Dust-Acoustic Nonlinear Waves in a Nanoparticle Fraction of Ultracold (2K) Multicomponent Dusty Plasma

The nonlinear dust-acoustic instability in the condensed submicron fraction of dust particles in the low-pressure glow discharge at ultra-low temperatures is experimentally and theoretically investigated. The main discharge parameters are estimated on the basisof the dust-acoustic wave analysis. In particular, the temperature and density of ions, as well as the Debye radius, are determined. It is shown that the ion temperature exceeds the temperature of the neutral gas. The drift characteristics of all plasma fractions are estimated. The reasons for the instability excitation are considered.

Evaluation of SARS-CoV-2 in semen, seminal plasma, and spermatozoa pellet of COVID-19 patients in the acute stage of infection

To date, there is limited information about the presence of SARS-CoV-2 in semen especially in the acute phase of the infection. While available data from cohort studies including a total of 342 patients in the acute or recovery phase of the infection are reassuring, one study mentioned detecting virus in the semen of 6/38 COVID-19 patients. Here we assessed SARS-CoV-2 presence in the semen of COVID-19 positive patients in the acute stage of infection, within 24 hours of the positive nasopharyngeal swabs. Semen, seminal plasma and spermatozoa pellet were screened for SARS-CoV-2 and manual or airborne contamination during semen sampling. Among the 32 COVID-19 volunteers, the median interval from the onset of symptoms to semen collection was 4 days [IQR: 0–8]. Only one presented positive SARS-CoV-2 PCR in semen and seminal plasma fractions, although the spermatozoa pellet was negative. Viral cultures were all negative. We observed slightly higher concentrations of bacterial DNA in the SARS-CoV-2 positive specimen than in all negative samples. The bacteria identified neither confirm nor rule out contamination by oropharyngeal secretions during collection. SARS-CoV-2 was rarely present in semen during the acute phase of the disease. This very rare situation could be connected to oral or manual contamination during semen collection. The possible presence of SARS-CoV-2 in semen calls for nasopharyngeal viral testing and strict hygiene protocols during semen collection before assisted reproductive attempts.

Apolipoprotein Signature of HDL and LDL from Atherosclerotic Patients in Relation with Carotid Plaque Typology: A Preliminary Report

In the past years, it has become increasingly clear that the protein cargo of the different lipoprotein classes is largely responsible for carrying out their various functions, also in relation to pathological conditions, including atherosclerosis. Accordingly, detailed information about their apolipoprotein composition and structure may contribute to the revelation of their role in atherogenesis and the understanding of the mechanisms that lead to atherosclerotic degeneration and toward vulnerable plaque formation. With this aim, shotgun proteomics was applied to identify the apolipoprotein signatures of both high-density and low-density lipoproteins (HDL and LDL) plasma fractions purified from healthy volunteers and atherosclerotic patients with different plaque typologies who underwent carotid endarterectomy. By this approach, two proteins with potential implications in inflammatory, immune, and hemostatic pathways, namely, integrin beta-2 (P05107) and secretoglobin family 3A member 2 (Q96PL1), have been confirmed to belong to the HDL proteome. Similarly, the list of LDL-associated proteins has been enriched with 21 proteins involved in complement and coagulation cascades and the acute-phase response, which potentially double the protein species of LDL cargo. Moreover, differential expression analysis has shown protein signatures specific for patients with “hard” or “soft” plaques.

Distinct Extracellular RNA Profiles in Different Plasma Components

Circulating extracellular RNAs (exRNAs) have great potential to serve as biomarkers for a wide range of diagnostic, therapeutic, and prognostic applications. So far, knowledge of the difference among different sources of exRNAs is limited. To address this issue, we performed a sequential physical and biochemical precipitation to collect four fractions (platelets and cell debris, the thrombin-induced precipitates, extracellular vesicles, and supernatant) from each of 10 plasma samples. From total RNAs of the 40 fractions, we prepared ligation-free libraries to profile full spectrum of all RNA species, without size selection and rRNA reduction. Due to complicated RNA composition in these libraries, we utilized a successive stepwise alignment strategy to map the RNA sequences to different RNA categories, including miRNAs, piwi-interacting RNAs, tRNAs, rRNAs, lincRNAs, snoRNAs, snRNAs, other ncRNAs, protein coding RNAs, and circRNAs. Our data showed that each plasma fraction had its own unique distribution of RNA species. Hierarchical cluster analyses using transcript abundance demonstrated similarities in the same plasma fraction and significant differences between different fractions. In addition, we observed various unique transcripts, and novel predicted miRNAs among these plasma fractions. These results demonstrate that the distribution of RNA species and functional RNA transcripts is plasma fraction-dependent. Appropriate plasma preparation and thorough inspection of different plasma fractions are necessary for an exRNA-based biomarker study.

Specific Seminal Plasma Fractions Are Responsible for the Modulation of Sperm–PMN Binding in the Donkey

While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. Remarkably, sperm interacts with PMN, but the presence of seminal plasma reduces that binding. As seminal plasma is a complex fluid made up of different molecules, including proteins, this study aimed to evaluate how different seminal plasma fractions, separated by molecular weight (<3, 3–10, 10–30, 30–50, 50–100, and >100 kDa), affect sperm–PMN binding. Sperm motility, viability, and sperm–PMN binding were evaluated after 0 h, 1 h, 2 h, 3 h, and 4 h of co-incubation at 38 °C. Two seminal plasma fractions, including 30–50 kDa or 50–100 kDa proteins, showed the highest sperm motility and viability. As viability of sperm not bound to PMN after 3 h of incubation was the highest in the presence of 30–50 and 50–100 kDa proteins, we suggest that both fractions are involved in the control of the jenny’s post-breeding inflammatory response. In conclusion, this study has shown for the first time that specific fractions rather than the entire seminal plasma modulate sperm–PMN binding within the donkey uterus. As several proteins suggested to be involved in the control of post-AI endometritis have a molecular weight between 30 and 100 kDa, further studies aimed at determining the identity of these molecules and evaluating their potential effect in vivo are much warranted.

Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids

Background: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that allows flexibility for high and mid–throughput analysis for three blood–based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and dried blood. Methods: Optimal conditions for sample preparation and DIA–MS analysis were established in plasma then automated and adapted for depleted plasma and whole blood. The MS workflow was modified to facilitate sensitive high–throughput or deep profile analysis with mid–throughput analysis. Analytical performance was evaluated from 5 complete workflows repeated over 3 days as well as a linearity analysis of a 5—6–point dilution curve. Result: Using our high-throughput workflow, 74%, 93%, 87% of peptides displayed an inter-day CV<30% in plasma, depleted plasma and whole blood. While the mid-throughput workflow had 67%, 90%, 78% of peptides in plasma, depleted plasma and whole blood meeting the CV<30% standard. Lower limits of detection and quantitation were determined for proteins and peptides observed in each biofluid and workflow. Combining the analysis of both high–throughput plasma fractions exceeded the number of reliably identified proteins for individual biofluids in the mid–throughput workflows. Conclusion: The workflow established here allowed for reliable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow on a large scale will facilitate the translation of candidate markers into clinical use.

THE STABILITY OF THE PACKED CELL VOLUME OF ANTICOAGULATED BOVINE BLOOD STORED AT REFRIGERATION TEMPERATURE.

Bovine blood samples with ethylene diamine tetra-acetate (EDTA) or oxalate as anticoagulant were stored at refrigeration temperature of 4°C. The packed cell volume (PCV) was determined daily. During storage, the PCV did not vary significantly. Complete haemolysis, suggested by failure of blood samples to separate into cellular and plasma fractions, started occuring in samples on days 28 and 17, and was observed to have occurred in all samples on days 41 and 24 in blood with EDTA and oxalate respectively.

PRAP1 is a novel lipid binding protein that promotes lipid absorption by facilitating MTTP-mediated lipid transport

Microsomal triglyceride transfer protein (MTTP) is an endoplasmic reticulum (ER) resident protein that is essential for the assembly and secretion of triglyceride (TG)-rich, apoB-containing lipoproteins. Although the function and structure of mammalian MTTP have been extensively studied, how exactly MTTP transfers lipids to lipid acceptors and whether there are other biomolecules involved in MTTP-mediated lipid transport remain elusive. Here we identify a role in this process for the poorly characterized protein PRAP1. We report that PRAP1 and MTTP are partially co-localized in the ER. We observe that PRAP1 directly binds to TG and facilitates MTTP-mediated lipid transfer. A single amino acid mutation at position 85 (E85V) impairs PRAP1's ability to form a ternary complex with TG and MTTP, as well as impairs its ability to facilitate MTTP-mediated apoB-containing lipoprotein assembly and secretion, suggesting that the ternary complex formation is required for PRAP1 to facilitate MTTP-mediated lipid transport. PRAP1 is detectable in chylomicron/VLDL-rich plasma fractions, suggesting that MTTP recognizes PRAP1-bound TG as a cargo and transfers TG along with PRAP1 to lipid acceptors. Both PRAP1 deficient and the E85V knock-in mutant mice fed a chow diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing lipid. Interestingly, both genetically modified mice gained significantly less body weight and fat mass when on high fat diets compared to littermate controls and were prevented from hepatosteatosis. Together, this study provides evidence that PRAP1 plays an important role in MTTP-mediated lipid transport and lipid absorption.

Determination of Expression Signature and Proportion of mtDNA in Plasma Fractions in Patients with Renal Cell Carcinoma

Renal Cell Carcinoma (RCC) is the third most common urologic malignancy, remaining one of the most lethal urological malignancies, preferably in developed countries. The incidence and mortality rates differ significantly according to sex, race, age and external factors such as smoking, obesity and hypertension increasing RCC risk. The use of novel predictive biomarkers is currently being increased as these improve the diagnosis, progression and prognosis of RCC. Since recent studies have demonstrated a promising association between mitochondrial DNA (mtDNA) copy number alteration in peripheral blood and the risk of developing RCC, we conducted a case-control study into a cohort of 15 controls and 13 patients to determine exosomes mtDNA content in plasma fractions as a potential novel non-invasive biomarker in liquid biopsy in order to monitor the RCC status in patients. In this way, plasma fractions highly purified in exosomes were obtained from blood samples from controls and RCC cases, and relative mtDNA content was measured by quantitative real-time polymerase chain reaction (qPCR). Our results show fragment size distribution profile and we observed that in phase F; with a higher content of exosomal mtDNA; p value shows statistically significant differences in mitochondrial genes HV long and CYB long