Human Antigen Research Articles

Molecular mechanism of lncRNA SNHG12 in immune escape of non-small cell lung cancer through the HuR/PD-L1/USP8 axis

BackgroundThe pivotal role of long noncoding RNAs (lncRNAs) in cancer immune responses has been well established. This study was conducted with the aim of exploring the molecular mechanism of lncRNA small nucleolar RNA host gene 12 (SNHG12) in immune escape of non-small cell lung cancer (NSCLC).MethodsExpression of lncRNA SNHG12, programmed cell death receptor ligand 1 (PD-L1), ubiquitin-specific protease 8 (USP8), and human antigen R (HuR) in NSCLC tissues and cells was measured, and their binding relationship was determined. NSCLC cell proliferation and apoptosis were assessed. Peripheral blood mononuclear cells (PBMCs) were co-cultured with NSCLC cells. The ratio of CD8+ T cells, PBMC proliferation, and inflammatory factors were determined. lncRNA SNHG12 localization was assessed via subcellular fractionation assay. The half-life period of mRNA was determined using actinomycin D. Xenograft tumor models were established to confirm the role of lncRNA SNHG12 in vivo.ResultsLncRNA SNHG12 was found to be prominently expressed in NSCLC tissues and cells, which was associated with a poor prognosis. Silencing lncRNA SNHG12 resulted in the reduction in proliferation and the promotion of apoptosis of NSCLC cells, while simultaneously increasing PBMC proliferation and the ratio of CD8+ T cells. Mechanically, the binding of lncRNA SNHG12 to HuR improved mRNA stability and expression of PD-L1 and USP8, and USP8-mediated deubiquitination stabilized the protein level of PD-L1. Overexpression of USP8 or PD-L1 weakened the inhibition of silencing lncRNA SNHG12 on the immune escape of NSCLC. Silencing lncRNA SNHG12 restricted tumor growth and upregulated the ratio of CD8+ T cells by decreasing USP8 and PD-L1.ConclusionLncRNA SNHG12 facilitated the immune escape of NSCLC by binding to HuR and increasing PD-L1 and USP8 levels.

The Role of Exposomes in the Pathophysiology of Autoimmune Diseases II: Pathogens.

In our continuing examination of the role of exposomes in autoimmune disease, we use this review to focus on pathogens. Infections are major contributors to the pathophysiology of autoimmune diseases through various mechanisms, foremost being molecular mimicry, when the structural similarity between the pathogen and a human tissue antigen leads to autoimmune reactivity and even autoimmune disease. The three best examples of this are oral pathogens, SARS-CoV-2, and the herpesviruses. Oral pathogens reach the gut, disturb the microbiota, increase gut permeability, cause local inflammation, and generate autoantigens, leading to systemic inflammation, multiple autoimmune reactivities, and systemic autoimmunity. The COVID-19 pandemic put the spotlight on SARS-CoV-2, which has been called “the autoimmune virus.” We explore in detail the evidence supporting this. We also describe how viruses, in particular herpesviruses, have a role in the induction of many different autoimmune diseases, detailing the various mechanisms involved. Lastly, we discuss the microbiome and the beneficial microbiota that populate it. We look at the role of the gut microbiome in autoimmune disorders, because of its role in regulating the immune system. Dysbiosis of the microbiota in the gut microbiome can lead to multiple autoimmune disorders. We conclude that understanding the precise roles and relationships shared by all these factors that comprise the exposome and identifying early events and root causes of these disorders can help us to develop more targeted therapeutic protocols for the management of this worldwide epidemic of autoimmunity.

69-Year-Old Man With Dysuria and Right Lower Abdominal Pain

69-Year-Old Man With Dysuria and Right Lower Abdominal Pain

A SARS-CoV-2 Spike Receptor Binding Motif Peptide Induces Anti-Spike Antibodies in Mice andIs Recognized by COVID-19 Patients.

The currently devastating pandemic of severe acute respiratory syndrome known as coronavirus disease 2019 or COVID-19 is caused by the coronavirus SARS-CoV-2. Both the virus and the disease have been extensively studied worldwide. A trimeric spike (S) protein expressed on the virus outer bilayer leaflet has been identified as a ligand that allows the virus to penetrate human host cells and cause infection. Its receptor-binding domain (RBD) interacts with the angiotensin-converting enzyme 2 (ACE2), the host-cell viral receptor, and is, therefore, the subject of intense research for the development of virus control means, particularly vaccines. In this work, we search for smaller fragments of the S protein able to elicit virus-neutralizing antibodies, suitable for production by peptide synthesis technology. Based on the analysis of available data, we selected a 72 aa long receptor binding motif (RBM436-507) of RBD. We used ELISA to study the antibody response to each of the three antigens (S protein, its RBD domain and the RBM436-507 synthetic peptide) in humans exposed to the infection and in immunized mice. The seroreactivity analysis showed that anti-RBM antibodies are produced in COVID-19 patients and immunized mice and may exert neutralizing function, although with a frequency lower than anti-S and -RBD. These results provide a basis for further studies towards the development of vaccines or treatments focused on specific regions of the S virus protein, which can benefit from the absence of folding problems, conformational constraints and other advantages of the peptide synthesis production.

Are we missing reverse TRALI? – A real world experience from a tertiary care oncology centre in India

Transfusion related acute lung injury (TRALI) is a rare but potentially fatal pulmonary complication of transfusion that presents as acute hypoxemia and non-cardiogenic pulmonary oedema, developing during or within six hours of transfusion. Majority of the cases reported are due to transfusion of plasma rich blood components containing antibodies to human leukocyte antigen (anti-HLA) or human neutrophil antigen (anti-HNA). Rarely, anti-HLA or anti-HNA in recipients against transfused donor leukocyte antigens, cause TRALI by a reverse mechanism. Herein, we report three cases of suspected TRALI following transfusions of buffy coat derived granulocytes and peripheral blood stem cells. Three patients with hematological malignancies developed pulmonary symptoms after transfusions of leukocyte rich blood components. All cases showed findings of bilateral pulmonary infiltrates at chest radiography and patients were managed accordingly; however, all three expired within seven days of transfusion due to progressive respiratory deterioration. The patients were transfusion dependent for a long time and had received multiple non-leukoreduced blood components in the past. Clinical findings in all three cases indicate the possibility of reverse TRALI. Although, patients’ anti-HLA or anti-HNA antibodies concordance with donors’ cognate antigens (HLA and HNA) was not confirmed; yet these three cases suggest that reverse pathogenesis of TRALI is not as infrequent as reported in the literature. However, reverse TRALI has not been confirmed as the presence and nature of antibodies in the transfused recipient were not investigated due to the non availability of immunodiagnostic tests in India.

Characterization of recombinant Ybgf protein for the detection of Coxiella antibodies in ruminants.

Q fever remains a One Health problem, posing a zoonotic threat and causing significant economic losses to the livestock industry. The advancement of detection tools is critical to the effective control of infection. In humans, laboratory investigations depend largely on the immunofluorescence assay, considered the gold standard. In contrast, serologic tools routinely used for veterinary screening have several gaps, resulting in interpretations that are frequently misleading. We investigated the potential application of recombinant Ybgf antigen (r-Ybgf), a periplasmic protein described as one of the most immunodominant antigens in humans, in an indirect ELISA. Following successful expression in the prokaryotic system and the preliminary evaluation of immunoreactivity in western blot, we used r-Ybgf to develop an in-house ELISA using serum samples from sheep, goats, and cattle, which were tested in parallel with an Idexx ELISA kit. The results obtained with the 2 tests were compared, and r-Ybgf performed favorably, with 81.8% sensitivity and 90.1% specificity and substantial agreement, as revealed by receiver operating characteristic analysis. Moreover, we evaluated the serologic response against phase I (PhI) and phase II (PhII) antigens, and r-Ybgf antigen induced by vaccination, using phase-specific ELISAs. The dynamics of antibody response showed a significant increase in reactivity against PhI and PhII, but not against r-Ybgf, antigens. This property may be very useful given the absence of a protocol for the differentiation of infected from vaccinated animals.

Pseudogene CLEC4GP1 modulates trophoblast cell apoptosis and invasion via IL-15 inhibition

Preeclampsia (PE) is a pregnancy-associated complication accompanied by gestational hypertension and proteinuria, affecting 2–8% of pregnancies globally. The placental trophoblast cell invasion of decidua and myometrium during early gestation is crucial for healthy placentation. Thus, trophoblast dysfunction might contribute to PE onset. Therefore, further investigations are needed to elucidate the underlying mechanism of trophoblast cell functions. In the present study, we identified a novel pseudogene named C-Type Lectin Domain Family 4 Member G Pseudogene 1 (CLEC4GP1), which was aberrantly expressed in PE placental tissues. In vitro analyses showed that CLEC4GP1 overexpression significantly increased the cell viability and invasiveness and decreased the apoptosis rate of HTR-8/SVneo and JEG-3 cells, while CLEC4GP1 knockdown exerted opposite effects, suggesting the beneficial role of CLEC4GP1 in trophoblast cells. Next, co-expression analysis found that CLEC4GP1 was negatively correlated with Interleukin 15 (IL-15). The expression of IL-15 dramatically increased in PE placental tissues. In HTR-8/SVneo and JEG-3 cells, IL-15 exhibited detrimental effects, opposite to CLEC4GP1, and they were negatively correlated. In addition, CLEC4GP1 attenuates the mRNA stability of IL-16 by inhibiting the binding between human antigen R (HuR) protein and IL-15 RNA. Finally, the obverse effects of CLEC4GP1 and IL-15 were investigated, and results showed that IL-15 reverted CLEC4GP1 induced cellular functions. In brief, these data suggest that CLEC4GP1/IL-15 axis might modulate the occurrence and progression of PE via influencing the trophoblast cell viability, apoptosis, and invasive capability. This study provided cognizance of targeting the CLEC4GP1/IL-15 axis as a novel therapeutic approach to mitigate PE progression.

Noninvasive Prenatal Testing in Immunohematology-Clinical, Technical and Ethical Considerations.

Hemolytic disease of the fetus and newborn (HDFN), as well as fetal and neonatal alloimmune thrombocytopenia (FNAIT), represent two important disease entities that are caused by maternal IgG antibodies directed against nonmaternally inherited antigens on the fetal blood cells. These antibodies are most frequently directed against the RhD antigen on red blood cells (RBCs) or the human platelet antigen 1a (HPA-1a) on platelets. For optimal management of pregnancies where HDFN or FNAIT is suspected, it is essential to determine the RhD or the HPA-1a type of the fetus. Noninvasive fetal RhD typing is also relevant for identifying which RhD-negative pregnant women should receive antenatal RhD prophylaxis. In this review, we will give an overview of the clinical indications and technical challenges related to the noninvasive analysis of fetal RBCs or platelet types. In addition, we will discuss the ethical implications associated with the routine administration of antenatal RhD to all pregnant RhD-negative women and likewise the ethical challenges related to making clinical decisions concerning the mother that have been based on samples collected from the (presumptive) father, which is a common practice when determining the risk of FNAIT.

Anticancer, Anticoagulant, Antioxidant and Antimicrobial Activities of Thevetia peruviana Latex with Molecular Docking of Antimicrobial and Anticancer Activities.

Natural origin molecules represent reliable and excellent sources to overcome some medicinal problems. The study of anticancer, anticoagulant, and antimicrobial activities of Thevetia peruviana latex were the aim of the current research. An investigation using high-performance liquid chromatography (HPLC) revealed that the major content of the flavonoids are rutin (11.45 µg/mL), quersestin (7.15 µg/mL), naringin (5.25 µg/mL), and hisperdin (6.07 µg/mL), while phenolic had chlorogenic (12.39 µg/mL), syringenic (7.45 µg/mL), and ferulic (5.07 µg/mL) acids in latex of T. peruviana. Via 1,1-diphenyl-2- picrylhydrazyl (DPPH) radical scavenging, the experiment demonstrated that latex had a potent antioxidant activity with the IC50 43.9 µg/mL for scavenging DPPH. Hemolysis inhibition was 58.5% at 1000 µg/mL of latex compared with 91.0% at 200 µg/mL of indomethacin as positive control. Negligible anticoagulant properties of latex were reported where the recorded time was 11.9 s of prothrombin time (PT) and 29.2 s of the activated partial thromboplastin time (APTT) at 25 µg/mL, compared with the same concentration of heparin (PT 94.6 s and APPT 117.7 s). The anticancer potential of latex was recorded against PC-3 (97.11% toxicity) and MCF-7 (96.23% toxicity) at 1000 μg/mL with IC50 48.26 μg/mL and 40.31 µg/mL, respectively. Disc diffusion assessment for antimicrobial activity recorded that the most sensitive tested microorganisms to latex were Bacillus subtilis followed by Escherichia coli, with an inhibition zone (IZ) of 31 mm with minimum inhibitory concentration (MIC) (10.2 μg/mL) and 30 mm (MIC, 12.51 μg/mL), respectively. Moreover, Candida albicans was sensitive (IZ, 28 mm) to latex, unlike black fungus (Mucor circinelloides). TEM examination exhibited ultrastructure changes in cell walls and cell membranes of Staphylococcus aureus and Pseudomonas aeruginosa treated with latex. Energy scores of the molecular docking of chlorogenic acid with E. coli DNA (7C7N), and Rutin with human prostate-specific antigen (3QUM) and breast cancer-associated protein (1JNX), result in excellent harmony with the experimental results. The outcome of research recommended that the latex is rich in constituents and considered a promising source that contributes to fighting cancer and pathogenic microorganisms.

Construction and evaluation of wild and mutant ofatumumab scFvs against the human CD20 antigen

Several monoclonal antibodies targeting the CD20 have been produced and Ofatumumab is a case in point. Although whole antibodies target cancer cells effectively, their applications are restricted in some ways. Single-chain fragment variable antibodies, rather than employing the entire structure of antibodies, have proven a practical approach for creating completely functional antigen-binding fragments. In current research, the DNA coding sequence of and of the wild and mutant forms of ofatumumab were joined with a flexible linker separately. Using the E. coli BL21 (DE3) expression system, the -linker- genes were cloned into the pET-28 a (+), and the associated recombinant proteins were produced. Purified and refolded scFvs (scFv-C and scFv-) represented a concentration of around 0.7 mg/ml from 1 L of initial E. coli culture with a molecular weight of about 27 kDa. Affinity measurement disclosed anti-CD20 scFv- possesses a higher affinity constant compared to anti-CD20 scFv- The recombinant scFvs exclusively attach to Raji cells but not to Jurkat cells, according to a cell-ELISA analysis. The MTT test signified anti-CD20 scFvs could affect cell viability in Raji cells but had no impact on Jurkat cells and also, Raji cells viability was affected more significantly by anti-CD20 scFv-

Integrated Network Pharmacology and Clinical Study to Reveal the Effects and Mechanisms of Bushen Huoxue Huatan Decoction on Polycystic Ovary Syndrome.

Objective Bushen Huoxue Huatan Decoction (BHHD) is a classic prescription for treating polycystic ovary syndrome (PCOS). This study aims to explore the effects and possible mechanisms of BHHD on PCOS by integrating network pharmacology and clinical study. Methods The components and potential drug targets of BHHD were analysed using the TCMSP platform, and the potential pathogenesis targets for PCOS were analysed using the GeneCards and OMIM databases. Subsequently, a disease-compound-target network diagram was established to identify the targets of BHHD treatment on PCOS. In addition, protein-protein interaction analysis, KEGG pathway analysis, and Gene Ontology biological analysis were carried out to reveal the mechanisms of BHHD. To further validate the analysis, a clinical trial involving 62 PCOS patients was conducted. All patients were treated with BHHD for 3 months and the ovulation rate, anthropometric indicators, clinical symptoms, and serological indicators were measured and compared before and after treatment. Results The network pharmacology analysis showed that quercetin, luteolin, and kaempferol are the most significant active components in BHHD; STAT3, Jun, AKT1, MAPK3, MAPK1, and TP53 are the most critical drug targets; regulating hormones, reversing insulin (INS) resistance, exerting anti-inflammatory effects, and improving fertility might be the most important mechanisms of BHHD in the treatment of PCOS. After BHHD administration, the cyclic ovulation rate and the clinical symptoms including acanthosis nigricans and acne of patients were obviously improved. The serum endocrine levels of luteinising hormone (LH), LH/follicle-stimulating hormone, testosterone, dehydroepiandrosterone sulphate, insulin (INS), and area under the INS curve were evidently reversed, and the serum inflammatory factors levels including human interleukin (IL)-18, IL-16, IL-1β, IL-8, macrophage migration inhibitory factor, and human leukocyte differentiation antigen CD40 ligand were greatly reduced. Conclusion BHHD has a good therapeutic effect on PCOS, and its mechanisms may be related to regulating hormone levels, improving insulin resistance, alleviating inflammation, and promoting pregnancy.

Comprehensive Landscape of STEAP Family Members Expression in Human Cancers: Unraveling the Potential Usefulness in Clinical Practice Using Integrated Bioinformatics Analysis

The human Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) family comprises STEAP1-4. Several studies have pointed out STEAP proteins as putative biomarkers, as well as therapeutic targets in several types of human cancers, particularly in prostate cancer. However, the relationships and significance of the expression pattern of STEAP1-4 in cancer cases are barely known. Herein, the Oncomine database and cBioPortal platform were selected to predict the differential expression levels of STEAP members and clinical prognosis. The most common expression pattern observed was the combination of the over- and underexpression of distinct STEAP genes, but cervical and gastric cancer and lymphoma showed overexpression of all STEAP genes. It was also found that STEAP genes’ expression levels were already deregulated in benign lesions. Regarding the prognostic value, it was found that STEAP1 (prostate), STEAP2 (brain and central nervous system), STEAP3 (kidney, leukemia and testicular) and STEAP4 (bladder, cervical, gastric) overexpression correlate with lower patient survival rate. However, in prostate cancer, overexpression of the STEAP4 gene was correlated with a higher survival rate. Overall, this study first showed that the expression levels of STEAP genes are highly variable in human cancers, which may be related to different patients’ outcomes.

Prucalopride might improve intestinal motility by promoting the regeneration of the enteric nervous system in diabetic rats.

The present study aimed to investigate whether prucalopride, as a 5-hydroxytryptamine 4 (5-HT4) receptor agonist, improved intestinal motility by promoting the regeneration of the enteric nervous system (ENS) in rats with diabetes mellitus (DM). A rat model of DM was established using an intraperitoneal injection of streptozotocin. The rats were randomly divided into four groups of 6 rats/group: Control, DM (DM model), DM + A (5 µg/kg prucalopride) and DM + B (10 µg/kg prucalopride). The rats in the Control group were given an equal volume of citric acid solvent. After successful model establishment, high blood glucose levels were maintained for 2 weeks before administration of prucalopride. The colonic transit time was measured using the glass bead discharge method. It was revealed that the colonic transit time of diabetic rats was the longest, and this was significantly shortened in the DM + B group. Subsequently, the colons were collected. The expression levels of Nestin, glial fibrillary acidic protein (GFAP), SOX10, RNA-binding protein human antigen D (HuD) and ubiquitin thiolesterase (PGP9.5) were determined via immunohistochemical analysis. Immunofluorescence double staining of 5-HT4 + Nestin and Ki67 + Nestin was performed. The 5-HT level was measured using ELISA. Compared with that in the control group, Nestin expression was significantly increased in the DM and DM + A groups, and it was concentrated in columnar epithelial cells and the mesenchyme. Furthermore, the expression levels of Nestin in the DM + A group were higher than those in the DM group. No difference was observed in the expression levels of Nestin between the DM + B group and the Control group. The expression levels of 5-HT protein were highest in the Control group; however, the expression levels of 5-HT protein in the DM group, DM + A group and DM + B group exhibited an increasing trend. Similar trends in the expression of 5-HT4 and Nestin were not observed; however, similar trends in the expression of Nestin and Ki67 were observed. The expression levels of GFAP, SOX10, PGP9.5 and Ki67 in the DM + A and DM + B groups were higher compared with those in the DM group. In the DM + A group, HuD expression was decreased compared with that in the Control group but it was markedly higher compared with that in the DM group. In conclusion, prucalopride may improve intestinal motility by promoting ENS regeneration in rats with DM.

MO230: De Novo Double Glomerulopathy (Membranous Nephropathy, Mn and Collapsing Focal Segmental Glomerulosclerosis, CFSGS) Associated to Positive Myeloperoxidase-O (MPO) Antibody Following Pfizer-Biontech MRNA Vaccination Covid 19

Abstract BACKGROUND AND AIMS In the COVID 19 pandemic era, anti SARS-CoV-2 vaccination showed high efficacy at preventing the infection and its most severe complications. The aim of this report is to describe an unusual double glomerulopathy related to anti SARS-CoV-2 vaccination and the good results obtained with the immunosoppressive treatment. METHOD An 80-year-old caucasian woman developed a nephrotic syndrome, progressive renal insufficiency and microhematuria. The patient presented a medical history of thrombocytopenic purpura treated and resolved by steroids in 2013, hypothyroidism, hypertension, ischaemic heart disease treated with surgical bypass in 2019 and pacemaker in 2020 for atrial ventricular block. Due to pandemic COVID 19 status, she received two doses of the Pfizer BioNTech mRNA COVID-19 vaccine in March 2021. Two weeks after the second dose her weight increased of 23 kg. The family physician added furosemide to her therapy for generalized edema with no diuretic effect. In April, creatinine was 1.38 mg/dL (versus 0.8 mg/dL 1 year before); urinalysis showed proteinuria (300 mg/dL) and microscopic hematuria; serum total cholesterol level was 218 mg/dL and triglycerides 178 mg/dL; then it was suggested to increase the doses of furosemide. In May 2021, creatinine resulted 2 mg/dL, serum albumin 2 g/dL, and urinalysis confirmed proteinuria and microscopic hematuria; proteinuria was 10 g/day. Abdomen ultrasound showed normal liver, kidneys and spleen, not ascites. Lower limb eco-Doppler showed right superficial femoral artery stenosis of 60% and absence of venous thrombosis. The physical examination evidenced anasarca. The patients were admitted to the nephrology unit; hepatitis B surface antigen, hepatitis C antibody and human immunodeficiency virus antigen and antibody were negative. Both complement C3 and C4 levels resulted within the normal range. Cryoglobulins were absent. Urinary Bence Jones, antinuclear antibody (ANA), anti-extractable nuclear antigen (ENA), anti-double stranded DNA (nDNA) antibodies were negative. Antineutrophil cytoplasm antibodies (ANCA) were 1:2560 with Perinuclear pattern and anti-MPO positivity (716 UA/mL); anti-proteinase-3 antibodies (PR3) were negative. Anti-phosholipase A2 receptor antibody (PLA2R Ab) was positive with high titre. A kidney biopsy was performed showing a double nephropathy: a focal segmental glomerulosclerosis (FSGS) with some collapsing features, superimposed on membranous glomerulonephritis (Fig. 1). RESULTS We started the Ponticelli regimen (alternate months steroids and cyclophosphamide). After the first month of therapy, blood tests revealed creatinine 1.7 mg/dL, haemoglobin 11.7 g/dL; serum albumin 2.7 g/dL and urinalysis without microscopic haematuria. At the third month of therapy, the patient developed atrial fibrillation and started anticoagulation; blood tests were as follows: creatinine 1.1 mg/dL, serum albumin 3.0 g/dL, Ab anti-MPO 7 UA/mL and PLA2R Ab was absent. A left ocular, frontal and parietal herpes zoster induced a short discontinuation of therapy and responded well to Acyclovir; then we concluded the fourth month of therapy. At the fifth month, a SARS CoV 2 RT PCR unexpectedly resulted positive; the patient remained asymptomatic, but we stopped definitively the therapy. One month later, blood tests showed: creatinine 1 mg/dL, serum albumin 4 g/dL, proteinuria 0.7 g/die, MPO 2 UA/mL and PLA2R Ab absent. CONCLUSION To our knowledge, this is the first case of nephrotic sindrome secondary to a de novo MN and FSGS, associated with positive MPO antibody, following Pfizer–BioNTech mRNA vaccination COVID 19; the patient responded well to immunosoppression going in remission and regaining renal function.

Molecular insights into binding dynamics of tandem RNA recognition motifs (tRRMs) of human antigen R (HuR) with mRNA and the effect of point mutations in impaired HuR-mRNA recognition

Human antigen R (HuR) is a key regulatory protein with prominent roles in RNA metabolism and post-transcriptional gene regulation. Many studies have shown the involvement of HuR in plethora of human diseases, which are often manifestations of impaired HuR-RNA interactions. However, the inherent complexities of highly flexible protein-RNA interactions have limited our understanding of the structural basis of HuR-RNA recognition. In this study, we dissect the underlying molecular mechanism of interaction between N-terminal tandem RNA-recognition motifs (tRRMs) of HuR and mRNA using molecular dynamics simulation. We have also explored the effect of point mutations (T90A, R97A and R136A) of three reported critical residues in HuR-mRNA binding specificity. Our findings show that N-terminal tRRMs exhibit conformational stability upon RNA binding. We further show that R136A and R97A mutants significantly lose their binding affinity owing to the loss of critical interactions with mRNA. This may be attributed to the larger domain rearrangements in the mutant complexes, especially the β2β3 loops in both the tRRMs, leading to unfavourable conformations and loss of binding affinity. We have identified critical binding residues in tRRMs of HuR, contributing favourable binding energy in mRNA recognition. This study contributes significantly to understand the molecular mechanism of RNA recognition by tandem RRMs and provides a platform to modulate binding affinities through mutations. This may further guide in future structure-based drug-therapies targeting impaired HuR-RNA interactions. Communicated by Ramaswamy H. Sarma

HuR Mediates Thermogenic Metabolism in Brown Adipose Tissue Through Control of Sarco‐Endoplasmic Calcium Cycling

Obesity is classified by the World Health Organization as one of eight principle causes of preventable chronic disease and is directly associated with decreased life expectancy due to increased risk of associated comorbidities. Brown adipose tissue (BAT) is a thermogenically active, endocrine organ regulating both glucose homeostasis and heat production, and the ability to induce BAT‐mediated energy expending represents a potential therapeutic avenue to reduce obesity.We recently showed that the RNA‐binding protein HuR (Human antigen R) plays a functional role in BAT‐mediated thermogenic metabolism. RNA sequencing analysis from mice with an adipocyte‐specific deletion of HuR (Adipo‐HuR‐/‐) revealed a BAT‐specific decrease in the expression of many genes responsible for sarco‐endoplasmic reticulum (SER) calcium cycling, including SERCA1a, RyR1, RyR2, and additional RyR complex proteins such as triadin, junctin, and CASR. Canonical thermogenesis in BAT occurs through uncoupling protein 1 (UCP1). However, several UCP1‐independent energy expending cycles, including intracellular calcium transport, have also been shown to regulate thermogenesis. SER calcium cycling has recently been established as a thermogenic mechanism in beige adipose tissue, but its functional contributions to thermogenesis in BAT have not yet been fully elucidated.To determine if the HuR‐dependent changes in SER calcium cycles manifest as a functional change in SER loading/cycling in BAT, we show that decreased cytosolic intracellular calcium levels in BAT isolated from Adipo‐HuR‐/‐mice. Additionally, we show that HuR is activated acutely downstream of beta‐adrenergic stimulation in BAT, and deletion of HuR blunts adrenergic‐induced increases in intracellular calcium. Furthermore, HuR directly binds RyR2 mRNA in brown adipocytes and regulates RyR2 expression through RNA stabilization. Taken together, this growing body of work suggests an important role for HuR in mediating thermogenesis through modulation of SER calcium cycling in brown adipose tissue.

Immortalized B Cells Transfected With mRNA of Antigen Fused to MITD (IBMAM): An Effective Immunological Tool for In-Vitro Immune-Monitoring of Antigen-specific T Cells

PURPOSE To overcome technical limitations of detecting low frequencies of antigen-specific-T cells in human patients, we sought to establish a highly sensitive platform for antigen-specific-T cell detection and activation using IBMAM (immortalized B cells transfected with mRNA of antigen fused to MHC-class-I-trafficking-domain [MITD]). METHODS We first established immortalized B cells in-vitro as antigen-presenting cells. B cells were isolated from CMV-seropositive normal healthy donors (NHDs), stimulated, and transduced with retrovirus containing BCL-6 and BCL-XL. Purified CD4+ T lymphocytes obtained from the NHDs PBMC were co-cultured in-vitro with autologous immortalized B cells transfected with CMVpp65 mRNA fused to MITD (IBMAM) as a model antigen and then measured for activation using IFN-g ELISA. We developed two pathways for IBMAM mediated antigen-specific T cell activation and detection. For the first pathway, if IFN-g is detected, T cells are isolated again from NHDs PBMC and expanded using rapid expansion procedures (REP). After the first REP, antigen-specific T cells are detected using IBMAM, sorted, and a second REP is performed. However, for the second pathway, if IFN-g is not detected, NHDs PBMC is stimulated twice using IBMAM and subsequently antigen-specific T cells are detected using IBMAM again. RESULTS B cells were successfully immortalized by transfecting at least 5*105 cells with retrovirus (1MOI) and co-cultured with CD40L-IL21 expressing human fibroblasts. We observed a significantly increased activation of antigen-specific CD4+ T cells when using IBMAM in comparison to controls transfected with CMVpp65 mRNA lacking the MITD fusion or a known CMVpp65 peptide pool. For pathway 1 and 2 of our platform, successful detection, activation, and expansion of CMVpp65-specific T cells were observed from low frequency NHDs samples using IBMAM. CONCLUSION We have thus established a highly sensitive method of immuno-monitoring that can be used for the detection of antigen-specific T cells in patient samples with low frequencies of antigen-specific-T cells, such as human cancer antigens.

Regulation of Glioblastoma Stem Cell Properties and Tumor Invasion by G⍺12 Signaling

Glioblastoma multiforme (GBM), the most common malignant brain tumor, is associated with upregulation of a number of G‐protein coupled receptors. TCGA analysis of glioblastoma reveals that Gα12 mRNA levels are elevated in 28% of GBM patient tumors, the highest rate for all tumor types surveyed. There are concomitant alterations in expression of a subset of GPCRs that couple to Gα12. Gα12 signals through activation of RhoA and its downstream effectors. Our earlier studies revealed that gene programs induced through the RhoA‐regulated transcriptional co‐activators YAP and MRTF‐A contribute to glioma stem cell properties and their in vivo growth as orthotopic (brain) tumors (Yu et al., Oncogene 2018). To determine if Gα12 signaling pathways transduce the effects of known and orphan GPCRs activated in the tumor environment we knocked down Gα12 using shRNA in two human GBM cancer stem cell lines, GSC‐23 and HK‐281. Gα12 mRNA levels decreased by 60‐80% without compensatory upregulation of mRNA for the homologous Gα13. Loss of Gα12 significantly attenuated the stem cell properties of GSC‐23 cells as determined by limiting dilution assays and also reduced mRNA levels for multiple canonical stem cells genes (CCND1, NANONG, OCT4, SOX2 and NESTIN) by 35% to 60%. Tumor growth of GSC‐23 control and Gα12 knockdown (KD) cells was examined following orthotopic injection into mouse brain. Surprisingly, Gα12 deletion had no evident effect on tumor size nor did it prolong mouse survival. On the other hand, histopathological analyses revealed striking differences at the tumor border, indicative of diminished invasive properties and this was confirmed by immunohistochemical staining for the human nucleoli antigen. RNA sequencing and DESeq2 analysis in WT and KD tumors identified a number of genes encoding extra and intracellular proteins related to cell adhesion and migration dynamics which were differentially expressed in Gα12 KDs. Thrombospondin‐1 (Thbs‐1) was one of the most repressed genes in the Gα12 knockdown tumors and it was previously identified as a YAP‐dependent gene in these cells. Expression of Thbs‐1 and Gα12 is highly correlated in the more aggressive mesenchymal‐types of GBM by Gliovis and IvyGAP tools, and clinical reports show elevated Thbs‐1 as a marker of poor prognosis in GBM patients. To assess the effect of Gα12 and Thbs‐1 on glioblastoma stem cell migration we performed in vitro Transwell‐based assays. These studies demonstrated that both Gα12 and its transcriptional target Thbs‐1 are required for agonist‐stimulated GSC‐23 cell migration and invasion. We also demonstrated through chemogenetic activation of Gα12 by DREADD expression that Gα12 signaling in GSC‐23 cells is sufficient to drive Thbs‐1 expression and migration. Our findings indicate that Gα12 is a focal point for GPCR control of transcriptional programs for stemness and invasiveness in glioma stem cells derived tumors and suggest targeting this site as a therapeutic intervention to disrupt tumor cell invasion.

Approaches to isolating and purifying nucleic acids from blood for genotyping human leukocytic antigen

Donorrecipient histocompatibility results from the presence on cell membranes of the main protein complex of histocompatibility and is a key condition for successful transplantation of cells, tissues, and organs. To determine histocompatibility, human leukocyte antigen is genotyped, and its accuracy relied significantly on the quality and quantity of nucleic acids obtained from the biomaterial. In laboratory practice, the most pure and intact deoxyribonucleic and ribonucleic acids are extracted from the blood; however, the choice of an accessible, effective, time-efficient, and viable method for their production remains challenging. The methods of isolation and purification of nucleic acids from the blood include organic extraction, salting out, and use of spin columns and magnetic particles (bidids), and their advantages and disadvantages, efficiency indicators, practicality, and cost were compared. The selection of the peripheral blood as a source of genetic material for genotyping human leukocyte antigen is justified. Experimental data comparing the pricequality ratio of commercial protocols for the extraction of deoxyribonucleic and ribonucleic acids from blood were analyzed. Prospects of modification of procedures for isolation and purification of nucleic acids from biomaterial for sequencing of genes of human leukocyte antigen classes I and II to increase efficiency of high-tech care are evaluated. Generally, the need for affordable and effective protocols for the extraction of deoxyribonucleic and ribonucleic acids from minimal biomaterial volumes stimulates the optimization and modification of existing protocols and the creation of new methods on new physicochemical principles.

Potential of RNA-binding protein human antigen R as a driver of osteogenic differentiation in osteoporosis

BackgroundEmerging evidence has correlated the human antigen R (HuR) with the low-density lipoprotein receptor-related protein 6 (LRP6) gene, an important therapeutic target for osteoporosis. Herein, we sought to probe the regulatory role of HuR in the LRP6 gene and their interaction in the progression of osteoporosis.MethodsHuR and downstream potential target genes were predicted by bioinformatics analysis to identify their potential functions in bone metabolism following osteoporosis. The effect of HuR on the osteoblastic differentiation and viability and apoptosis of mouse embryo osteoblast precursor cells (MC3T3-E1) was evaluated after artificial modulation of HuR expression.ResultsBone phenotypes were observed in ovariectomized mice in response to adenovirus-mediated HuR overexpression. Poor expression of HuR was identified in the bone tissues of ovariectomized mice. Silencing of HuR inhibited the osteoblastic differentiation of MC3T3-E1 cells, as evidenced by decreased expression of Runx2 and Osterix along with reduced ALP activity. Mechanistically, HuR stabilized LRP6 mRNA and promoted its translation by binding to the 3'UTR of LRP6 mRNA, leading to activation of the downstream Wnt pathway. By this mechanism, osteoblastic differentiation of MC3T3-E1 cells was induced. In ovariectomized mice, overexpression of HuR alleviated osteoporosis-related phenotypes.ConclusionOverall, these data together support the promoting role of HuR in the osteoblastic differentiation, highlighting a potential novel strategy for osteoporosis treatment.