A simplified LC-MS/MS method for the quantification of the cardiovascular disease biomarker trimethylamine-N-oxide and its precursors

Trimethylamine-N-oxide(TMAO)has emerged as a potential biomarker for atherosclerosis and the development of cardiovascular diseases(CVDs).Although several clinical studies have shown striking associations of TMAO levels with atherosclerosis and CVDs,TMAO determinations are not clinical routine yet.The current methodology relies on isotope-labeled internal standards,which adds to pre-analytical complexity and costs for the quantification of TMAO and its precursors carnitine,betaine or choline.Here,we report a liquid chromatography-tandem mass spectrometry based method that is fast(throughput up to 240 samples/day),consumes low sample volumes(e.g.,from a finger prick),and does not require isotope-labeled standards.We circumvented the analytical problem posed by the presence of endogenous TMAO and its precursors in human plasma by using an artificial plasma matrix for calibration.We cross-validated the results obtained using an artificial matrix with those using mouse plasma matrix and demonstrated that TMAO,carnitine,betaine and choline were accurately quantified in’reallife’human plasma samples from healthy volunteers,obtained either from a finger prick or from venous puncture.Additionally,we assessed the stability of samples stored at-20℃and room temperature.Whereas all metabolites were stable at-20℃,increasing concentrations of choline were determined when stored at room temperature.Our method will facilitate the establishment of TMAO as a routine clinical biomarker in hematology in order to assess the risk for CVDs development,or to monitor disease progression and intervention effects.

Innate immune recognition and modulation in hepatitis D virus infection

Hepatitis D virus(HDV)is a global health threat with more than 15 million humans affected.Current treatment options are largely unsatisfactory leaving chronically infected humans at high risk to develop liver cirrhosis and hepatocellular carcinoma.HDV is the only human satellite virus known.It encodes only two proteins,and requires Hepatitis B virus(HBV)envelope protein expression for productive virion release and spread of the infection.How HDV could evolve and why HBV was selected as a helper virus remains unknown.Since the discovery of Na+-taurocholate co-transporting polypeptide as the essential uptake receptor for HBV and HDV,we are beginning to understand the interactions of HDV and the immune system.While HBV is mostly regarded a stealth virus,that escapes innate immune recognition,HBV-HDV coinfection is characterized by a strong innate immune response.Cytoplasmic RNA sensor melanoma differentiation antigen 5 has been reported to recognize HDV RNA replication and activate innate immunity.Innate immunity,however,seems not to impair HDV replication while it inhibits HBV.In this review,we describe what is known up-to-date about the interplay between HBV as a helper and HDV’s immune evasion strategy and identify where additional research is required.

Immunological aspects of liver cell transplantation

Within the field of regenerative medicine, the liver is of major interest for adoption of regenerative strategies due to its well-known and unique regenerative capacity. Whereas therapeutic strategies such as liver resection and orthotopic liver transplantation(OLT) can be considered standards of care for the treatment of a variety of liver diseases, the concept of liver cell transplantation(LCTx) still awaits clinical breakthrough. Success of LCTx is hampered by insufficient engraftment/long-term acceptance of cellular allografts mainly due to rejection of transplanted cells. This is in contrast to the results achieved for OLT where longterm graft survival is observed on a regular basis and, hence, the liver has been deemed an immuneprivileged organ. Immune responses induced by isolated hepatocytes apparently differ considerably from those observed following transplantation of solid organs and, thus, LCTx requires refined immunological strategies to improve its clinical outcome. In addition, clinical usage of LCTx but also related basic research efforts are hindered by the limited availability of high quality liver cells, strongly emphasizing the need for alternative cell sources. This review focuses on the various immunological aspects of LCTx summarizing data available not only for hepatocyte transplantation but also for transplantation of non-parenchymal liver cells and liver stem cells.

Countrywide Survey for MERS-Coronavirus Antibodies in Dromedaries and Humans in Pakistan

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic pathogen capable of causing severe respiratory disease in humans. Although dromedary camels are considered as a major reservoir host, the MERS-CoV infection dynamics in camels are not fully understood. Through surveillance in Pakistan, nasal (n = 776) and serum (n = 1050)samples were collected from camels between November 2015 and February 2018. Samples were collected from animal markets, free-roaming herds and abattoirs. An in-house ELISA was developed to detect IgG against MERS-CoV. A total of 794 camels were found seropositive for MERS-CoV. Prevalence increased with the age and the highest seroprevalence was recorded in camels aged [ 10 years (81.37%) followed by those aged 3.1–10 years (78.65%) and B 3 years (58.19%).Higher prevalence was observed in female (78.13%) as compared to male (70.70%). Of the camel nasal swabs, 22 were found to be positive by RT-qPCR though with high Ct values. Moreover, 2,409 human serum samples were also collected from four provinces of Pakistan during 2016–2017. Among the sampled population, 840 humans were camel herders.Although we found a high rate of MERS-CoV antibody positive dromedaries (75.62%) in Pakistan, no neutralizing antibodies were detected in humans with and without contact to camels.

Membrane-destabilizing ionizable phospholipids: Novel components for organ-selective mRNA delivery and CRISPR-Cas gene editing

In a new study published in Nature Materials,Liu et al.1 report a novel design of lipid nanoparticles(LNPs)in which multi-tailed ionizable phospholipids(iPhos)constitute the active component,and which facilitates endosomal escape and thus improves delivery of mRNA and/or single-guide(sg)RNA for in vivo gene editing.LNPs composed of the best-performing iPhos and different helper lipids_zwitterionic lipids,ionizable cationic lipids and permanently cationic lipids-achieved selective organ targeting(SORT)and organ-specific CRISPR-Cas9 gene editing in spleen,liver,and lungs of mice,respectively.

Cytotoxic FCER1G^(+) innate-like T cells:new potential for tumour immunotherapy

In a recent study published in Nature,Chou and colleagues define a new evolutionarily conserved class of tumour-elicited immune response mediated by a distinct population of T cell receptor(TCR)-positive FCER1G-expressing innate-like T cells with high cytotoxic potential(αβILTCKs).1 Targeted immunotherapies and most prominently immune checkpoint blockade(ICB)therapies,brought clinical benefits to tumour patients that were inconceivable 15 years ago.2 These ICBs target inhibitory receptors such as PD-1 on tumour infiltrating CD8+cytotoxic T lymphocytes(CTLs)that can recognise mutated cancer cell antigens and thereby enable tumour cell killing.Yet,a significant cohort of cancer patients are non-responsive to ICB treatment and therefore,there is a strong need to discover additional anti-cancer immunomechanisms.Recent work in Nature by Chou and colleagues identifies a population ofαβILTCKs that exhibit reactivity to unmutated tumour antigens.

What are the pathways between poverty and malaria in sub-Saharan Africa?A systematic review of mediation studies

Background Malaria remains a major burden in sub-Saharan Africa(SSA).While an association between poverty and malaria has been demonstrated,a clearer understanding of explicit mechanisms through which socioeconomic position(SEP)influences malaria risk is needed to guide the design of more comprehensive interventions for malaria risk mitigation.This systematic review provides an overview of the current evidence on the mediators of socioeconomic disparities in malaria in SSA.Methods We searched PubMed and Web of Science for randomised controlled trials,cohort,case-control and cross-sectional studies published in English between January 1,2000 to May 31,2022.Further studies were identified following reviews of reference lists of the studies included.We included studies that either(1)conducted a formal mediation analysis of risk factors on the causal pathway between SEP and malaria infections or(2)adjusted for these potential mediators as confounders on the association between SEP and malaria using standard regression models.At least two independent reviewers appraised the studies,conducted data extraction,and assessed risk of bias.A systematic overview is presented for the included studies.Results We identified 41 articles from 20 countries in SSA for inclusion in the final review.Of these,30 studies used cross-sectional design,and 26 found socioeconomic inequalities in malaria risk.Three formal mediation analyses showed limited evidence of mediation of food security,housing quality,and previous antimalarial use.Housing,education,insecticide-treated nets,and nutrition were highlighted in the remaining studies as being protective against malaria independent of SEP,suggesting potential for mediation.However,methodological limitations included the use of cross-sectional data,insufficient confounder adjustment,heterogeneity in measuring both SEP and malaria,and generally low or moderate-quality studies.No studies considered exposure mediator interactions or considered identifiability assumptions.Conclusions Few studies have conducted formal mediation analyses to elucidate pathways between SEP and malaria.Findings indicate that food security and housing could be more feasible(structural)intervention targets.Further research using well-designed longitudinal studies and improved analysis would illuminate the current sparse evidence into the pathways between SEP and malaria and adduce evidence for more potential targets for effective intervention.

CXCR5^(+)PD-1^(++)CD4^(+)T cells colonize infant intestines early in life and promote B cell maturation

Gastrointestinal infections are a major cause for serious clinical complications in infants.The induction of antibody responses by B cells is critical for protective immunity against infections and requires CXCR5+PD-1++CD4+T cells(TFH cells).We investigated the ontogeny of CXCR5+PD-1++CD4+T cells in human intestines.While CXCR5+PD-1++CD4+T cells were absent in fetal intestines,CXCR5+PD-1++CD4+T cells increased after birth and were abundant in infant intestines,resulting in significant higher numbers compared to adults.These findings were supported by scRNAseq analyses,showing increased frequencies of CD4+T cells with a TFH gene signature in infant intestines compared to blood.Co-cultures of autologous infant intestinal CXCR5+PD-1+/−CD4+T cells with B cells further demonstrated that infant intestinal TFH cells were able to effectively promote class switching and antibody production by B cells.Taken together,we demonstrate that functional TFH cells are numerous in infant intestines,making them a promising target for oral pediatric vaccine strategies.

Harnessing naturally occurring mutations for T-cell therapy:a potential new avenue to enhance treatment efficacy

In a recent study published in Nature,Garcia et al.use a sophisticated approach to identify fitness-enhancing mutations for T cells that was inspired by cancer evolution.1 The identified CARD11-PIK3R3 gene fusion enhanced tumor rejection and persistence of engineered T cells in multiple tumor models and might have the potential to improve efficacy of adoptive T-cell therapies in cancer patients.

HBV Integration Induces Complex Interactions between Host and Viral Genomic Functions at the Insertion Site

Hepatitis B virus(HBV),one of the well-known DNA oncogenic viruses,is the leading cause of hepatocellular carcinoma(HCC).In infected hepatocytes,HBV DNA can be integrated into the host genome through an insertional mutagenesis process inducing tumorigenesis.Dissection of the genomic features surrounding integration sites will deepen our understanding of mechanisms underlying integration.Moreover,the quantity and biological activity of integration sites may reflect the DNA damage within affected cells or the potential survival benefits they may confer.The wellknown human genomic features include repeat elements,particular regions(such as telomeres),and frequently interrupted genes(e.g.,telomerase reverse transcriptase[i.e.TERT],lysine methyltransferase 2B[i.e.KMT2B],cyclin E1[CCNE1],and cyclin A2[CCNA2]).Consequently,distinct genomic features within diverse integrations differentiate their biological functions.Meanwhile,accumulating evidence has shown that viral proteins produced by integrants may cause cell damage even after the suppression of HBV replication.The integration-derived gene products can also serve as tumor markers,promoting the development of novel therapeutic strategies for HCC.Viral integrants can be single copy or multiple copies of different fragments with complicated rearrangement,which warrants elucidation of the whole viral integrant arrangement in future studies.All of these considerations underlie an urgent need to develop novel methodology and technology for sequence characterization and function evaluation of integration events in chronic hepatitis B-associated disease progression by monitoring both host genomic features and viral integrants.This endeavor may also serve as a promising solution for evaluating the risk of tumorigenesis and as a companion diagnostic for designing therapeutic strategies targeting integration-related disease complications.

Hepatitis B and Delta Virus: Advances on Studies about Interactions between the Two Viruses and the Infected Hepatocyte

The mechanisms determining persistence of hepatitis B virus (HBV) infection and long-term pathogenesis of HBV-associated liver disease appear to be multifactorial.Although viral replication can be efficiently suppressed by the antiviral treatments currently available,viral clearance is generally not achieved since HBV has developed unique replication strategies,enabling persistence of its genome within the infected hepatocytes.Moreover,no direct antiviral therapy exists for the more than 15 million people worldwide that are also coinfected with the hepatitis delta virus (HDV),a defective virus that needs the HBV envelope proteins for propagation.The limited availability of robust HBV and HDV infection systems has hindered the understanding of the complex network of virus-virus and virushost interactions that are established in the course of infection and slowed down progress in drug development.Since chronic HBV/HDV coinfection leads to the most severe form of chronic viral hepatitis,elucidation of the molecular mechanisms regulating virus-host interplay and pathogenesis are urgently needed.This article summarizes the current knowledge regarding the interactions among HBV,HDV,and the infected target cell and discusses the dependence of HDV on HBV activity and possible future therapeutic approaches.

Low serum neutralizing anti-SARS-CoV-2 S antibody levels in mildly affected COVID-19 convalescent patients revealed by two different detection methods

Neutralizing antibodies targeting the receptor-binding domain(RBD)of the SARS-CoV-2 spike(S)block severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)entry into cells via surface-expressed angiotensin-converting enzyme 2(ACE2).We used a surrogate virus neutralization test(sVNT)and SARS-CoV-2 S protein-pseudotyped vesicular stomatitis virus(VSV)vector-based neutralization assay(pVNT)to assess the degree to which serum antibodies from coronavirus disease 2019(COVID-19)convalescent patients interfere with the binding of SARS-CoV-2 S to ACE2.Both tests revealed neutralizing anti-SARS-CoV-2 S antibodies in the sera of ~90% of mildly and 100% of severely affected COVID-19 convalescent patients.Importantly,sVNT and pVNT results correlated strongly with each other and to the levels of anti-SARS-CoV-2 S1 IgG and IgA antibodies.Moreover,levels of neutralizing antibodies correlated with the duration and severity of clinical symptoms but not with patient age.Compared to pVNT,sVNT is less sophisticated and does not require any biosafety labs.Since this assay is also much faster and cheaper,sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.

Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin

The papain-like protease(PL^(pro)) of Middle-East respiratory syndrome coronavirus(MERS-CoV) has proteolytic,deubiquitinating,and de ISGylating activities.The latter two are involved in the suppression of the antiviral innate immune response of the host cell.To contribute to an understanding of this process,we present here the X-ray crystal structure of a complex between MERS-CoV PL^(pro) and human ubiquitin(Ub) that is devoid of any covalent linkage between the two proteins.Five regions of the PL^(pro) bind to two areas of the Ub.The C-terminal five residues of Ub,RLRGG,are similar to the P5–P1 residues of the polyprotein substrates of the PL^(pro) and are responsible for the major part of the interaction between the two macromolecules.Through sitedirected mutagenesis,we demonstrate that conserved Asp165 and non-conserved Asp164 are important for the catalytic activities of MERS-CoV PL^(pro).The enzyme appears not to be optimized for catalytic efficiency; thus,replacement of Phe269 by Tyr leads to increased peptidolytic and deubiquitinating activities.Ubiquitin binding by MERS-CoV PL^(pro) involves remarkable differences compared to the corresponding complex with SARS-CoV PL^(pro).The structure and the mutational study help understand common and unique features of the deubiquitinating activity of MERS-CoV PL^(pro).

Crystal structure of the C-terminal fragment of NS1 protein from yellow fever virus

Dear Editor,Yellow fever(YF),a mosquito-borne flavivirus disease,is endemic in tropical areas of Africa and Central and South America.YF is transmitted via the bite of infected Aedes aegypti or Haemogogus mosquitoes and mainly affects humans and nonhuman primates.The clinical course of infection in humans shows a wide spectrum of severity including no symptoms,mild illness,and severe disease including

Costs and cost-effectiveness of HIV early infant diagnosis in low- and middle-income countries: a scoping review

Background:Continuing progress in the global pediatric human immunodeficiency virus(HIV)response depends on timely identification and care of infants with HIV.As countries scale-out improvements to HIV early infant diagnosis(EID),economic evaluations are needed to inform program design and implementation.This scoping review aimed to summarize the available evidence and discuss practical implications of cost and cost-effectiveness analyses of HIV EID.Methods:We systematically searched bibliographic databases(Embase,MEDLINE and EconLit)and grey literature for economic analyses of HIV EID in low-and middle-income countries published between January 2008 and June 2021.We extracted data on unit costs,cost savings,and incremental cost-effectiveness ratios as well as outcomes related to health and the HIV EID care process and summarized results in narrative and tabular formats.We converted unit costs to 2021 USD for easier comparison of costs across studies.Results:After title and abstract screening of 1278 records and full-text review of 99 records,we included 29 studies:17 cost analyses and 12 model-based cost-effectiveness analyses.Unit costs were 21.46-51.80 USD for point-of-care EID tests and 16.21-42.73 USD for laboratory-based EID tests.All cost-effectiveness analyses stated at least one of the interventions evaluated to be cost-effective.Most studies reported costs of EID testing strategies;however,few studies assessed the same intervention or reported costs in the same way,making comparison of costs across studies challenging.Limited data availability of context-appropriate costs and outcomes of children with HIV as well as structural heterogeneity of cost-effectiveness modelling studies limits generalizability of economic analyses of HIV EID.Conclusions:The available cost and cost-effectiveness evidence for EID of HIV,while not directly comparable across studies,covers a broad range of interventions and suggests most interventions designed to improve EID are cost-effective or cost-saving.Further studies capturing costs and benefits of EID services as they are delivered in real-world settings are needed.

Neutralization of the SARS-CoV-2 Delta variant after heterologous and homologous BNT162b2 or ChAdOx1 nCoV-19 vaccination

Since the beginning of the COVID-19 pandemic,divergent variants of concern(VoCs)of SARS-CoV-2 have evolved and become the most prevalent SARS-CoV-2 variants in distinct locations at different times.Currently,the Delta variant(B.1.617.2)dominates infection events in large parts of the world.Immunization campaigns,however,still use SARS-CoV-2 vaccines based on the spike(S)protein of the original Wuhan virus.