Identification of the novel HLA-A*02:01:01:251 allele in a candidate bone marrow donor by next-generation sequencing.

A single nucleotide mismatch within intron 1 differentiates HLA-A*02:01:01:251 from the HLA-A*02:01:01:01 allele.

Human Vγ9Vδ2 T cell expansion and their cytotoxic responses against cholangiocarcinoma.

Human Vγ9Vδ2 T lymphocytes are regarded as promising effector cells for cancer immunotherapy since they have the ability to eliminate several tumor cells through non-peptide antigen recognition. However, the cytotoxic function and the mechanism of Vγ9Vδ2 T cells leading to specific killing of cholangiocarcinoma cells are yet to be confirmed. In this study, we established a protocol for ex vivo expansion of Vγ9Vδ2 T cells from healthy donors' peripheral blood mononuclear cells by culture with zoledronate and addition of IL-2, and IL-15 or IL-18 or neither. Testing the cytotoxic capacity of cultured Vγ9Vδ2 T cells against cholangiocarcinoma cell lines showed higher reactivity than against control cells. Surface expression of CD107 was detected on the Vγ9Vδ2 T cells, suggesting that these cells limit in vitro growth of cholangiocarcinoma cells via degranulation of the perforin and granzyme pathway. Analysis of molecular signaling was used to demonstrate expression of pro- and anti-survival genes and a panel of cytokine genes in Vγ9Vδ2 T cells. We found that in the presence of either IL-15 or IL-18, levels of caspase 3 were significantly reduced. Also, IL-15 and IL-18 stimulated cells contained cytotoxicity against cholangiocarcinoma cells, suggesting that stimulated Vγ9Vδ2 T cells may provide a feasible therapy for cholangiocarcinoma.

KIR2DS1 and KIR2DL1-C245 Dominantly Repress NK Cell Degranulation Triggered by Monoclonal or Bispecific Antibodies, whereas Education by Uptuning Inhibitory Killer Ig-related Receptors Exerts No Advantage in Ab-dependent Cellular Cytotoxicity.

NK cell responsiveness to target cells is tuned by interactions between inhibitory NK cell receptors and their cognate HLA class I ligands in a process termed "NK cell education." Previous studies addressing the role for NK cell education in Ab-dependent cellular cytotoxicity (ADCC) show ambiguous results and do not encompass full educational resolution. In this study, we systematically characterized human NK cell CD16-triggered degranulation toward defined human tumor cell lines in the presence of either the mAb rituximab or a recently developed CD34xCD16 bispecific killer engager. Despite positive correlation between killer Ig-related receptor (KIR)-mediated education and CD16 expression, NK cells educated by one or even two inhibitory KIRs did not perform better in terms of ADCC than uneducated NK cells in either missing-self or KIR-ligand matched settings at saturating Ab concentrations. Instead, NKG2A+ NK cells consistently showed more potent ADCC in the missing-self context despite lower levels of CD16 expression. KIR2DS1+ NK cells demonstrated dampened ADCC in both the missing-self and KIR-ligand matched settings, even in the presence of its ligand HLA C2. The lower response by KIR2DS1+ NK cells was also observed when stimulated with a bispecific killer engager. Surprisingly, repression of ADCC was also observed by NKG2A+ NK cells coexpressing the inhibitory KIR2DL1-C245 receptor that confers weak education. In conclusion, our study suggests that NK cell education by inhibitory KIRs does not augment ADCC per se, whereas expression of KIR2DS1 and KIR2DL1-C245 dominantly represses ADCC. These insights add to the fundamental understanding of NK cells and may have implications for their therapeutic use.

Deep histopathology genotype-phenotype analysis of focal cortical dysplasia type II differentiates between the GATOR1-altered autophagocytic subtype IIa and MTOR-altered migration deficient subtype IIb.

Focal cortical dysplasia type II (FCDII) is the most common cause of drug-resistant focal epilepsy in children. Herein, we performed a deep histopathology-based genotype-phenotype analysis to further elucidate the clinico-pathological and genetic presentation of FCDIIa compared to FCDIIb. Seventeen individuals with histopathologically confirmed diagnosis of FCD ILAE Type II and a pathogenic variant detected in brain derived DNA whole-exome sequencing or mTOR gene panel sequencing were included in this study. Clinical data were directly available from each contributing centre. Histopathological analyses were performed from formalin-fixed, paraffin-embedded tissue samples using haematoxylin-eosin and immunohistochemistry for NF-SMI32, NeuN, pS6, p62, and vimentin. Ten individuals carried loss-of-function variants in the GATOR1 complex encoding genes DEPDC5 (n = 7) and NPRL3 (n = 3), or gain-of-function variants in MTOR (n = 7). Whereas individuals with GATOR1 variants only presented with FCDIIa, i.e., lack of balloon cells, individuals with MTOR variants presented with both histopathology subtypes, FCDIIa and FCDIIb. Interestingly, 50% of GATOR1-positive cases showed a unique and predominantly vacuolizing phenotype with p62 immunofluorescent aggregates in autophagosomes. All cases with GATOR1 alterations had neurosurgery in the frontal lobe and the majority was confined to the cortical ribbon not affecting the white matter. This pattern was reflected by subtle or negative MRI findings in seven individuals with GATOR1 variants. Nonetheless, all individuals were seizure-free after surgery except four individuals carrying a DEPDC5 variant. We describe a yet underrecognized genotype-phenotype correlation of GATOR1 variants with FCDIIa in the frontal lobe. These lesions were histopathologically characterized by abnormally vacuolizing cells suggestive of an autophagy-altered phenotype. In contrast, individuals with FCDIIb and brain somatic MTOR variants showed larger lesions on MRI including the white matter, suggesting compromised neural cell migration.

Selection of mutant Listeria phages under food-relevant conditions can enhance application potential.

Bacteriophages are viruses that infect and kill bacteria. Currently, phage products are available for the control of the pathogen Listeria monocytogenes in food products in the United States. In this study, we explore whether experimental evolution can be used to generate phages with improved abilities to function under specific food-relevant conditions. Ultra-pasteurized oat and whole milk were chosen as test matrices as they represent different food groups, yet have similar physical traits and macronutrient composition. We showed that (i) wild-type phage LP-125 infection kinetics are different in the two matrices and (ii) LP-125 has a significantly higher burst size in oat milk. From this, we attempted to evolve LP-125 to have improved infection kinetics in whole milk. Ancestral LP-125 was passaged through 10 rounds of amplification in milk conditions. Plaque-purified DNA samples from milk-selected phages were isolated and sequenced, and mutations present in the isolated phages were identified. We found two nonsynonymous substitutions in LP125_108 and LP125_112 genes, which encode putative baseplate-associated glycerophosphoryl diester phosphodiesterase and baseplate protein, respectively. Protein structural modeling showed that the substituted amino acids in the mutant phages are predicted to localize to surface-exposed helices on the corresponding structures, which might affect the surface charge of proteins and their interaction with the bacterial cell. The phage containing the LP125_112 mutation adsorbed significantly faster than the ancestral phage in both oat and whole milk. Follow-up experiments suggest that fat content may be a key factor for the expression of the phenotype of this mutation. IMPORTANCE Bacteriophages are one of the tools available to control the foodborne pathogen, Listeria monocytogenes. Phage products must work under a broad range of food conditions to be an effective control for L. monocytogenes. Here, we show that the experimental evolution of phages can be used to generate new phages with phenotypes useful under specific conditions. We used this approach to select for a mutant phage that more efficiently binds to L. monocytogenes that is grown in whole milk and oat milk. We show that the fat content of these milks is necessary for the expression of this phenotype. Our findings show that experimental evolution can be used to select for improved phages with better performance under specific conditions. This approach has the potential to support the development of condition-specific phage-based biocontrols in the food industry.

The role of creatine kinase in distinguishing generalized tonic-clonic seizures from psychogenic non-epileptic seizures (PNES) and syncope: a retrospective study and meta-analysis of 1300 patients.

BACKGROUND/AIM: As the clinical differentiation between epileptic seizures, psychogenic non-epileptic seizures (PNES), and syncope depends mainly on a detailed report of the event, which may not be available, an objective assessment of a potential biochemical analysis is needed. We aimed to investigate whether serum creatine kinase (CK) could be used to differentiate epileptic seizure from PNES and syncope and to assess the strength of evidence present. METHODS: We directed a retrospective cohort study coupled with a systematic review and meta-analysis of studies that measured CK in patients with epilepsy, PNES, syncope, and healthy controls. RESULTS: The cohort study, which traced 202 patients, showed that the CK level was significantly higher 48 h after the event in the epilepsy group versus patients with syncope (p < 0.01) Along with 1086 patients obtained through a database search for meta-analysis, CK level compared to different types of seizures from PNES was higher in epileptic seizure patients with a mean difference of 568.966 mIU/ml (95% CI 166.864, 971.067). The subgroup analysis of CK showed that it was higher in GTCS compared to syncope with a mean difference of 125.39 mIU/ml (95% CI 45.25, 205.52). DISCUSSION: Increased serum levels of CK have been associated mainly with epileptic seizures in relation to non-epileptic events. However, further studies would try to explore the variation in measurements and any other potential diagnostic marker. CONCLUSION: The cohort study shows that the CK level in epilepsy seizures is higher after 48 h from the event compared to syncope. Moreover, the meta-analysis results show the present diagnostic utility of CK and its importance to be used in accordance with a detailed report of the event.

More than mcr: canonical plasmid- and transposon-encoded mobilized colistin resistance genes represent a subset of phosphoethanolamine transferases.

Mobilized colistin resistance genes (mcr) may confer resistance to the last-resort antimicrobial colistin and can often be transmitted horizontally. mcr encode phosphoethanolamine transferases (PET), which are closely related to chromosomally encoded, intrinsic lipid modification PET (i-PET; e.g., EptA, EptB, CptA). To gain insight into the evolution of mcr within the context of i-PET, we identified 69,814 MCR-like proteins present across 256 bacterial genera (obtained by querying known MCR family representatives against the National Center for Biotechnology Information [NCBI] non-redundant protein database via protein BLAST). We subsequently identified 125 putative novel mcr-like genes, which were located on the same contig as (i) ≥1 plasmid replicon and (ii) ≥1 additional antimicrobial resistance gene (obtained by querying the PlasmidFinder database and NCBI's National Database of Antibiotic Resistant Organisms, respectively, via nucleotide BLAST). At 80% amino acid identity, these putative novel MCR-like proteins formed 13 clusters, five of which represented putative novel MCR families. Sequence similarity and a maximum likelihood phylogeny of mcr, putative novel mcr-like, and ipet genes indicated that sequence similarity was insufficient to discriminate mcr from ipet genes. A mixed-effect model of evolution (MEME) indicated that site- and branch-specific positive selection played a role in the evolution of alleles within the mcr-2 and mcr-9 families. MEME suggested that positive selection played a role in the diversification of several residues in structurally important regions, including (i) a bridging region that connects the membrane-bound and catalytic periplasmic domains, and (ii) a periplasmic loop juxtaposing the substrate entry tunnel. Moreover, eptA and mcr were localized within different genomic contexts. Canonical eptA genes were typically chromosomally encoded in an operon with a two-component regulatory system or adjacent to a TetR-type regulator. Conversely, mcr were represented by single-gene operons or adjacent to pap2 and dgkA, which encode a PAP2 family lipid A phosphatase and diacylglycerol kinase, respectively. Our data suggest that eptA can give rise to "colistin resistance genes" through various mechanisms, including mobilization, selection, and diversification of genomic context and regulatory pathways. These mechanisms likely altered gene expression levels and enzyme activity, allowing bona fide eptA to evolve to function in colistin resistance.

Escherichia coli B-Strains Are Intrinsically Resistant to Colistin and Not Suitable for Characterization and Identification of mcr Genes.

Antimicrobial resistance is an increasing threat to human and animal health. Due to the rise of multi-, extensive, and pandrug resistance, last resort antibiotics, such as colistin, are extremely important in human medicine. While the distribution of colistin resistance genes can be tracked through sequencing methods, phenotypic characterization of putative antimicrobial resistance (AMR) genes is still important to confirm the phenotype conferred by different genes. While heterologous expression of AMR genes (e.g., in Escherichia coli) is a common approach, so far, no standard methods for heterologous expression and characterization of mcr genes exist. E. coli B-strains, designed for optimum protein expression, are frequently utilized. Here, we report that four E. coli B-strains are intrinsically resistant to colistin (MIC 8-16 µg/mL). The three tested B-strains that encode T7 RNA polymerase show growth defects when transformed with empty or mcr-expressing pET17b plasmids and grown in the presence of IPTG; K-12 or B-strains without T7 RNA polymerase do not show these growth defects. E. coli SHuffle T7 express carrying empty pET17b also skips wells in colistin MIC assays in the presence of IPTG. These phenotypes could explain why B-strains were erroneously reported as colistin susceptible. Analysis of existing genome data identified one nonsynonymous change in each pmrA and pmrB in all four E. coli B-strains; the E121K change in PmrB has previously been linked to intrinsic colistin resistance. We conclude that E. coli B-strains are not appropriate heterologous expression hosts for identification and characterization of mcr genes. IMPORTANCE Given the rise in multidrug, extensive drug, and pandrug resistance in bacteria and the increasing use of colistin to treat human infections, occurrence of mcr genes threatens human health, and characterization of these resistance genes becomes more important. We show that three commonly used heterologous expression strains are intrinsically resistant to colistin. This is important because these strains have previously been used to characterize and identify new mobile colistin resistance (mcr) genes. We also show that expression plasmids (i.e., pET17b) without inserts cause cell viability defects when carried by B-strains with T7 RNA polymerase and grown in the presence of IPTG. Our findings are important as they will facilitate improved selection of heterologous strains and plasmid combinations for characterizing AMR genes, which will be particularly important with a shift to Culture-independent diagnostic tests where bacterial isolates become increasingly less available for characterization.

Clinical Features, Neuropathology, and Surgical Outcome in Patients With Refractory Epilepsy and Brain Somatic Variants in the SLC35A2 Gene.

BACKGROUND AND OBJECTIVES: The SLC35A2 gene, located at chromosome Xp11.23, encodes for a uridine diphosphate-galactose transporter. We describe clinical, genetic, neuroimaging, EEG, and histopathologic findings and assess possible predictors of postoperative seizure and cognitive outcome in 47 patients with refractory epilepsy and brain somatic SLC35A2 gene variants. METHODS: This is a retrospective multicenter study where we performed a descriptive analysis and classical hypothesis testing. We included the variables of interest significantly associated with the outcomes in the generalized linear models. RESULTS: Two main phenotypes were associated with brain somatic SLC35A2 variants: (1) early epileptic encephalopathy (EE, 39 patients) with epileptic spasms as the predominant seizure type and moderate to severe intellectual disability and (2) drug-resistant focal epilepsy (DR-FE, 8 patients) associated with normal/borderline cognitive function and specific neuropsychological deficits. Brain MRI was abnormal in all patients with EE and in 50% of those with DR-FE. Histopathology review identified mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy in 44/47 patients and was inconclusive in 3. The 47 patients harbored 42 distinct mosaic SLC35A2 variants, including 14 (33.3%) missense, 13 (30.9%) frameshift, 10 (23.8%) nonsense, 4 (9.5%) in-frame deletions/duplications, and 1 (2.4%) splicing variant. Variant allele frequencies (VAFs) ranged from 1.4% to 52.6% (mean VAF: 17.3 ± 13.5). At last follow-up (35.5 ± 21.5 months), 30 patients (63.8%) were in Engel Class I, of which 26 (55.3%) were in Class IA. Cognitive performances remained unchanged in most patients after surgery. Regression analyses showed that the probability of achieving both Engel Class IA and Class I outcomes, adjusted by age at seizure onset, was lower when the duration of epilepsy increased and higher when postoperative EEG was normal or improved. Lower brain VAF was associated with improved postoperative cognitive outcome in the analysis of associations, but this finding was not confirmed in regression analyses. DISCUSSION: Brain somatic SLC35A2 gene variants are associated with 2 main clinical phenotypes, EE and DR-FE, and a histopathologic diagnosis of MOGHE. Additional studies will be needed to delineate any possible correlation between specific genetic variants, mutational load in the epileptogenic tissue, and surgical outcomes.

Gamma delta T-cell reconstitution after allogeneic HCT: A platform for cell therapy.

Allogeneic Hematopoietic stem cell transplantation (allo-HCT) is a curative platform for several hematological diseases. Despite its therapeutic benefits, the profound immunodeficiency associated with the transplant procedure remains a major challenge that renders patients vulnerable to several complications. Today, It is well established that a rapid and efficient immune reconstitution, particularly of the T cell compartment is pivotal to both a short-term and a long-term favorable outcome. T cells expressing a TCR heterodimer comprised of gamma (γ) and delta (δ) chains have received particular attention in allo-HCT setting, as a large body of evidence has indicated that γδ T cells can exert favorable potent anti-tumor effects without inducing severe graft versus host disease (GVHD). However, despite their potential role in allo-HCT, studies investigating their detailed reconstitution in patients after allo-HCT are scarce. In this review we aim to shed lights on the current literature and understanding of γδ T cell reconstitution kinetics as well as the different transplant-related factors that may influence γδ reconstitution in allo-HCT. Furthermore, we will present data from available reports supporting a role of γδ cells and their subsets in patient outcome. Finally, we discuss the current and future strategies to develop γδ cell-based therapies to exploit the full immunotherapeutic potential of γδ cells in HCT setting.

SARS-CoV-2 (COVID-19)-specific T cell and B cell responses in convalescent rheumatoid arthritis: Monozygotic twins pair case observation.

Rheumatoid arthritis (RA) patients present higher risk of SARS-CoV-2 infection (COVID-19), and proper management of the disease in this population requires a better understanding of how the immune system controls the virus. We analyzed the T cell and B cell phenotypes, and their repertoire in a pair of monozygotic twins with RA mismatched for COVID-19 infection. Twin- was not infected, while Twin+ was infected and effectively controlled the infection. We found no significant changes on the αß T cell composition, while γδ T cells and B cells presented considerable expansion of memory population in Twin+ and robust T/B cell responses to several SARS-CoV-2 peptides. T cell receptor ß/γ-chain and immunoglobulin heavy chain next-generation sequencing depicted a remarkable higher diversity in Twin+ compared with Twin-, despite no significant changes being found in variable/joining family usage. Repertoire overlap analyses showed that, although being identical twins, very few clones were shared between them, indicating that COVID-19 may lead to deep changes on the immune cell repertoire in RA patients. Altogether, our results indicate that RA patients may develop robust and persistent COVID-19-specific T/B cell responses; γδ T cells and B cells may play a key role in the management of COVID-19 in RA, and the infection may lead to a profound reshaping of immune cell receptor specificities.

Monitoring the Microevolution of Salmonella enterica in Healthy Dairy Cattle Populations at the Individual Farm Level Using Whole-Genome Sequencing.

Livestock represent a possible reservoir for facilitating the transmission of the zoonotic foodborne pathogen Salmonella enterica to humans; there is also concern that strains can acquire resistance to antimicrobials in the farm environment. Here, whole-genome sequencing (WGS) was used to characterize Salmonella strains (n = 128) isolated from healthy dairy cattle and their associated environments on 13 New York State farms to assess the diversity and microevolution of this important pathogen at the level of the individual herd. Additionally, the accuracy and concordance of multiple in silico tools are assessed, including: (i) two in silico serotyping tools, (ii) combinations of five antimicrobial resistance (AMR) determinant detection tools and one to five AMR determinant databases, and (iii) one antimicrobial minimum inhibitory concentration (MIC) prediction tool. For the isolates sequenced here, in silico serotyping methods outperformed traditional serotyping and resolved all un-typable and/or ambiguous serotype assignments. Serotypes assigned in silico showed greater congruency with the Salmonella whole-genome phylogeny than traditional serotype assignments, and in silico methods showed high concordance (99% agreement). In silico AMR determinant detection methods additionally showed a high degree of concordance, regardless of the pipeline or database used (≥98% agreement among susceptible/resistant assignments for all pipeline/database combinations). For AMR detection methods that relied exclusively on nucleotide BLAST, accuracy could be maximized by using a range of minimum nucleotide identity and coverage thresholds, with thresholds of 75% nucleotide identity and 50-60% coverage adequate for most pipeline/database combinations. In silico characterization of the microevolution and AMR dynamics of each of six serotype groups (S. Anatum, Cerro, Kentucky, Meleagridis, Newport, Typhimurium/Typhimurium variant Copenhagen) revealed that some lineages were strongly associated with individual farms, while others were distributed across multiple farms. Numerous AMR determinant acquisition and loss events were identified, including the recent acquisition of cephalosporin resistance-conferring bla CMY- and bla CTX-M-type beta-lactamases. The results presented here provide high-resolution insight into the temporal dynamics of AMR Salmonella at the scale of the individual farm and highlight both the strengths and limitations of WGS in tracking zoonotic pathogens and their associated AMR determinants at the livestock-human interface.

Clinical characteristics and postoperative seizure outcome in patients with mild malformation of cortical development and oligodendroglial hyperplasia.

OBJECTIVE: We describe for the first time clinical characteristics in a series of 20 pre-surgically investigated patients with mild malformation of cortical development with oligodendroglial hyperplasia (MOGHE) who were operated on in our epilepsy center. We aimed to better diagnose this entity and help surgical planning. METHODS: Data on 20 patients with histologically confirmed MOGHE were retrospectively evaluated as to age at epilepsy onset and operation, seizure semiology, magnetic resonance imaging (MRI) localization, electroencephalography (EEG) patterns, extent of the operative resection, and postoperative seizure outcome. RESULTS: Epilepsy began mainly in early childhood; however, symptoms did not manifest until adolescence or adulthood in 30% of patients. All patients had pathologic MRI findings. In 45% of patients the lesion was initially overlooked. Most commonly, the lesion was seen in the frontal lobe. Seizure semiology was characterized as follows: (1) epileptic spasms at epilepsy onset were common and (2) nocturnal hyperkinetic seizures during the course of the disease were rare. EEG always showed frequent interictal epileptic discharges. Two peculiar patterns were observed: (1) during sleep stage I-II, sub-continuous repetitive (0.5-1.5/s) unilateral plump spike/polyspike slow waves were seen and (2) during wakefulness, unilateral paroxysms of 2-2.5/s spike-wave complexes occurred. In total, 60% of patients were seizure-free 1 year postoperatively. Postoperative seizure outcome was positively correlated with the extent of resection, age at epilepsy onset, and age at operation. Postoperative long-term outcomes remained stable in patients undergoing larger operations. SIGNIFICANCE: MRI, EEG, and semiology already contribute to the diagnosis of probable MOGHE preoperatively. Because postoperative seizure outcomes depend on the extent of the resection, prior knowledge of a probable MOGHE helps to plan the resection and balance the risks and benefits of such an intervention. In patients undergoing larger operations, epilepsy surgery achieved good postoperative results; the first long-term outcome data were stable in these patients.

Development of a database and standardized approach for rpoB sequence-based subtyping and identification of aerobic spore-forming Bacillales.

Aerobic spore-forming Bacillales are a highly diverse and ubiquitous group that includes organisms that cause foodborne illnesses and food spoilage. Classical microbiological and biochemical identification of members of the order Bacillales represents a challenge due to the diversity of organisms in this group as well as the fact that the phenotypic-based taxonomic assignment of some named species in this group is not consistent with their phylogenomic characteristics. DNA-sequencing-based tools, on the other hand, can be fast and cost-effective, and can provide for a more reliable identification and characterization of Bacillales isolates. In comparison to 16S rDNA, rpoB was shown to better discriminate between Bacillales isolates and to allow for improved taxonomic assignment to the species level. However, the lack of a publicly accessible rpoB database, as well as the lack of standardized protocols for rpoB-based typing and strain identification, is a major challenge. Here, we report (i) the curation of a DNA sequence database for rpoB-based subtype classification of Bacillales isolates; (ii) the development of standardized protocols for generating rpoB sequence data, and a scheme for rpoB-based initial taxonomic identification of Bacillales isolates at the species level; and (iii) the integration of the database in a publicly accessible online platform that allows for the analysis of rpoB sequence data from uncharacterized Bacillales isolates. Specifically, we curated a database of DNA sequences for a 632-nt internal variable region within the rpoB gene from representative Bacillales reference type strains and a large number of isolates that we have previously isolated and characterized through multiple projects. As of May 21, 2021, the rpoB database contained more than 8350 rpoB sequences representing 1902 distinct rpoB allelic types that can be classified into 160 different genera. The database also includes 1129 rpoB sequences for representative Bacillales reference type strains as available on May 21, 2021 in the NCBI database. The rpoB database is integrated into the online Food Microbe Tracker platform (www.foodmicrobetracker.com) and can be queried using the integrated BLAST tool to initially subtype and taxonomically identify aerobic and facultative anaerobic spore-formers. While whole-genome sequencing is increasingly used in bacterial taxonomy, the rpoB sequence-based identification scheme described here provides a valuable tool as it allows for rapid and cost-effective initial isolate characterization, which can help to identify and characterize foodborne pathogens and food spoilage bacteria. In addition, the database and primers described here can also be adopted for metagenomics approaches that include rpoB as a target, improving discriminatory power and identification over what can be achieved using 16S rDNA as a target.

Vitamin D status and healthy Egyptian adolescents: Where do we stand?

ABSTRACT: Vitamin D deficiency is a worldwide public health problem. Low vitamin D and its consequences among children and adolescents could be considered as one of the most important health-related problems. This study aimed to estimate the prevalence of vitamin D deficiency in healthy Egyptian adolescents and investigate factors associated with vitamin D status.A cross-sectional study was conducted on 572 school children (270 males and 302 females) aged 14 to 18 years, who were randomly selected from high schools in one governorate in Egypt. Data were collected through a self-administered questionnaire. Vitamin D level, serum calcium, phosphorus, alkaline phosphates were measured.Vitamin D deficiency was almost present in all the studied Egyptian healthy adolescents (99%), 94.8% had vitamin D deficiency and 4.2% had vitamin D insufficiency. Girls had a higher prevalence of vitamin D deficiency than boys. There was a significant association between lack of physical activity, sun exposure, and vitamin D deficiency.Vitamin D deficiency and insufficiency are highly prevalent. In sunny countries, the special pattern of conservative clothing and the lack of outdoor physical activity might be the underlying factors for the high prevalence in females. Vitamin D supplementation seems to be mandatory to halt the problem.

Revisiting the Role of γδ T Cells in Anti-CMV Immune Response after Transplantation.

Gamma delta (γδ) T cells form an unconventional subset of T lymphocytes that express a T cell receptor (TCR) consisting of γ and δ chains. Unlike conventional αß T cells, γδ T cells share the immune signature of both the innate and the adaptive immunity. These features allow γδ T cells to act in front-line defense against infections and tumors, rendering them an attractive target for immunotherapy. The role of γδ T cells in the immune response to cytomegalovirus (CMV) has been the focus of intense research for several years, particularly in the context of transplantation, as CMV reactivation remains a major cause of transplant-related morbidity and mortality. Therefore, a better understanding of the mechanisms that underlie CMV immune responses could enable the design of novel γδ T cell-based therapeutic approaches. In this regard, the advent of next-generation sequencing (NGS) and single-cell TCR sequencing have allowed in-depth characterization of CMV-induced TCR repertoire changes. In this review, we try to shed light on recent findings addressing the adaptive role of γδ T cells in CMV immunosurveillance and revisit CMV-induced TCR reshaping in the era of NGS. Finally, we will demonstrate the favorable and unfavorable effects of CMV reactive γδ T cells post-transplantation.

Alternative σ Factors Regulate Overlapping as Well as Distinct Stress Response and Metabolic Functions in Listeria monocytogenes under Stationary Phase Stress Condition.

Listeria monocytogenes can regulate and fine-tune gene expression, to adapt to diverse stress conditions encountered during foodborne transmission. To further understand the contributions of alternative sigma (σ) factors to the regulation of L. monocytogenes gene expression, RNA-Seq was performed on L. monocytogenes strain 10403S and five isogenic mutants (four strains bearing in-frame null mutations in three out of four alternative σ factor genes, ΔCHL, ΔBHL, ΔBCL, and ΔBCH, and one strain bearing null mutations in all four genes, ΔBCHL), grown to stationary phase. Our data showed that 184, 35, 34, and 20 genes were positively regulated by σB, σL, σH, and σC (posterior probability > 0.9 and Fold Change (FC) > 5.0), respectively. Moreover, σB-dependent genes showed the highest FC (based on comparisons between the ΔCHL and the ΔBCHL strain), with 44 genes showing an FC > 100; only four σL-dependent, and no σH- or σC-dependent genes showed FC >100. While σB-regulated genes identified in this study are involved in stress-associated functions and metabolic pathways, σL appears to largely regulate genes involved in a few specific metabolic pathways, including positive regulation of operons encoding phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs). Overall, our data show that (i) σB and σL directly and indirectly regulate genes involved in several energy metabolism-related functions; (ii) alternative σ factors are involved in complex regulatory networks and appear to have epistatic effects in stationary phase cells; and (iii) σB regulates multiple stress response pathways, while σL and σH positively regulate a smaller number of specific pathways.

Characterization of the roles of activated charcoal and Chelex in the induction of PrfA regulon expression in complex medium.

The foodborne pathogen Listeria monocytogenes is able to survive across a wide range of intra- and extra-host environments by appropriately modulating gene expression patterns in response to different stimuli. Positive Regulatory Factor A (PrfA) is the major transcriptional regulator of virulence gene expression in L. monocytogenes. It has long been known that activated charcoal is required to induce the expression of PrfA-regulated genes in complex media, such as Brain Heart Infusion (BHI), but not in chemically defined media. In this study, we show that the expression of the PrfA-regulated hly, which encodes listeriolysin O, is induced 5- and 8-fold in L. monocytogenes cells grown in Chelex-treated BHI (Ch-BHI) and in the presence of activated charcoal (AC-BHI), respectively, relative to cells grown in BHI medium. Specifically, we show that metal ions present in BHI broth plays a role in the reduced expression of the PrfA regulon. In addition, we show that expression of hly is induced when the levels of bioavailable extra- or intercellular iron are reduced. L. monocytogenes cells grown Ch-BHI and AC-BHI media showed similar levels of resistance to the iron-activated antibiotic, streptonigrin, indicating that activated charcoal reduces the intracellular labile iron pool. Metal depletion and exogenously added glutathione contributed synergistically to PrfA-regulated gene expression since glutathione further increased hly expression in metal-depleted BHI but not in BHI medium. Analyses of transcriptional reporter fusion expression patterns revealed that genes in the PrfA regulon are differentially expressed in response to metal depletion, metal excess and exogenous glutathione. Our results suggest that metal ion abundance plays a role in modulating expression of PrfA-regulated virulence genes in L. monocytogenes.

The Bacillus subtilis monothiol bacilliredoxin BrxC (YtxJ) and the Bdr (YpdA) disulfide reductase reduce S-bacillithiolated proteins.

The bacterial cytosol is generally a reducing environment with protein cysteine residues maintained in their thiol form. The low molecular weight thiol bacillithiol (BSH) serves as a general thiol reductant, analogous to glutathione, in a wide range of bacterial species. Proteins modified by disulfide bond formation with BSH (S-bacillithiolation) are reduced by the action of bacilliredoxins, BrxA and BrxB. Here, the YtxJ protein is identified as a monothiol bacilliredoxin, renamed BrxC, and is implicated in BSH removal from oxidized cytosolic proteins, including the glyceraldehyde 3-phosphate dehydrogenases GapA and GapB. BrxC can also debacillithiolate the mixed disulfide form of the bacilliredoxin BrxB. Bdr is a thioredoxin reductase-like flavoprotein with bacillithiol-disulfide (BSSB) reductase activity. Here, Bdr is shown to additionally function as a bacilliredoxin reductase. Bdr and BrxB function cooperatively to debacillithiolate OhrR, a transcription factor regulated by S-bacillithiolation on its sole cysteine residue. Collectively, these results expand our understanding of the BSH redox network comprised of three bacilliredoxins and a BSSB reductase that serve to counter the widespread protein S-bacillithiolation that results from conditions of disulfide stress.

Characterization of ascites- and tumor-infiltrating γδ T cells reveals distinct repertoires and a beneficial role in ovarian cancer.

The role of γδ T cells in antitumor immunity has been under investigation for the past two decades, but little is known about their contribution to clinical outcomes in patients. Here, we set out to define the clonotypic, phenotypic, and functional features of γδ T cells in peripheral blood, ascites, and metastatic tumor tissue from patients with advanced epithelial ovarian cancer. T cell receptor (TCR) sequencing of the γ chain revealed that tumor-infiltrating γδ T cells have a unique and skewed repertoire with high TCR diversity and low clonality. In contrast, ascites-derived γδ T cells presented a lower TCR diversity and higher clonality, suggesting a TCR-dependent clonal focusing at this site. Further investigation showed that tumor samples had abundant γδ T cells with a tissue-resident, activation-associated phenotype, less usage of Vγ9 and an impaired response to adaptive-associated stimuli, implying an innate-like activation pathway, rather than an adaptive TCR-engaging pathway, at these tumor sites. Furthermore, high γδ T cell cytokine responsiveness upon stimulation was associated with a favorable outcome for patients in terms of both overall survival and reduced residual tumor burden after primary surgery. Last, the functionality of γδ T cells and patient survival were negatively affected by the proportions of CD39-expressing T cells, highlighting the potential of CD39 as a target to improve γδ T cell responses and unleash their antitumor capabilities.