Human Ia Research Articles

An Efficient and Cost-Effective Approach to Generate Functional Human Inducible Pluripotent Stem Cell-Derived Astrocytes.

Human inducible pluripotent stem cell (hiPSC)-derived astrocytes (iAs) are critical to study astrocytes in health and disease. They provide several advantages over human fetal astrocytes in research, which include consistency, availability, disease modeling, customization, and ethical considerations. The generation of iAs is hampered by the requirement of Matrigel matrix coating for survival and proliferation. We provide a protocol demonstrating that human iAs cultured in the absence of Matrigel are viable and proliferative. Further, through a side-by-side comparison of cultures with and without Matrigel, we show significant similarities in astrocyte-specific profiling, including morphology (shape and structure), phenotype (cell-specific markers), genotype (transcriptional expression), metabolic (respiration), and functional aspects (glutamate uptake and cytokine response). In addition, we report that, unlike other CNS cell types, such as neuronal progenitor cells and neurons, iAs can withstand the absence of Matrigel coating. Our study demonstrates that Matrigel is dispensable for the culture of human iPSC-derived astrocytes, facilitating an easy, streamlined, and cost-effective method of generating these cells.

R-loops, type I topoisomerases and cancer.

R-loops are abundant and dynamic structures ubiquitously present in human cells both in the nuclear and mitochondrial genomes. They form in cis in the wake of transcription complexes and in trans apart from transcription complexes. In this review, we focus on the relationship between R-loops and topoisomerases, and cancer genomics and therapies. We summarize the topological parameters associated with the formation and resolution of R-loops, which absorb and release high levels of genomic negative supercoiling (Sc-). We review the deleterious consequences of excessive R-loops and rationalize how human type IA (TOP3B) and type IB (TOP1) topoisomerases regulate and resolve R-loops in coordination with helicase and RNase H enzymes. We also review the drugs (topoisomerase inhibitors, splicing inhibitors, G4 stabilizing ligands) and cancer predisposing genes (BRCA1/2, transcription, and splicing genes) known to induce R-loops, and whether stabilizing R-loops and thereby inducing genomic damage can be viewed as a strategy for cancer treatment.

The Medawar Prize Acceptance Speech 2022.

The Medawar Prize Acceptance Speech 2022.

Endothelial cell malfunction in unruptured intracranial aneurysm lesions revealed using a 3D-casted mold.

Intracranial aneurysms (IA) are major causes of devastating subarachnoid hemorrhages. They are characterized by a chronic inflammatory process in the intracranial arterial walls triggered and modified by hemodynamic force loading. Because IA lesion morphology is complex, the blood flow conditions loaded on endothelial cells in each portion of the lesion in situ vary greatly. We created a 3D-casted mold of the human unruptured IA lesion and cultured endothelial cells on this model; it was then perfused with culture media to model physiological flow conditions. Gene expression profiles of endothelial cells in each part of the IA lesion were then analyzed. Comprehensive gene expression profile analysis revealed similar gene expression patterns in endothelial cells from each part of the IA lesion but gene ontology analysis revealed endothelial cell malfunction within the IA lesion. Histopathological examination, electron microscopy, and immunohistochemical analysis indicated that endothelial cells within IA lesions are damaged and dysfunctional. Thus, our findings reveal endothelial cell malfunction in IA lesions and provided new insights into IA pathogenesis.

The stability of the metabolic turnover of arachidonic acid in human unruptured intracranial aneurysmal walls is sustained

ObjectiveIntracranial aneurysm (IA) is considered a chronic inflammatory condition that affects intracranial arteries. Cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) are considered potential targets of specific medical treatment for IAs. Previous studies have reported the elevated COX2 expression in the IA wall. However, not much has been studied about the upstream regulation of COX2 and PGE2, and the metabolism of arachidonic acid (AA) in human IAs. In this study, we aimed to elucidate the distribution of fatty acids in human IA walls for the first time. MethodsSamples from 6 ruptured and 5 unruptured human IAs were surgically resected after the aneurysmal clipping and analyzed using desorption electrospray ionization imaging mass spectrometry. ResultsAA and AA-containing phospholipids were not detected in the unruptured IA walls. On the contrast, significantly larger amounts of AA and AA-containing phospholipids were detected in the ruptured IA walls compared to unruptured IA walls. ConclusionsThis study showed for the first time that AA was not detected in unruptured human IA walls. Our findings suggest that the stability of the turnover of AA in human unruptured IA walls is sustained. In contrast, this study showed that larger amounts of AA and AA-containing phospholipids were detected in the ruptured IA walls. More cases and further analysis are necessary to interpret our present results.

1231P - Master checkpoint Cbl-b inhibition: Anti-tumour efficacy in a murine colorectal cancer model following siRNA-based cell therapy

BackgroundThe E3 ubiquitin ligase Cbl-b is a key master immune checkpoint regulator that limits immune activation critical for anti-tumor immunity. We first reported that Cbl-b deficiency in mice confers spontaneous in vivo rejection of tumor cells. This checkpoint role in anti-tumor immunity was confirmed in multiple mouse and human studies. Further discovery efforts led to the advancement of APN401, an ex vivo human Cbl-b siRNA-based autologous cellular therapy, currently in clinical development in patients with advanced solid tumors. APN401 has been target validated and its preclinical efficacy was established in various mouse syngeneic tumor models. We present here the potent anti-tumor efficacy of APN401 immunotherapy of Cbl-b silenced murine T cells in a syngeneic MC38 colorectal tumor model. MethodsT cells isolated from MC38 tumor-bearing donor C57Bl/6 mice were silenced ex vivo with APN401 murine Cbl-b specific siRNA vs control siRNA, adoptively transferred into MC38 tumor-bearing recipient mice, and tumor growth was monitored using an in vivo imaging system. ResultsMurine APN401 treatment resulted in a significant MC38 tumor growth inhibition of 63% (p=0.008) vs controls after just a single dose application. Profound anti-tumor efficacy induced by Cbl-b-silenced T cells strongly correlated with enhanced production of TH1 cytokines IL-2 and IFN-γ. Furthermore, in vivo tracking of fluorescently labeled and silenced T cells revealed migration to relevant lymphoid organs and local tumor sites. Murine APN401 treatment was safe and well-tolerated similar to recent human APN401 clinical phase Ia study results in patients with advanced solid tumors. ConclusionsIn a model of murine colon cancer, Cbl-b-silencing induced vigorous in vivo anti-tumorigenic immune responses. The treatment modality was safe and well tolerated. These data provide direct preclinical proof of concept that siRNA-based silencing of Cbl-b provides a novel, effective and tunable approach as cellular immunotherapy. Targeting Cbl-b through human APN401 cell therapy is a promising novel therapeutic approach for the treatment of solid cancers, also highlighted by our fast-tracked global APN401 clinical development. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

A Complex Relationship: Dietary Folate, Arsenic Metabolism, and Insulin Resistance in Mice.

A Complex Relationship: Dietary Folate, Arsenic Metabolism, and Insulin Resistance in Mice.

SPARC induces phenotypic modulation of human brain vascular smooth muscle cells via AMPK/mTOR-mediated autophagy

Secreted protein acidic and rich in cysteine (SPARC) was widely expressed in VSMCs of human IAs and could reduce the capability of self-repair. This indicates that SPARC may play a role in the promotion of IAs formation and progression, but the mechanism remains unclear. In this study, we further investigated whether SPARC could induce phenotypic modulation of Human Brain Vascular Smooth Muscle Cells (HBVSMCs) and sought to elucidate the role of SPARC-mediated autophagy involved in it. The results demonstrated that SPARC inhibited the expression of contractile genes in HBVSMCs and induced a synthetic phenotype. More importantly, SPARC significantly up-regulated multiple proteins including autophagy marker microtubule-associated protein light chain 3-II (LC3-II), Beclin-1, and autophagy-related gene 5(ATG5). Furthermore, SPARC could promote p62 degradation. The autophagy inhibitor 3- methyladenine (3-MA) significantly blocked SPARC-induced phenotypic modulation of HBVSMCs. We further sought to elucidate the molecular mechanism involved in SPARC-induced autophagy, and found that SPARC could activate the AMPK/mTOR signaling pathway in HBVSMCs. AMPK could be pharmacologically inhibited by Compound C (CC), which significantly decreased the phosphorylation of AMPK into p-AMPK, increased the phosphorylation of mTOR into p-mTOR, and decreased LC3-II, Beclin-1 and ATG5 levels. This suggested that activated AMPK/ mTOR signaling is related to SPARC-mediated autophagy. These results indicated that SPARC plays a role in the phenotypic modulation of HBVSMCs through autophagy activation by AMPK/mTOR signaling pathway.

Assessment of structurally and functionally high-risk nsSNPs impacts on human bone morphogenetic protein receptor type IA (BMPR1A) by computational approach

BMPR1A (BMP type 1 receptor) is a transmembrane cell-surface receptor also known as ALK3 (activin-like kinases-3) encodes for a type I serine/threonine kinase receptor and a member of the transforming growth-factor β–receptor (TGF-β) super family. The BMPR1A has a significant interaction with BMP-2 for protein activity and also has a low affinity with growth and differentiation factor 5 (GDF5); positively regulates chondrocyte differentiation. The genetic variations can alter the structure and function of the BMPR1A gene that causes several diseases such as juvenile polyposis syndrome or hereditary cancer-predisposing syndrome. The current study was carried out to identify potential deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) in BMPR1A by implementing different computational algorithms such as SIFT, PolyPhen2, SNAP2, PROVEAN, PhD-SNP, SNPs&GO, nsSNPAnalyzer, and P-Mut. From 205 nsSNPs in BMPR1A, 7 nsSNPs (C76Y, C124R, C124Y, C376Y, R443C, R480W, and W487R) were predicted as deleterious in 8 prediction algorithms. The Consurf analysis showed that selected 7 nsSNPs were present in the highly conserved regions. Molecular dynamics simulation analysis also performed to explore conformational changes in the variant structure with respect to its native structure. According to the MDS result, all variants flexibility and rigidity were unbalanced, which may alter the structural and functional behavior of the native protein. Although, three nsSNPs i.e., C124R, C376Y, and R443C have already been reported in patients associated with JPS, but their structural and functional molecular studies remain uncharacterized. Therefore, the findings of this study can provide a better understanding of uncharacterized nsSNPS and to find their association with disease susceptibility and also facilitate to the researchers for designing or developing the target dependent drugs.

Long-term complications of glycogen storage disease type Ia in the canine model treated with gene replacement therapy.

Glycogen storage disease type Ia (GSD Ia) in dogs closely resembles human GSD Ia. Untreated patients with GSD Ia develop complications associated with glucose-6-phosphatase (G6Pase) deficiency. Survival of human patients on intensive nutritional management has improved; however, long-term complications persist including renal failure, nephrolithiasis, hepatocellular adenomas (HCA), and a high risk for hepatocellular carcinoma (HCC). Affected dogs fail to thrive with dietary therapy alone. Treatment with gene replacement therapy using adeno-associated viral vectors (AAV) expressing G6Pase has greatly prolonged life and prevented hypoglycemia in affected dogs. However, long-term complications have not been described to date. Five GSD Ia-affected dogs treated with AAV-G6Pase were evaluated. Dogs were euthanized due to reaching humane endpoints related to liver and/or kidney involvement, at 4 to 8years of life. Necropsies were performed and tissues were analyzed. Four dogs had liver tumors consistent with HCA and HCC. Three dogs developed renal failure, but all dogs exhibited progressive kidney disease histologically. Urolithiasis was detected in two dogs; uroliths were composed of calcium oxalate and calcium phosphate. One affected and one carrier dog had polycystic ovarian disease. Bone mineral density was not significantly affected. Here, we show that the canine GSD Ia model demonstrates similar long-term complications as GSD Ia patients in spite of gene replacement therapy. Further development of gene therapy is needed to develop a more effective treatment to prevent long-term complications of GSD Ia.

Rv1495 toxin as a model for inhibitors of Mycobacterium tuberculosis DNA topoisomerase I

Topoisomerases overcome topological barriers encountered during DNA replication, transcription, recombination and repair. The genomes of mycobacteria encode only one type IA topoisomerase; Mycobacterium tuberculosis DNA topoisomerase I (Mtb Top I) is thus essential for cellular survival and proliferation, and has been validated as a novel TB drug target. Inhibitors of Mtb Top I may be useful for development of much needed new therapy for drug resistant TB.Rv1495 is a M. tuberculosis toxin that belongs to the MazEF family (MazE is the antitoxin and MazF is the antitoxin), with endoribonuclease activity. Besides being an endoribonuclease, Rv1495 (MazF homolog in M. tuberculosis) toxin has been shown to interact directly with the C‐terminal domain of Mtb Top I for mutual inhibition of catalytic activity. The C‐terminal domains of bacterial topoisomerase I have diverged in evolution with zinc ribbon domains found in most bacterial topoisomerase I as well as human type IA topoisomerases, while mycobacterial topoisomerase I have C‐terminal domains do not bind Zn(II). The mode of Mtb Top I inhibition by Rv1495 could be informative for the design and discovery of small molecule drugs that can target topoisomerase I in M. tuberculosis and pathogenic NTM (non‐tuberculosis mycobacteria).Previous studies have revealed that inhibition of Mtb Top I is due to the direct physical interaction of the N‐terminal domain of Rv1495 toxin with the 33 kDa C‐terminal domain of Mtb Top I. However, no molecular details about this interaction have been reported. We have established an in vivo genetic selection scheme to identify specific amino acid residues in Mtb Top I required for this protein‐protein interaction and inhibition by Rv1495. E. coli AS17 cells have a temperature sensitive mutation in topA coding for topoisomerase I. Recombinant Mtb Top I expressed by plasmid pLICMTOPI can complement the topAts mutation for growth of AS17 at the non‐permissive temperature of 42°C. This complementation is abolished by co‐transformation of AS17 with a second plasmid pBADRv1495 expressing Rv1495. We have constructed a library of pLICMTOPI plasmid with random mutations in Mtb Top I coding sequence. Following transformation of AS17/pBADRv1495, mutated Mtb Top I clones that can complement growth of AS17 at 42°C in the presence of Rv1495 expression have been isolated successfully. These mutants retain the catalytic function of Mtb Top I to support viability at 42°C, but are resistant to inhibition by Rv1495 toxin. The results from analysis of the mutations involved should allow identification of a potential binding site on Mtb Top I for small molecules that can target mycobacteria topoisomerase I specifically.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Improved TaqMan real-time assays for detecting hepatitis A virus

Rapid advancement in genomics and bioinformatics in recent years holds great promise for research and development in many disciplines including public health. For the detection of pathogens, methods based on nucleic acid amplification need to be re-evaluated periodically to ensure the validity of signature primers and probes as more and more outbreak strains are sequenced and collected into databases in public domains. In this study, a previous assay designed computationally for detecting hepatitis A virus (HAV) was re-examined. Alignment of 57 complete or near complete HAV genomes allowed identification of conserved sequences for developing new primers and TaqMan probes. Two sets of real-time reverse transcription PCR reagents were developed by targeting highly conserved regions with primers and probes having optimal melting temperatures and minimum secondary structures. These two assays had 10 to 1000 fold lower detection limits than the previous assay when tested using representative human HAV genotypes IA, IB, and IIIA. The better of the two improved assays had a detection limit of 3.7 × 10−2 to 6.6 × 10−2 TCID50 or less. The improved detection sensitivity was likely due to improvement in the following four areas: 1) The Gardner1 probe has a single nucleotide mismatch at the 5′ end in all 19 strains of genotypes IIIA and IIIB. 2) For the Gardner1 forward primer, there is a mismatch corresponding to the 3′ end of the oligonucleotides in two strains belonging to genotype IA. 3) The Gardner1 probe had a melting temperature of 66.2 °C, which is less than the optimum of 68–70 °C (Dorak, 2006). 4) The Gardner1 forward and reverse primers had high potential of forming primer dimers.The improved HAV detection assays developed in this study would support better food safety surveillance initiatives and response to disease outbreaks of viral food-borne illness.

Synapsin-antibodies in psychiatric and neurological disorders: Prevalence and clinical findings

ObjectiveTo study the prevalence of autoantibodies to synapsin in patients with psychiatric and neurological disorders and to describe clinical findings in synapsin antibody positive patients. MethodsSera of 375 patients with different psychiatric and neurological disorders and sera of 97 healthy controls were screened (dilution 1:320) for anti-synapsin IgG using HEK293 cells transfected with rat synapsin Ia. Positive sera were further analyzed by immunoblots with brain tissue from wild type and synapsin knock out mice and with HEK293 cells transfected with human synapsin Ia and Ib. Binding of synapsin IgG positive sera to primary neurons was studied using murine hippocampal neurons. ResultsIgG in serum from 23 (6.1%) of 375 patients, but from none of the 97 healthy controls (p=0.007), bound to rat synapsin Ia transfected cells with a median (range) titer of 1:1000 (1:320–1:100,000). Twelve of the 23 positive sera reacted with a protein of the molecular size of synapsin I in immunoblots of wild type but not of synapsin knock out mouse brain tissue. Out of 19/23 positive sera available for testing, 13 bound to human synapsin Ia and 16 to human synapsin Ib transfected cells. Synapsin IgG positive sera stained fixed and permeabilized murine hippocampal neurons. Synapsin IgG positive patients had various psychiatric and neurological disorders. Tumors were documented in 2 patients (melanoma, small cell lung carcinoma); concomitant anti-neuronal or other autoantibodies were present in 8 patients. ConclusionsAutoantibodies to human synapsin Ia and Ib are detectable in a proportion of sera from patients with different psychiatric and neurological disorders, warranting further investigation into the potential pathophysiological relevance of these antibodies.

A sphingosine-1-phosphate receptor type 1 agonist, ASP4058, suppresses intracranial aneurysm through promoting endothelial integrity and blocking macrophage transmigration.

Background and PurposeIntracranial aneurysm (IA), common in the general public, causes lethal subarachnoid haemorrhage on rupture. It is, therefore, of utmost importance to prevent the IA from rupturing. However, there is currently no medical treatment. Recent studies suggest that IA is the result of chronic inflammation in the arterial wall caused by endothelial dysfunction and infiltrating macrophages. The sphingosine‐1‐phosphate receptor type 1 (S1P1 receptor) is present on the endothelium and promotes its barrier function. Here we have tested the potential of an S1P1 agonist, ASP4058, to prevent IA in an animal model.Experimental ApproachThe effects of a selective S1P1 agonist, ASP4058, on endothelial permeability and migration of macrophages across an endothelial cell monolayer were tested in vitro using a Transwell system, and its effects on the size of IAs were evaluated in a rat model of IA.Key ResultsS1P1 receptor was expressed in endothelial cells of human IA lesions and control arterial walls. ASP4058 significantly reduced FITC‐dextran leakage through an endothelial monolayer and suppressed the migration of macrophages across the monolayer in vitro. Oral administration of ASP4058 reduced the vascular permeability, macrophage infiltration and size of the IAs by acting as an S1P1 agonist in the rat model. This effect was mimicked by another two structurally‐unrelated S1P1 agonists.Conclusion and ImplicationsA selective S1P1 agonist is a strong drug candidate for IA treatment as it promotes the endothelial cell barrier and suppresses the trans‐endothelial migration of macrophages in IA lesions.

Genetic and pathogenic difference between Streptococcus agalactiae serotype Ia fish and human isolates

BackgroundStreptococcus agalactiae (GBS) is a common pathogen to infect newborn, woman, the elderly, and immuno-compromised human and fish. 37 fish isolates and 554 human isolates of the GBS in 2007–2012 were investigated in serotypes, antibiotic susceptibility, genetic difference and pathogenicity to tilapia.ResultsPCR serotyping determined serotype Ia for all fish GBS isolates and only in 3.2 % (3–4.2 %) human isolates. For fish isolates, all consisted a plasmid less than 6 kb and belonged to ST7 type, which includes mainly pulsotypes I and Ia, with a difference in a deletion at the largest DNA fragment. These fish isolates were susceptible to all antimicrobials tested in 2007 and increased in non-susceptibility to penicillin, and resistance to clindamycin and ceftriaxone in 2011. Differing in pulsotype and lacking plasmid from fish isolates, human serotype Ia isolates were separated into eight pulsotypes II–IX. Main clone ST23 included pulsotypes II and IIa (50 %) and ST483 consisted of pulsotype III. Human serotype Ia isolates were all susceptible to ceftriaxone and penicillin and few were resistant to erythromycin, azithromycin, clindamycin, levofloxacin and moxifloxacine with the resistant rate of 20 % or less. Using tilapia to analyze the pathogenesis, fish isolates could cause more severe symptoms, including hemorrhage of the pectoral fin, hemorrhage of the gill, and viscous black and common scites, and mortality (>95 % for pulsotype I) than the human isolates (<30 %); however, the fish pulostype Ia isolate 912 with deletion caused less symptoms and the lowest mortality (<50 %) than pulsotype I isolates.ConclusionGenetic, pathogenic, and antimicrobial differences demonstrate diverse origin of human and fish serotype Ia isolates. The pulsotype Ia of fish serotype Ia isolates may be used as vaccine strains to prevent the GBS infection in fish.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0794-4) contains supplementary material, which is available to authorized users.

In Vivo and In Vitro Arsenic Exposition Induces Oxidative Stress in Anterior Pituitary Gland.

Inorganic arsenic (iAs) is at the top of toxic metalloids. Inorganic arsenic-contaminated water consumption is one of the greatest environmental health threats worldwide. Human iAs exposure has been associated with cancers of several organs, neurological disorders, and reproductive problems. Nevertheless, there are no reports describing how iAs affects the anterior pituitary gland. The aim of this study was to investigate the mechanisms involved in iAs-mediated anterior pituitary toxicity both in vivo and in vitro. We showed that iAs administration (from 5 to 100 ppm) to male rats through drinking water increased messenger RNA expression of several oxidative stress-responsive genes in the anterior pituitary gland. Serum prolactin levels diminished, whereas luteinizing hormone (LH) levels were only affected at the higher dose tested. In anterior pituitary cells in culture, 25 µmol/L iAs significantly decreased prolactin release in a time-dependent fashion, whereas LH levels remained unaltered. Cell viability was significantly reduced mainly by apoptosis evidenced by morphological and phosphatidylserine externalization studies. This process is characterized by early depolarization of mitochondrial membrane potential and increased levels of reactive oxygen species. Expression of some key oxidative stress-responsive genes, such as heme oxygenase-1 and metallothionein-1, was also stimulated by iAs exposure. The antioxidant N-acetyl cysteine prevented iAs-induced effects on the expression of oxidative stress markers, prolactin release, and apoptosis. In summary, the present work demonstrates for the first time that iAs reduces prolactin release both in vivo and in vitro and induces apoptosis in anterior pituitary cells, possibly resulting from imbalanced cellular redox status.

Maternal and fetal human leukocyte antigen class Ia and II alleles in severe preeclampsia and eclampsia.

A line of investigations indicate that genes in the human leukocyte antigen (HLA) complex are involved in a successful acceptance of the semiallogeneic fetus during pregnancy. In this study, associations between specific HLA class Ia (HLA-A and -B) and class II (HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1) alleles and the risk of developing severe preeclampsia/eclampsia were investigated in a detailed and large-scale study. In total, 259 women diagnosed with severe preeclampsia or eclampsia and 260 matched control women with no preeclampsia, together with their neonates, were included in the study. HLA genotyping for mothers and neonates was performed using next-generation sequencing. The HLA-DPB1*04:01:01G allele was significantly more frequent (Pc=0.044) among women diagnosed with severe preeclampsia/eclampsia compared with controls, and the DQA1*01:02:01G allele frequency was significantly lower (Pc=0.042) among newborns born by women with severe preeclampsia/eclampsia compared with controls. In mothers with severe preeclampsia/eclampsia, homozygosity was significantly more common compared with controls at the HLA-DPB1 locus (Pc=0.0028). Although the current large study shows some positive results, more studies, also with a functional focus, are needed to further clarify a possible role of the classical HLA genes in preeclampsia.

Are Human Digit Muscles Devoid of Recurrent Inhibition?

Are Human Digit Muscles Devoid of Recurrent Inhibition?

Detection of enzyme activities and their relation to serotypes of bovine and human group B streptococci.

Enzymatic properties of group B streptococci (GBS) serotypes from bovine milk and human routine vaginal specimens were investigated. Out of the 56 human and 66 bovine GBS, 35 and 30 could be classified serologically by a co-agglutination test with type-specific antisera, respectively. Hyaluronidase (HYAL), streptokinase (SK) and protease activities were detected using culture media. HYAL activity was observed mostly in typable human GBS, and serotypes Ia, Ic and II comprised 77.3% of the typable strains producing HYAL. Bovine GBS serotypes II, III and VII comprised 87.5% of typable bovine strains exhibiting HYAL activity. SK activity was detected only in three human GBS. Human GBS serotypes Ia, Ic, II, III, VII and almost all typable bovine GBS strains showed protease activity. β-D-glucosidase activity was frequently observed in human GBS, whereas N-acetyl-β-D-glucosaminidase activity was mostly detected in non-typable GBS from humans. These results indicate that different GBS serotypes could vary in their virulence properties, and bovine and human GBS isolates could not be differentiated by their enzyme activities. Use of the culture media appeared to be a simple-to-apply and useful method for the detection of extracellular enzyme activity such as HYAL, protease and SK.

Identification and validation of a novel microRNA-like molecule derived from a cytoplasmic RNA virus antigenome by bioinformatics and experimental approaches.

BackgroundIt is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs (miRNAs) due to nucleus inaccessibility. Recent studies have described cytoplasmic RNA virus genome-derived miRNAs in West Nile virus (WNV) and Dengue virus (DENV). However, naturally occurring miRNAs derived from the antigenome of a cytoplasmic RNA virus have not been described.MethodsHepatitis A virus (HAV) was served as a model virus to investigate whether the antigenome of a cytoplasmic RNA virus would be processed into miRNAs or miRNA-like small RNAs upon infection. HAV antigenome was queried for putative miRNA precursors (pre-miRNA) with the VMir analyzer program. Mature miRNA prediction was performed using MatureBayes and Bayes-SVM-MiRNA web server v1.0. Finally, multiple experimental approaches, including cloning and sequencing-, RNAi-, plasmid-based miRNA expression- and luciferase reporter assays, were performed to identify and validate naturally occurring viral antigenome-derived miRNAs.ResultsUsing human HAV genotype IA (isolate H2) (HAVH2), a virally encoded miRNA-like small RNA was detected on the antigenome and named hav-miR-N1-3p. Transcription of viral pre-miRNA in KMB17 and HEK293T cells led to mature hav-miR-N1-3p production. In addition, silencing of the miRNA-processing enzyme Dicer or Drosha caused a dramatic reduction in miRNA levels. Furthermore, artificial target of hav-miR-N1-3p was silenced by synthesized viral miRNA mimics and the HAVH2 naturally-derived hav-miR-N1-3p.ConclusionThese results suggested that the antigenome of a cytoplasmic RNA virus could be processed into functional miRNAs. Our findings provide new evidence supporting the hypothesis that cytoplasmic RNA viruses naturally encode miRNAs through cellular miRNA processing machinery.