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Here the authors describe a small antibody-like protein that can prevent infection by diverse SARS-CoV-2 variants in cell culture and in mice that were intranasally treated with this inhibitor before or shortly after being exposed to the virus.The emergence of increasingly immunoevasive SARS-CoV-2 variants emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Intranasal administration of neutralizing antibodies has shown encouraging protective potential but there remains a need for SARS-CoV-2 blocking agents that are less vulnerable to mutational viral variation and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and stable trimeric antibody-mimetic sherpabody targeted against a conserved region of the viral spike glycoprotein. TriSb92 potently neutralizes SARS-CoV-2, including the latest Omicron variants like BF.7, XBB, and BQ.1.1. In female Balb/c mice intranasal administration of just 5 or 50 micrograms of TriSb92 as early as 8 h before but also 4 h after SARS-CoV-2 challenge can protect from infection. Cryo-EM and biochemical studies reveal triggering of a conformational shift in the spike trimer as the inhibitory mechanism of TriSb92. The potency and robust biochemical properties of TriSb92 together with its resistance against viral sequence evolution suggest that TriSb92 could be useful as a nasal spray for protecting susceptible individuals from SARS-CoV-2 infection.

STUDY QUESTION Does an estradiol-based combined oral contraceptive (COC) have a milder effect on the serum proteome than an ethinylestradiol (EE)-based COC or dienogest (DNG) only? SUMMARY ANSWER The changes in serum proteome were multifold after the use of a synthetic EE-based COC compared to natural estrogen COC or progestin-only preparation. WHAT IS KNOWN ALREADY EE-based COCs widely affect metabolism, inflammation, hepatic protein synthesis and blood coagulation. Studies comparing serum proteomes after the use of COCs containing EE and natural estrogens are lacking. STUDY DESIGN, SIZE, DURATION This was a spin-off from a randomized, controlled, two-center clinical trial. Women (n = 59) were randomized to use either EE + DNG, estradiol valerate (EV) + DNG or DNG only continuously for 9 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS Participants were healthy, young, white volunteer women. Serum samples were collected before and after 9 weeks of hormonal exposure. Samples from 44 women were available for analysis (EE + DNG n = 14, EV + DNG n = 16 and DNG only n = 14). Serum proteins were analyzed by quantitative, discovery-type label-free proteomics. MAIN RESULTS AND THE ROLE OF CHANCE Altogether, 446 proteins/protein families with two or more unique peptides were detected and quantified. The number of proteins/families that altered over the 9-week period within the study groups was 121 for EE + DNG and 5 for EV + DNG, while no changes were detected for DNG only. When alterations were compared between the groups, significant differences were detected for 63 proteins/protein families, of which 58 were between the EE + DNG and EV + DNG groups. The most affected functions during the use of EE + DNG were the complement system, acute phase response signaling, metabolism and the coagulation system. The results were validated by fetuin-B and cortisol-binding globulin ELISA and sex hormone-binding globulin immunoassay. LARGE SCALE DATA Data are available via ProteomeXchange with identifiers PXD033617 (low abundance fraction) and PXD033618 (high abundance fraction). LIMITATIONS, REASONS FOR CAUTION The power analysis of the trial was not based on the proteomic analysis of this spin-off study. In the future, targeted proteomic analysis with samples from another trial should be carried out in order to confirm the results. WIDER IMPLICATIONS OF THE FINDINGS The EE-based COC exerted a broader effect on the serum proteome than the EV-based COC or the DNG-only preparation. These results demonstrate that the effects of EE in COCs go far beyond the established endpoint markers of estrogen action, while the EV combination is closer to the progestin-only preparation. The study indicates that EV could provide a preferable option to EE in COCs in the future and signals a need for further studies comparing the clinical health outcomes of COCs containing EE and natural estrogens. STUDY FUNDING/COMPETING INTEREST(S) Funding for this researcher-initiated study was obtained from the Helsinki University Hospital research funds, the Hospital District of Helsinki and Uusimaa, the Sigrid Juselius Foundation, the Academy of Finland, the Finnish Medical Association, the University of Oulu Graduate School, the Emil Aaltonen Foundation, the Swedish Cultural Foundation in Finland, the Novo Nordisk Foundation, Orion Research Foundation and the Northern Ostrobothnia Regional Fund. The funders had no role in study design, data collection and analysis, publishing decisions or manuscript preparation. T.P. has received honoraria for lectures, consultations and research grants from Exeltis, Gedeon Richter, MSD, Merck, Pfizer, Roche, Stragen and Mithra Pharmaceuticals. O.H. occasionally serves on advisory boards for Bayer AG and Gedeon Richter and has designed and lectured at educational events for these companies. The other authors have nothing to disclose. O.H. occasionally serves on advisory boards for Bayer AG and Gedeon Richter and has designed and lectured at educational events for these companies. The other authors have nothing to disclose. TRIAL REGISTRATION NUMBER ClinicalTrials.gov NCT02352090 TRIAL REGISTRATION DATE 27 January 2015 DATE OF FIRST PATIENT’S ENROLMENT 1 April 2015

Cells shape their extracellular milieu by secreting intracellular products into the environment including extracellular vesicles which are lipid-bilayer limited membrane particles. These vesicles carry out a range of functions, including regulation of coagulation, via multiple contributor mechanisms. Urinary extracellular vesicles are secreted by various cells, lining the urinary space, including the nephron and bladder. They are known to have procoagulant properties, however, the details of this function, beyond tissue factor are not well known. The aim of the study was to access the role of urinary extracellular vesicles in impacting coagulation upon supplementation to plasma. This could indicate their physiological function upon kidney injury or pathology. Supplementation to standard human plasma and plasmas deficient in various coagulation factors was used for this purpose, and calibrated automated thrombogram (CAT®) was the major technique applied. We found that these vesicles contain multiple coagulation-related factors, and their lipid composition affects coagulation activities of plasma upon supplementation. Remarkably, these vesicles can restore thrombin generation in FVII, FVIII, FIX and FXI -deficient plasmas. This study explores the multiple roles of urinary extracellular vesicles in coagulation in in vitro blood coagulation and implies their importance in its regulation by several mechanisms.

Funding Information: Open Access funding provided by University of Helsinki. The study has been supported by The Finnish Dental Society Apollonia (grant for MP and LL), the Helsinki and Uusimaa Hospital District (HUS, grant for LL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The data of this study has not been presented elsewhere. Publisher Copyright: © 2022, The Author(s). We examined the usefulness of dried spot blood and saliva samples in SARS-CoV-2 antibody analyses. We analyzed 1231 self-collected dried spot blood and saliva samples from healthcare workers. Participants filled in a questionnaire on their COVID-19 exposures, infections, and vaccinations. Anti-SARS-CoV-2 IgG, IgA, and IgM levels were determined from both samples using the GSP/DELFIA method. The level of exposure was the strongest determinant of all blood antibody classes and saliva IgG, increasing as follows: (1) no exposure (healthy, non-vaccinated), (2) exposed, (3) former COVID-19 infection, (4) one vaccination, (5) two vaccinations, and (6) vaccination and former infection. While the blood IgG assay had a 99.5% sensitivity and 75.3% specificity to distinguish participants with two vaccinations from all other types of exposure, the corresponding percentages for saliva IgG were 85.3% and 65.7%. Both blood and saliva IgG-seropositivity proportions followed similar trends to the exposures reported in the questionnaires. Self-collected dry blood and saliva spot samples combined with the GSP/DELFIA technique comprise a valuable tool to investigate an individual’s immune response to SARS-CoV-2 exposure or vaccination. Saliva IgG has high potential to monitor vaccination response wane, since the sample is non-invasive and easy to collect.

Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]x0PxxP, the hydrophobic residue 0 being proline or leucine. We have studied the unusual 0 = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity. We also compared the affinities of EEEV nsP3 peptide, its mutants, and cellular ligands to SNX9 SH3. These data suggest that nsP3 has evolved to minimize reduction of conformational entropy upon binding, hence acquiring stronger affinity, enabling takeover of SNX9. The RxAPxxP motif was also found in human T cell leukemia virus-1 (HTLV-1) Gag polyprotein. We found that this motif was required for efficient HTLV-1 infection, and that the specificity of SNX9 SH3 for the RxAPxxP core binding motif was importantly involved in this process.

The populational heterogeneity of a disease, in part due to comorbidity, poses several complexities. Individual comorbidity profiles, on the other hand, contain useful information to refine phenotyping, prognostication, and risk assessment, and they provide clues to underlying biology. Nevertheless, the spectrum and the implications of the diagnosis profiles remain largely uncharted. Here we mapped comorbidity patterns in 100 common diseases using 4-year retrospective data from 526,779 patients and developed an online tool to visualize the results. Our analysis exposed disease-specific patient subgroups with distinctive diagnosis patterns, survival functions, and laboratory correlates. Computational modeling and real-world data shed light on the structure, variation, and relevance of populational comorbidity patterns, paving the way for improved diagnostics, risk assessment, and individualization of care. Variation in outcomes and biological correlates of a disease emphasizes the importance of evaluating the generalizability of current treatment strategies, as well as considering the limitations that selective inclusion criteria pose on clinical trials.

Nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), is an acute febrile illness caused by Puumala orthohantavirus (PUUV). NE manifests typically with acute kidney injury (AKI), with a case fatality rate of about 0.1%. The treatment and management of hantavirus infections are mainly supportive, although neutralizing monoclonal antibodies and immune sera therapeutics are under investigation. In order to assess the potential use of antibody therapeutics in NE, we sought to determine the relationship between circulating PUUV neutralizing antibodies, PUUV nucleocapsid protein (N) IgG antibodies, and viral loads with markers of disease severity. The study included serum samples of extensively characterized patient cohorts (n = 116) from Tampere University Hospital, Finland. The results showed that upon hospitalization, most patients already had considerable neutralizing and anti-PUUV-N IgG antibody levels. However, contrary to expectations, neutralizing antibody titers from the first day of hospitalization did not appear to protect from AKI or correlate with more favorable disease outcomes. This indicates that further studies are needed to investigate the applicability of neutralizing antibodies as a therapy for hospitalized NE patients. publishedVersion

Background Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by biliary strictures, cholestasis, and a markedly increased risk of cholangiocarcinoma. New markers for the screening and differential diagnosis of PSC are needed. In this pilot study, we have analyzed both the bile and serum proteomic profiles of 80 PSC patients and non-PSC controls (n = 6 for bile and n = 18 for serum). Aim The aim of this study was to discover candidates for new biomarkers for the differential diagnosis of PSC. Methods Bile and serum samples were processed and subsequently analyzed using ultra performance liquid chromatography-ultra definition mass spectrometry (UPLC-UDMSE). Further analysis included statistical analyses such as receiver operating characteristic curve analysis as well as pathway analysis using Ingenuity Pathway Analysis. Results and conclusions In bile, we discovered 64 proteins with significantly different levels between the groups, with fold changes of up to 129. In serum, we discovered 112 proteins with significantly different levels. Receiver operating characteristic curve analysis found multiple proteins with high area under the curve values, up to 0.942, indicating that these serum proteins are of value as new non-invasive classifiers of PSC. Pathway analysis revealed multiple canonical pathways that were enriched in the dataset, which have roles in bile homeostasis and metabolism. We present several serum proteins that could serve as new blood-based markers for the diagnosis of PSC after further validation. The measurement of serum levels of these proteins could be of use in the screening of patients with suspected PSC.

Publisher Copyright: © 2021 The Authors Background: The pathophysiological changes related to brain death may affect the quality of the transplanted organs and expose the recipients to risks. We probed systemic changes reflected in donor plasma proteome and investigated their relationship to heart transplant outcomes. Methods: Plasma samples from brain-dead multi-organ donors were analyzed by label-free protein quantification using high-definition mass spectrometry. Unsupervised and supervised statistical models were used to determine proteome differences between brain-dead donors and healthy controls. Proteome variation and the corresponding biological pathways were analyzed and correlated with transplant outcomes. Results: Statistical models revealed that donors had a unique but heterogeneous plasma proteome with 237 of 463 proteins being changed compared to controls. Pathway analysis showed that coagulation, gluconeogenesis, and glycolysis pathways were upregulated in donors, while complement, LXR/RXR activation, and production of nitric oxide and reactive oxygen species in macrophages pathways were downregulated. In point-biserial correlation analysis, lysine-specific demethylase 3A was moderately correlated with any grade and severe PGD. In univariate and multivariate Cox regression analyses myosin Va and proteasome activator complex subunit 2 were significantly associated with the development of acute rejections with hemodynamic compromise within 30 days. Finally, we found that elevated levels of lysine-specific demethylase 3A and moesin were identified as predictors for graft-related 1-year mortality in univariate analysis. Conclusions: We show that brain death significantly changed plasma proteome signature Donor plasma protein changes related to endothelial cell and cardiomyocyte function, inflammation, and vascular growth and arteriogenesis could predict transplant outcome suggesting a role in donor evaluation.

Non

Introduction: Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for 10% of the global cancer burden. Rectal cancer accounts for around 30% of CRC cases, and patients with resectable rectal cancer are often given preoperative radiotherapy (PRT) to reduce the rate of local recurrence. The human plasma proteome is an exceptionally complex proteome and ideal to study due to its ability to reflect the presence of diseases such as cancer and the ease of obtaining blood samples. Previous proteomic studies involving rectal cancer patients have mostly focused on the identification of proteins involved in resistance to radiotherapy. Objective: The aim of this study was to investigate the overall effects of PRT on plasma protein expression in rectal cancer patients, as there is a lack of such studies. Methods: Here, we have used mass spectrometry and subsequent statistical analyses to analyze the plasma samples of 30 rectal cancer patients according to PRT status (positive or negative) and tumor stage (II or III). Results and Conclusions: We discovered 42 proteins whose levels differed significantly between stage II and III rectal cancer patients who did or did not receive PRT. This study shows that PRT, although localized to the pelvis, leads to measurable, tumor stage-specific changes in plasma protein expression. Future studies of plasma proteins should, when relevant, take this into account and be aware of the widespread effects that PRT has on the plasma proteome.

Evaluate computed tomography (CT) signs that predict need for revision endoscopic sinus surgery (ESS) of chronic rhinosinusitis (CRS).CRS patients (n = 48) underwent routine sinus CT scans and baseline ESS in 2006-2011. Lund-Mackay (LM) scores and 43 other CT signs were analysed blinded from both sides. Patients filled in a questionnaire during the day of CT scanning. Follow-up data were collected from hospital records until January 2018. Associations were analysed by Fisher's exact, Mann Whitney U, Kaplan-Meier method with logrank test and Cox's proportional hazard model.Total LM score was not significantly associated with the need for revision ESS. The best predictive model was a sum of CT signs of non-detectable anatomy of inferior/middle turbinates, obstructed frontal recess, and previous sinus surgery. Using these CT findings, we formed a Radiological Score (RS) (min-max, 0-3 points). Having at least one RS point was significantly associated with the need for revision ESS during the average follow-up of 10.7 years (p = 0.008, Logrank test).We identified a radiologic score that was able to predict the need for revision ESS, which is probably useful in predicting CRS outcomes.Score radiologico della Tac in grado di predire la revisione chirurgica nei pazienti affetti da rinosinusite cronica.Valutare i segni della tomografia computerizzata (TC) predittivi della necessità di un intervento di revisione endoscopica dei seni paranasali (ESS) nella rinosinusite cronica (CRS).I pazienti con CRS (n = 48) sono stati sottoposti a TC dei seni paranasali e ESS dal 2006 al 2011. I punteggi di Lund-Mackay (LM) e altri 43 segni TC sono stati analizzati in cieco. I pazienti hanno compilato un questionario di valutazione al momento dell’esecuzione della TC. Sono stati revisionati i dati di follow-up dei registri ospedalieri fino a Gennaio 2018. Le analisi sono state effettuate mediante test di Fisher, Mann Whitney U, Kaplan-Meier con il test di logrank e il modello di rischio proporzionale di Cox.Il punteggio LM totale non era significativamente associato alla necessità di revisione mediante ESS. Il miglior modello predittivo è risultato la somma dei seguenti reperti TC: turbinati inferiori/medi non rilevabili, recesso frontale ostruito ed esiti di precedenti interventi chirurgici nasosinusali. Utilizzando questi risultati TC, abbiamo creato un punteggio radiologico (RS) (min-max, 0-3 punti). Un punteggio RS minimo di uno, è significativamente associato alla necessità di revisione ESS durante il follow-up medio di 10,7 anni (p = 0,008, test di Logrank).Abbiamo identificato un punteggio radiologico in grado di prevedere la necessità di revisione ESS e quindi probabilmente utile nel predire gli esiti della CRS.

Background and PurposeRepurposing of currently available drugs is a valuable strategy to tackle the consequences of COVID-19. Recently, several studies have investigated the effect of psychoactive drugs on SARS-CoV-2 in cell culture models as well as in clinical practice. Our aim was to expand these studies and test some of these compounds against newly emerged variants.Experimental ApproachSeveral antidepressant drugs and antipsychotic drugs with different primary mechanisms of action were tested in ACE2/TMPRSS2-expressing human embryonic kidney cells against the infection by SARS-CoV-2 spike protein-dependent pseudoviruses. Some of these compounds were also tested in human lung epithelial cell line, Calu-1, against the first wave (B.1) lineage of SARS-CoV-2 and the variants of concern, B.1.1.7 and B.1.351.Key ResultsSeveral clinically used antidepressants, including fluoxetine, citalopram, reboxetine, imipramine, as well as antipsychotic compounds chlorpromazine, flupenthixol, and pimozide inhibited the infection by pseudotyped viruses with minimal effects on cell viability. The antiviral action of several of these drugs was verified in Calu-1 cells against the (B.1) lineage of SARS-CoV-2. By contrast, the anticonvulsant carbamazepine, and novel antidepressants ketamine and its derivatives as well as MAO and phosphodiesterase inhibitors phenelzine and rolipram, respectively, showed no activity in the pseudovirus model. Furthermore, fluoxetine remained effective against pseudo viruses with N501Y, K417N, and E484K spike mutations, and the VoC-1 (B.1.1.7) and VoC-2 (B.1.351) variants of SARS-CoV-2.Conclusion and ImplicationsOur study confirms previous data and extends information on the repurposing of these drugs to counteract SARS-CoV-2 infection including different variants of concern.

Colorectal cancer (CRC) stands for 10% of the worldwide cancer burden and has recently become the second most common cause of cancer death. The 5-year survival rate depends mainly on stage at diagnosis. Mass spectrometric proteomic analysis is widely used to study the plasma proteome, which is complex and contains multitudes of proteins. In this study, we have used Ultra Performance Liquid Chromatography-Ultra Definition Mass Spectrometry (UPLC-UDMSE)-based proteomics to analyze plasma samples from 76 CRC patients. We identified several plasma proteins, such as CP, TVP23C, FETUB, and IGFBP3, of which altered levels led to significant differences in survival, as seen by Cox regression and Kaplan-Meier analysis. Additionally, during Cox regression analysis, samples were adjusted for age and/or tumor stage, enabling stringent analysis. These proteins, although in need of further validation, could be of use during patient follow-up, as their levels can non-invasively be measured from blood samples, and could be of use in predicting patient outcome. Several of these proteins additionally have roles in metabolism and inflammation, two processes central to the development and progression of cancer, further indicating their importance in cancer.

Objectives Many comorbidities are associated with adult asthma and may exacerbate the asthma burden of disease. This study aims to investigate the risk for major oral diseases or oral-manifesting diseases in asthmatic compared with non-asthmatic adults. Design We conducted a population-based matched cohort study with a 13.8-year follow-up. Setting A baseline questionnaire was completed by participants in 1997 and follow-up data were extracted from the national hospital discharge registry of the National Institute for Health and Welfare in Finland from 1997 to 2014. Participants A total of 1394 adults with asthma were matched with 2398 adults without asthma based on sex, age and area of residence. Asthmatic adults were identified from the Drug Reimbursement Register of the Finnish Social Insurance Institution based on a special drug reimbursement right resulting from asthma. Participants without asthma were identified from the Population Register. Main outcomes and measures Oral health-related primary diagnoses were retrieved using codes from the International Classification of Diseases, 10th edition and divided into groups of diseases. Cox's proportional hazards models stratified by matching unit and models matched and adjusted for pack-years, education level and body mass index (when possible) were used to evaluate the matched and further adjusted HRs for diseases comparing asthmatic and non-asthmatic cohorts. Results Adult asthma was associated with a higher risk for any oral-manifesting disease (adjusted HR 1.41, 95% CI 1.11 to 1.80), herpes zoster (adjusted HR 6.18, 95% CI 1.21 to 31.6), benign tumours of the oral cavity and pharynx (matched HR 1.94, 95% CI 1.05 to 3.56) and dermatological diseases (pemphigus, pemphigoid, dermatitis herpetiformis, psoriasis and lichen planus, HR 1.67, 95% CI 1.01 to 2.78). Conclusions In this study, adult asthmatics experienced a higher risk for a major oral disease or oral-manifesting disease. publishedVersion

Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. However, the development of mouse-adapted virus strains has revealed key mutations in the SARS-CoV-2 spike protein that increase infectivity, and interestingly, many of these mutations are also present in naturally occurring SARS-CoV-2 variants of concern. This suggests that these variants might have the ability to infect common laboratory mice. Herein we show that the SARS-CoV-2 beta variant attains infectibility to BALB/c mice and causes pulmonary changes within 2-3 days post infection, consistent with results seen in other murine models of COVID-19, at a reasonable virus dose (2 x 10(5) PFU). The findings suggest that common laboratory mice can serve as the animal model of choice for testing the effectiveness of antiviral drugs and vaccines against SARS-CoV-2.

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy—termed the "R-clamp”—that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution., Author summary Viral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein. Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the R-clamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, and have implications on therapeutic development aiming to interfere with SH3 binding of Nef.

The prevalence of allergic diseases and asthma is increasing rapidly worldwide, with environmental and lifestyle behaviors implicated as a reason. Epidemiological studies have shown that children who grow up on farms are at lower risk of developing childhood atopic disease, indicating the presence of a protective “farm effect”. The Old Order Mennonite (OOM) community in Upstate New York have traditional, agrarian lifestyles, a low rate of atopic disease, and long periods of exclusive breastfeeding. Human milk proteins are heavily glycosylated, although there is a paucity of studies investigating the milk glycoproteome. In this study, we have used quantitative glycoproteomics to compare the N-glycoprotein profiles of 54 milk samples from Rochester urban/suburban and OOM mothers, two populations with different lifestyles, exposures, and risk of atopic disease. We also compared N-glycoprotein profiles according to the presence or absence of atopic disease in the mothers and, separately, the children. We identified 79 N-glycopeptides from 15 different proteins and found that proteins including immunoglobulin A1, polymeric immunoglobulin receptor, and lactotransferrin displayed significant glycan heterogeneity. We found that the abundances of 38 glycopeptides differed significantly between Rochester and OOM mothers and also identified four glycopeptides with significantly different abundances between all comparisons. These four glycopeptides may be associated with the development of atopic disease. The findings of this study suggest that the differential glycosylation of milk proteins could be linked to atopic disease.

Background Colorectal cancer (CRC) is the third most common cancer worldwide, and its incidence is expected to increase to over 2.2 million new cases in 2030. Stage II CRC is classified as localized disease, while stage III CRC has spread to regional lymph nodes. The 5-year survival rate is over 80% for patients with stage II CRC, but less than 60% for patients with stage III CRC. Proteins, especially plasma proteins that are detectable in easily obtained blood samples, that differ between stage II and III CRC could be useful for predicting and monitoring disease progression. CRC displays differences depending on primary tumor location (right colon, left colon, or rectum), and how plasma protein expression changes during CRC progression from stage II to III depending on primary tumor location is not well-characterized. Methods In this study, we have used Ultra Performance Liquid Chromatography-Ultra Definition Mass Spectrometry (UPLC-UDMSE)-based proteomics to analyze plasma samples from 83 patients with stage II or III CRC, followed by statistical and pathway analysis (data are available via ProteomeXchange). The patients were divided into groups according to tumor stage (II or III) and changes in plasma protein expression between stage II and III (localized and regional disease) samples were studied both regardless of primary tumor location and also within each primary tumor location (right colon, left colon, rectum). Results We discovered differences in plasma protein expression within all groups analyzed and identified proteins whose levels changed in one, two, or all three primary tumor locations between stage II and III CRC. Proteins were identified that could separate the groups compared and pathway analysis by IPA discovered altered pathways involved in lipid metabolism and inflammation, among others. Conclusions Plasma protein expression changes significantly as CRC progresses from stage II to III. While the levels of certain plasma proteins changed during cancer progression in only one or two primary tumor locations, the levels of 13 proteins changed in all primary tumor locations and are therefore common to CRC progression.

Background: Orexin-A and -B are neuropeptides involved in sleep-wake regulation. In human narcolepsy type 1, this cycle is disrupted due to loss of orexin-producing neurons in the hypothalamus. Cerebrospinal fluid (CSF) orexin-A measurement is used in the diagnosis of narcolepsy type 1. Currently available immunoassays may lack specificity for accurate orexin quantification. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for CSF orexin-A and B.Methods: We used CSF samples from narcolepsy type 1 (n=22) and type 2 (n=6) and non-narcoleptic controls (n=44). Stable isotope-labeled orexin-A and -B internal standards were added to samples before solid-phase extraction and quantification by LC-MS/MS. The samples were also assayed by commercial radioimmunoassay (RIA, n=42) and enzymatic immunoassay (EIA, n=72) kits. Stability of orexins in CSF was studied for 12 months.Results: Our assay has a good sensitivity (10 pmol/L = 35 pg/mL) and a wide linear range (35-3500 pg/mL). Added orexin-A and -B were stable in CSF for 12 and 3 months, respectively, when frozen. The median orexin-A concentration in CSF from narcolepsy type 1 patients was Conclusions: Orexin-A concentrations measured by our LC-MS/MS assay were lower in narcolepsy type 1 patients as compared to controls. RIA yielded on average higher concentrations than LC-MS/MS.

Designing smarter anticancer T cells Biological signaling systems can exhibit a large, nonlinear—or “ultrasensitive”—response, which would be useful to engineer into therapeutic T cells to allow for better discrimination between cancer cells and normal tissues. Hernandez-Lopez et al. modified human T cells using a two-step mechanism that allowed them to kill cells expressing large amounts of cancer marker protein but not cells expressing a small amount of the same protein. A first synthetic receptor recognized the antigen with low affinity. That receptor signaled to increase expression of a chimeric antigen receptor (CAR) with high affinity for the same antigen. The circuit proved effective in cell culture and mouse cancer models, offering hope of extending the CAR T cell strategy against solid tumors. Science , this issue p. 1166

Funding Information: The study was supported in part by research grants from Finnish Medical Foundation, the Finnish Society of Allergology and Immunology, the Jane and Aatos Erkko Foundation, the Finnish Cultural Foundation, Hospital District of Helsinki and Uusimaa (TYH2018103, TYH2019322), Paulo Foundation, the Tampere Tuberculosis Foundation, the Väinö and Laina Kivi Foundation, the Finnish ORL-HNS Foundation. Acknowledgements Publisher Copyright: © 2020, The Author(s). Genome wide association studies (GWASs) have revealed several airway disease-associated risk loci. Their role in the onset of asthma, allergic rhinitis (AR) or chronic rhinosinusitis (CRS), however, is not yet fully understood. The aim of this review is to evaluate the airway relevance of loci and genes identified in GWAS studies. GWASs were searched from databases, and a list of loci associating significantly (p

Until now, cheese peptidomics approaches have been criticised for their lower throughput. Namely, analytical gradients that are most commonly used for mass spectrometric detection are usually over 60 or even 120 min. We developed a cheese peptide mapping method using nano ultra-high-performance chromatography data-independent acquisition high-resolution mass spectrometry (nanoUHPLC-DIA-HRMS) with a chromatographic gradient of 40 min. The 40 min gradient did not show any sign of compromise in milk protein coverage compared to 60 and 120 min methods, providing the next step towards achieving higher-throughput analysis. Top 150 most abundant peptides passing selection criteria across all samples were cross-referenced with work from other publications and a good correlation between the results was found. To achieve even faster sample turnaround enhanced DIA methods should be considered for future peptidomics applications.

Colorectal cancer (CRC) includes tumors in the right colon, left colon, and rectum, although they differ significantly from each other in aspects such as prognosis and treatment. Few previous mass spectrometry‐based studies have analyzed differences in protein expression depending on the tumor location. In this study, we have used mass spectrometry‐based proteomics to analyze plasma samples from 83 CRC patients to study if differences in plasma protein expression can be seen depending on primary tumor location (right colon, left colon, or rectum). Differences were studied between the groups both regardless of and according to tumor stage (II or III). Large differences in plasma protein expression were seen, and we found that plasma samples from patients with rectal cancer separated from samples from patients with colon cancer when analyzed by principal component analysis and hierarchical clustering. Samples from patients with cancer in the right and left colon also tended to separate from each other. Pathway analysis discovered canonical pathways involved in lipid metabolism and inflammation to be enriched. This study will help to further define CRC as distinct entities depending on tumor location, as shown by the widespread differences in plasma protein profile and dysregulated pathways., Colorectal cancer (CRC) includes tumors in the right colon, left colon, and rectum, although they differ significantly from each other in aspects such as prognosis and treatment. In this study, we have used mass spectrometry‐based proteomics to analyze plasma samples from 83 CRC patients. We subsequently show that plasma protein expression differs significantly between patients with cancer in the colon and rectum.

Idiopathic pulmonary fibrosis (IPF) is a lung parenchymal disease of unknown cause usually occurring in older adults. It is a chronic and progressive condition with poor prognosis and diagnosis is largely clinical. Currently, there exist few biomarkers that can predict patient outcome or response to therapies. Together with lack of markers, the need for novel markers for the detection and monitoring of IPF, is paramount. We have performed label-free plasma proteomics of thirty six individuals, 17 of which had confirmed IPF. Proteomics data was analyzed by volcano plot, hierarchical clustering, Partial-least square discriminant analysis (PLS-DA) and Ingenuity pathway analysis. Univariate and multivariate statistical analysis overlap identified haptoglobin-related protein as a possible marker of IPF when compared to control samples (Area under the curve 0.851, ROC-analysis). LXR/RXR activation and complement activation pathways were enriched in t-test significant proteins and oxidative regulators, complement proteins and protease inhibitors were enriched in PLS-DA significant proteins. Our pilot study points towards aberrations in complement activation and oxidative damage in IPF patients and provides haptoglobin-related protein as a new candidate biomarker of IPF.

Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.

HIV-Nef activates the myeloid cell-typical tyrosine kinase Hck, but its molecular role in the viral life cycle is not entirely understood. We found that HIV plasma extracellular vesicles (HIV pEV) containing/10 proteases and Nef also harbor Hck, and analyzed its role in the context of HIV pEV secretion. Myeloid cells required Hck for the vesicle-associated release of ADAM17. This could be induced by the introduction of Nef and implied that HIV targeted Hck for vesicle-associated ADAM17 secretion from a myeloid compartment. The other contents of HIV-pEV, however, including miRNA and effector protein profiles, as well as the presence of haptoglobin suggested hepatocytes as a possible cellular source. HIV liver tissue analysis supported this assumption, revealing induction of Hck translation, evidence for ADAM protease activation and HIV infection. Our findings suggest that HIV targets Hck to induce pro-inflammatory vesicles release and identifies hepatocytes as a possible host cell compartment., Highlights • Hck is found along with HIV Nef and ADAM17 in plasma extracellular vesicles (pEV) from HIV-infected individuals. • Hck is required for the secretion of ADAM17 via pEV from myeloid cells and hepatocytes. • Liver tissue from HIV infected individuals revealed induction of Hck expression and presence of Nef.

Purpose The purpose of this study is to elucidate the effect of excess body weight and liver fat on the plasma proteome without interference from genetic variation. Experimental Design The effect of excess body weight is assessed in young, healthy monozygotic twins from pairs discordant for body mass index (intrapair difference (Delta) in BMI > 3 kg m(-2), n = 26) with untargeted LC-MS proteomics quantification. The effect of liver fat is interrogated via subgroup analysis of the BMI-discordant twin cohort: liver fat discordant pairs (Delta liver fat > 2%, n = 12) and liver fat concordant pairs (Delta liver fat

Background Intraductal Papillary Mucinous Neoplasia (IPMN) are potentially malignant cystic tumors of the pancreas. IPMN can progress from low to moderate to high grade dysplasia and further to IPMN associated carcinoma. Often the difference between benign and malignant nature of the IPMN is not clear preoperatively. We aim to elucidate molecular expression patterns of various grades of IPMN and pancreatic carcinoma. Additionally we suggest potential novel biomarkers to differentiate IPMN from healthy individuals and pancreatic carcinoma to enable early detection as well as help in differential diagnosis in future. Methods We have performed retrospective label-free proteomic analysis of the serum samples from 44 patients with various grades of benign IPMN or IPMN associated carcinoma and 11 healthy controls. Proteomic data was further analyzed by various multivariate statistical methods. Four groups of samples (low-grade, high-grade IPMN, pancreatic carcinoma and age- and sex-matched healthy controls) were compared with ANOVA. Orthogonal projections to latent structures-discriminant analysis (OPLS-DA) modeling gave S-plot for feature selection. Stringently selected potential markers were further evaluated with ROC curve analysis and area under the curve was calculated. Differentially expressed proteins were used for pathway analysis. Linear trend analysis (Mann Kendall test) was used for identifying significant increasing or decreasing trends from healthy-low grade-high grade IPMN-pancreatic carcinoma. Results Based on protein expression (436 proteins quantified), PCA separated most sample groups from each other. S-Plot selected biomarker panels with moderate to very high AUC values for differentiating controls from Low-, High-Grade IPMN and carcinoma. Linear trend analysis identified 12 proteins which were consistently increasing or decreasing trend among the groups. We found potential biomarkers to differentiate healthy controls from different degrees of dysplasia and pancreatic carcinoma. These biomarkers can classify IPMN, carcinoma and healthy controls from each other which is an unmet clinical need. Data are available via ProteomeXchange with identifier PXD009139. Conclusion Kininogen-1 was able to differentiate healthy persons from low and high-grade IPMN. Retinol binding protein-4 could classify the low-grade IPMN from pancreatic carcinoma. Twelve proteins including apolipoproteins and complement proteins had significantly increasing or decreasing trends from healthy to low to high-grade IPMN to pancreatic carcinoma.

Purpose Despite recent advances in donation after circulatory death, transplants from brain-dead donors remain the sole source in heart transplantation (HTx) worldwide. Due to organ shortage, marginal donors are increasingly used and the utilization of transplants becomes markedly more challenging. They undergo invariably brain death that induces a systemic cytokine and catecholamine storm that lead to systemic inflammation, labile hemodynamics, and organ hypoperfusion. Together, these can damage the heart and aggravate later occurring graft injury, and ultimately, compromise graft function. However, the effect of donor brain death on allografts is not well understood. Methods In a separate prospective, blinded single-center trial, we collected donor plasma samples and relevant clinical patient data from 50 HTx brain-dead donors and as controls plasma samples from age- and gender-matched 23 healthy volunteers. Quantitative label-free proteomics in high definition MSE mode (HDMSE) was carried out on the samples. Various statistical analyses were performed on quantitative proteomics data to obtain the most reliably distinguishing proteins, which classify the donors vs controls. Results With two or more unique proteins per identification, 463 proteins were quantified in our pilot study. A complete separation between donors and controls based on a set of 278 proteins (p-value Conclusion To our knowledge, we are the first one elucidating the proteomic signature of brain-death in human blood samples with open-label proteomics. We show that brain death alters protein expression, and that these changes are dependent on the donor demographics. The molecular pathway analyses characterize these changes in a systemic perspective. We found a set of plasma proteins that we suggest as a diagnostic blood biomarker panel to detect high risk heart transplants and we believe that we may identify novel treatment targets induced in noxious pathways after brain death.

In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV). Non

International audience; Puumala virus is an RNA virus hosted by the bank vole (Myodes glareolus) and is today present in most European countries. Whilst it is generally accepted that hantaviruses have been tightly co-evolving with their hosts, Puumala virus (PUUV) evolutionary history is still controversial and so far has not been studied at the whole European level. This study attempts to reconstruct the phylogeographical spread of modern PUUV throughout Europe during the last postglacial period in the light of an upgraded dataset of complete PUUV small (S) segment sequences and by using most recent computational approaches. Taking advantage of the knowledge on the past migrations of its host, we identified at least three potential independent dispersal routes of PUUV during postglacial recolonization of Europe by the bank vole. From the Alpe-Adrian region (Balkan, Austria, and Hungary) to Western European countries (Germany, France, Belgium, and Netherland), and South Scandinavia. From the vicinity of Carpathian Mountains to the Baltic countries and to Poland, Russia, and Finland. The dissemination towards Denmark and North Scandinavia is more hypothetical and probably involved several independent streams from south and north Fennoscandia.

Polycystic ovary syndrome (PCOS) is the most common endocrinological disorder of fertile-aged women. Several adverse pregnancy outcomes and abnormalities of the placenta have been associated with PCOS. By using quantitative label-free proteomics we investigated whether changes in the plasma proteome of pregnant women with PCOS could elucidate the mechanisms behind the pathologies observed in PCOS pregnancies. A total of 169 proteins with ≥2 unique peptides were detected to be differentially expressed between women with PCOS (n = 7) and matched controls (n = 20) at term of pregnancy, out of which 35 were significant (p-value

Pancreatic ductal adenocarcinoma is the most common and aggressive type of pancreatic cancer, with a 5-year survival rate that is less than 10%. New biomarkers to aid in predicting the prognosis of pancreatic ductal adenocarcinoma patients are needed. Previous proteomic studies have to a great extent focused on finding proteins of value for the diagnosis of pancreatic ductal adenocarcinoma. There is a lack of studies that have profiled the serum or plasma proteome in order to discover candidates for new prognostic biomarkers. In this study, we have used ultra-performance liquid chromatography–ultra-definition mass spectrometry to analyze the serum samples of 21 pancreatic ductal adenocarcinoma patients with short or long survival. Statistical analysis discovered 31 proteins whose expression differed significantly between pancreatic ductal adenocarcinoma patients with short or long survival. Pathway analysis discovered multiple canonical pathways enriched in this data set, with several pathways having roles in inflammation and lipid metabolism. The serum proteins identified here, which include complement components and several enzymes, could be of value as candidates for new noninvasive prognostic markers.

Background: Serotonin (5-hydroxytyramine) is a mediator of gastrointestinal smooth muscle contraction, and is secreted by neuroendocrine neoplasms (NENs). We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for serum serotonin to be used in NEN diagnostics and follow-up. Methods: We used serum samples from healthy volunteers (n = 31) and patients suspected or monitored for NEN (n = 98). Serotonin-D-4 internal standard was added to samples before solid phase extraction (SPE) and quantification by LC-MS/MS. The effects of sample handling and preparation on serotonin stability were studied. Finally, we established a provisional reference range for serum serotonin and compared our assay with serum 5hydroxyindoleacetic acid (5-HIAA) for detection of NENs. Results: Our assay is sensitive and has a wide linear range (10-10,000 nmo1/1). Serum serotonin is stable for 7 days at room temperature and for 3 months at -20 degrees C. Sampling temperature is not critical. Normal range for serum serotonin was 270-1490 nmo1/1. We found that serum serotonin and 5-HIAA performed equally well as diagnostic tests for NENs. Conclusions: Our LC-MS/MS assay for serum serotonin is well suited for clinical research and patient diagnostics. Our results confirm that it can complement 5-HIAA in diagnosis of NENs.

Over 1.4 million people are diagnosed with colorectal cancer (CRC) each year, making it the third most common cancer in the world. Increased screening and therapeutic modalities including improved combination treatments have reduced CRC mortality, although incidence and mortality rates are still increasing in some areas. Serum-based biomarkers are mainly used for follow-up of cancer, and are ideal due to the ease and minimally invasive nature of sample collection. Unfortunately, CEA and other serum markers have too low sensitivity for screening and preoperative diagnostic purposes. Increasing interest is focused on the possible use of biomarkers for predicting treatment response and prognosis in cancer. In this study, we have performed mass spectrometry analysis (UPLC-UDMSE) of serum samples from 19 CRC patients. Increased levels of C-reactive protein (CRP), which occur during local inflammation and the presence of a systemic inflammatory response, have been linked to poor prognosis in CRC patients. We chose to analyze samples according to CRP values by dividing them into the categories CRP 30, and, separately, according to short and long 5-year survival. The aim was to discover differentially expressed proteins associated with poor prognosis and shorter survival. We quantified 256 proteins and performed detailed statistical analyses and pathway analysis. We discovered multiple proteins that are up- or downregulated in patients with CRP >30 as compared to CRP

Although most of Arvicolinae associated hantaviruses can not cause disease in humans, hemorrhagic fever with renal syndrome (HFRS) cases caused by Tula virus (TULV) have been described in Europe since 2002. In addition to Europe, TULV was also identified in the Microtus arvalis obscurus voles sampled from Kazakhstan, which shares borders with China. To gain more insight into the molecular epidemiology of TULV, a total of 365 rodents representing 7 species of 4 subfamily (Arvicolinae, Murinae, Gerbillinae, and Cricetinae) were captured in Qapqal county, Xinjiang, northwest China. Hantavirus RNA was recovered from 40 lung tissue samples of M. arvalis obscurus, with the prevalence of 10.96%. Genetic analysis revealed that all recovered viral sequences were most closely related to those of TULV, but exhibited >11% nucleotide differences from all currently known TULV, suggesting that they may represent a new subtype of TULV. In the S tree, the newly identified viruses formed a distinct lineage and showed a close evolutionary relationship with those sampled from Southwestern Siberia and Kazakhstan. However, they exhibited a different clustering pattern in both the M and the L trees, suggesting the possibility of genetic reassortment. Finally, the recombination event was also observed in Xinjiang TULV viruses. In sum, all these data reveal a complex evolutionary history of TULV in Central Asia.

BackgroundPotato virus Y (PVY, genus Potyvirus) causes substantial economic losses in solanaceous plants. Routine screening for PVY is an essential part of seed potato certification, and serological assays are often used. The commercial, commonly used monoclonal antibodies, MAb1128, MAb1129, and MAb1130, recognize the viral coat protein (CP) of PVY and distinguish PVYN strains from PVYO and PVYC strains, or detect all PVY strains, respectively. However, the minimal epitopes recognized by these antibodies have not been identified.Methodology/principal findingsSPOT peptide array was used to map the epitopes in CP recognized by MAb1128, MAb1129, and MAb1130. Then alanine replacement as well as N- and C-terminal deletion analysis of the identified peptide epitopes was done to determine critical amino acids for antibody recognition and the respective minimal epitopes. The epitopes of all antibodies were located within the 30 N-terminal-most residues. The minimal epitope of MAb1128 was 25NLNKEK30. Replacement of 25N or 27N with alanine weakened the recognition by MAb1128, and replacement of 26L, 29E, or 30K nearly precluded recognition. The minimal epitope for MAb1129 was 16RPEQGSIQSNP26 and the most critical residues for recognition were 22I and 23Q. The epitope of MAb1130 was defined by residues 5IDAGGS10. Mutation of residue 6D abrogated and mutation of 9G strongly reduced recognition of the peptide by MAb1130. Amino acid sequence alignment demonstrated that these epitopes are relatively conserved among PVY strains. Finally, recombinant CPs were produced to demonstrate that mutations in the variable positions of the epitope regions can affect detection with the MAbs.Conclusions/significanceThe epitope data acquired can be compared with data on PVY CP-encoding sequences produced by laboratories worldwide and utilized to monitor how widely the new variants of PVY can be detected with current seed potato certification schemes or during the inspection of imported seed potatoes as conducted with these MAbs.

Background A nef gene is present in all primate lentiviral genomes and is important for high viral loads and progression to AIDS in human or experimental macaque hosts of HIV or SIV, respectively. In these hosts, infection of the thymus results in a decreased output of naive T cells that may contribute to the development of immunodeficiency. We have previously shown that HIV-1 subtype B Nef proteins can block human T-cell development. However, the underlying mechanism(s) and the conservation of this Nef function between different groups of HIV and SIV remained to be determined. Results We investigated whether reduction of thymic output is a conserved function of highly divergent lentiviral Nef proteins including those from both types of human immunodeficiency viruses (HIV-1 and HIV-2), their direct simian counterparts (SIVcpz, SIVgor and SIVsmm, respectively), and some additional SIV strains. We found that expression of most of these nef alleles in thymocyte progenitors impaired T-cell development and reduced thymic output. For HIV-1 Nef, binding to active p21 protein (Cdc42/Rac)-activated kinase (PAK2) was a major determinant of this function. In contrast, selective disruption of PAK2 binding did not eliminate the effect on T-cell development of SIVmac239 Nef, as was shown by expressing mutants in a newly discovered PAK2 activating structural motif (PASM) constituted by residues I117, H121, T218 and Y221, as well as previously described mutants. Rather, down-modulation of cell surface CD3 was sufficient for reduced thymic output by SIVmac Nef, while other functions of SIV Nefs contributed. Conclusions Our results indicate that primate lentiviral Nef proteins impair development of thymocyte precursors into T cells in multiple ways. The interaction of HIV-1 Nef with active PAK2 by HIV-1 seem to be most detrimental, and downregulation of CD3 by HIV-2 and most SIV Nef proteins sufficient for reduced thymic output. Since the reduction of thymic output by Nef is a conserved property of divergent lentiviruses, it is likely to be relevant for peripheral T-cell depletion in poorly adapted primate lentiviral infections.

Background Longquan City, Zhejiang province, China, has been seriously affected by hemorrhagic fever with renal syndrome (HFRS) since the first cases were registered in 1974. To understand the epidemiology and emergence of HFRS in Longquan, which may be indicative of large parts of rural China, we studied long-term incidence patterns and performed a molecular epidemiological investigation of the causative hantaviruses in human and rodent populations. Method/Principal Findings During 1974–2011, 1866 cases of HFRS were recorded in Longquan, including 20 deaths. In 2011, the incidence of HFRS remained high, with 19.61 cases/100,000 population, despite the onset of vaccination in 1997. During 1974–1998, HFRS cases in Longquan occurred mainly in winter, while in the past decade the peak of HFRS has shifted to the spring. Notably, the concurrent prevalence of rodent-borne hantaviruses in the region was also high. Phylogenetic analyses of viral sequences recovered from rodents in Longquan revealed the presence of novel genetic variants of Gou virus (GOUV) in Rattus sp. rats and Hantaan virus (HTNV) in the stripe field mice, respectively. Strikingly, viral sequences sampled from infected humans were very closely related to those from rodents. Conclusions/Significance HFRS represents an important public health problem in Longquan even after years of preventive measures. Our data suggest that continual spillover of the novel genetic variant of GOUV and the new genetic lineage of HTNV are responsible for the high prevalence of HFRS in humans. In addition, this is the first report of GOUV associated with human HFRS cases, and our data suggest that GOUV is now the major cause of HFRS in this region., Author Summary Hemorrhagic fever with renal syndrome (HFRS) is a major public health problem in China despite human vaccination. We investigated the epidemiology and emergence of HFRS in Longquan (Zhejiang Province), a rural area with a high incidence of HFRS. During 1974–2011, a total of 1866 cases of HFRS were recorded in Longquan, including 20 deaths. Strikingly, phylogenetic analyses of viral sequences sampled from local rodents in Longquan revealed the presence of novel variants of Gou virus (GOUV) in Rattus sp. rats and Hantaan virus (HTNV) in the stripe field mice, respectively. Moreover, viral sequences sampled from infected humans in Longquan were very closely related to those from rodents. Overall, these data indicate that there is a continual spillover GOUV and HTNV from rodents to humans in Longquan, and this might be responsible for the high prevalence of HFRS. As well as highlighting the importance of the human-animal interface, these data also suggest that GOUV is now the major cause of HFRS in this region.

Förster resonance energy transfer (FRET) is a phenomenon widely utilized in biomedical research of macromolecular interactions. In FRET energy is transferred between two fluorophores, the donor and the acceptor. Herein we describe a novel approach utilizing time-resolved FRET (TR-FRET) for the detection of antibodies not only in a solution-phase homogenous assay but also in single- and two-step solid-phase assays. Our method is based on the principle that the Y-shaped immunoglobulin G molecule is able to simultaneously bind two identical antigen molecules. Hence, if a specific IgG is mixed with donor- and acceptor-labeled antigens, the binding of antigens can be measured by TR-FRET. Using donor- and acceptor-labeled streptavidins (SAs) in conjunction with a polyclonal and a monoclonal anti-SA antibody we demonstrate that this approach is fully functional. In addition we characterize the immune complexes responsible for the TR-FRET signal using density gradient ultracentrifugation and solid-phase immunoassays. The homogenous TR-FRET assay described provides a rapid and robust tool for antibody detection, with a wide potential in medical diagnostics.

Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects., Author Summary Some bacterial species are specialists and adapted to a single niche, while others are generalists and able to grow in various environmental conditions. Lactobacillus rhamnosus is a generalist and its members can often be found in different human cavities but also in various artisanal and industrial dairy products. To gain insights into the genetic complexity and ecological versatility of this species, we collected 100 L. rhamnosus strains from different niches. Genomic and functional analysis of these revealed a dichotomy within the species that reflected its adaptation to particular niches. The variable regions identified in the L. rhamnosus genome encode lifestyle traits that allowed us to demonstrate that some L. rhamnosus isolates possibly resided in multiple habitats. Our work brings valuable data on the ecological dynamics and adaptability of the species and provides a basis for a model explaining the ecology of L. rhamnosus in an anthropocentric perspective. Finally, we observed that a set of pheno-genomic markers, i.e. CRISPR oligotyping or carbohydrate metabolism, would be sufficient and among the best ways to differentiate the L. rhamnosus strains, providing a general approach to select the highest diversity in these and other bacterial species.

Background Y. enterocolitica biotype (BT) 1A strains are often isolated from human clinical samples but their contribution to disease has remained a controversial topic. Variation and the population structure among the clinical Y. enterocolitica BT 1A isolates have been poorly characterized. We used multi-locus sequence typing (MLST), 16S rRNA gene sequencing, PCR for ystA and ystB, lipopolysaccharide analysis, phage typing, human serum complement killing assay and analysis of the symptoms of the patients to characterize 298 clinical Y. enterocolitica BT 1A isolates in order to evaluate their relatedness and pathogenic potential. Results A subset of 71 BT 1A strains, selected based on their varying LPS patterns, were subjected to detailed genetic analyses. The MLST on seven house-keeping genes (adk, argA, aroA, glnA, gyrB, thrA, trpE) conducted on 43 of the strains discriminated them into 39 MLST-types. By Bayesian analysis of the population structure (BAPS) the strains clustered conclusively into two distinct lineages, i.e. Genetic groups 1 and 2. The strains of Genetic group 1 were more closely related (97% similarity) to the pathogenic bio/serotype 4/O:3 strains than Genetic group 2 strains (95% similarity). Further comparison of the 16S rRNA genes of the BT 1A strains indicated that altogether 17 of the 71 strains belong to Genetic group 2. On the 16S rRNA analysis, these 17 strains were only 98% similar to the previously identified subspecies of Y. enterocolitica. The strains of Genetic group 2 were uniform in their pathogenecity-related properties: they lacked the ystB gene, belonged to the same LPS subtype or were of rough type, were all resistant to the five tested yersiniophages, were largely resistant to serum complement and did not ferment fucose. The 54 strains in Genetic group 1 showed much more variation in these properties. The most commonly detected LPS types were similar to the LPS types of reference strains with serotypes O:6,30 and O:6,31 (37%), O:7,8 (19%) and O:5 (15%). Conclusions The results of the present study strengthen the assertion that strains classified as Y. enterocolitica BT 1A represent more than one subspecies. Especially the BT 1A strains in our Genetic group 2 commonly showed resistance to human serum complement killing, which may indicate pathogenic potential for these strains. However, their virulence mechanisms remain unknown.

Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1 × 10(-3) to 1.7 × 10(-4 )mol/L. This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins.

Introduction Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome called nephropathia epidemica (NE). The aim of the present study was to evaluate plasma cell-free DNA (cf-DNA) levels and urinary cf-DNA excretion in acute NE as well as their associations with the severity of the disease. Methods Total plasma cf-DNA was quantified directly in plasma of 61 patients and urine of 20 patients with acute NE. We also carried out a qualitative high-sensitivity lab-on-a-chip DNA assay in 20 patients to elucidate the appearance of cf-DNA in plasma and urine. Results The maximum plasma cf-DNA values taken during acute NE were significantly higher than the control values taken after the hospitalization period (median 1.33 µg/ml, range 0.94–3.29 µg/ml vs. median 0.77 µg/ml, range 0.55–0.99 µg/ml, P

Mosquitoes collected in northern Italy were screened for flavivirus RNA. Positive amplicons were sequenced and found most similar to insect flavivirus (ISF), Usutu virus (USUV) and surprisingly also to Japanese encephalitis virus (JEV). The sequence (167 bp), obtained from one pool of Culex pipiens, was found identical to JEV strains from bats in China. Unfortunately additional sequence data or virus isolations were not obtained in this study. Confirmation of potential introduction of JEV to Italy and other European countries is urgently needed.