesterone, sex hormone binding globulin, growth hormone, insulin-like growth factor-I and its principal binding protein, and prolactin measured. Mammograms were digitised and measured using a computer-assisted method. After adjustment for other risk factors, we found in premenopausal women that serum insulin-like growth factor-I levels, and in postmenopausal women, serum levels of prolactin, were both significantly and positively associated with per cent density. Total oestradiol and progesterone levels were unrelated to per cent density in both groups. In postmenopausal women, free oestradiol (negatively), and sex hormone binding globulin (positively), were significantly related to per cent density. These data show an association between blood levels of breast mitogens and mammographic density, and suggest a biological basis for the associated risk of breast cancer

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers

Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly