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A diverse T cell receptor (TCR) repertoire is essential for protection against a variety of pathogens, and TCR repertoire size is believed to decline with age. However, the precise size of human TCR repertoires, in both total and subsets of T cells, as well as their changes with age, are not fully characterized. We conducted a longitudinal analysis of the human blood TCR[alpha] and TCR[beta] repertoire of [CD4.sup.+] and [CD8.sup.+] T cell subsets using a unique molecular identifier-based (UMI-based) RNA-seq method. Thorough analysis of 1.9 x [10.sup.8] T cells yielded the lower estimate of TCR repertoire richness in an adult at 3.8 x [10.sup.8]. Alterations of the TCR repertoire with age were observed in all 4 subsets of T cells. The greatest reduction was observed in naive [CD8.sup.+] T cells, while the greatest clonal expansion was in memory [CD8.sup.+] T cells, and the highest increased retention of TCR sequences was in memory [CD8.sup.+] T cells. Our results demonstrated that age-related TCR repertoire attrition is subset specific and more profound for [CD8.sup.+] than [CD4.sup.+] T cells, suggesting that aging has a more profound effect on cytotoxic as opposed to helper T cell functions. This may explain the increased susceptibility of older adults to novel infections., Introduction Naive T cells produced by the thymus have the potential to recognize any pathogen, whereas memory T cells are generated from a past immunological response and offer long-lasting protection [...]

Abstract Background E. coli B (BL21), unlike E.coli K-12 (JM109) is insensitive to glucose concentration and, therefore, grows faster and produces less acetate than E. coli K-12, especially when growing to high cell densities at high glucose concentration. By performing genomic analysis, it was demonstrated that the cause of this difference in sensitivity to the glucose concentration is the result of the differences in the central carbon metabolism activity. We hypothesized that the global transcription regulator Cra (FruR) is constitutively expressed in E. coli B and may be responsible for the different behaviour of the two strains. To investigate this possibility and better understand the function of Cra in the two strains, cra - negative E. coli B (BL21) and E. coli K-12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and real-time PCR. Results The deletion of the cra gene in E. coli B (BL21) minimally affected the growth and maximal acetate accumulation, while the deletion of the same gene in E.coli K-12 (JM109) caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. ppsA (gluconeogenesis gene), aceBA (the glyoxylate shunt genes) and poxB (the acetate producing gene) were down-regulated in both strains, while acs (acetate uptake gene) was down-regulated only in E.coli B (BL21). These transcriptional differences had little effect on acetate and pyruvate production. Additionally, it was found that the lower growth of E. coli K-12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producing bet operon (betABT). Conclusions The transcriptional changes caused by the deletion of cra gene did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone; rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that cra deletion caused transcription inhibition of the bet operon in E. coli K-12 (JM109) but did not affect this operon transcription in E. coli B (BL21). This property, together with the insensitivity to high glucose concentrations, makes this the E. coli B (BL21) strain more resistant to environmental changes.

The signal that triggers the expression of the ace operon and, in turn, the transition of central metabolism's architecture from acetogenic to gluconeogenic in Escherichia coli remains elusive despite extensive research both in vivo and in vitro. Here, with the aid of flux analysis together with measurements of the enzymic activity of isocitrate lyase (ICL) and its aceA -messenger ribonucleuc acid (mRNA) transcripts, we provide credible evidence suggesting that the expression of the ace operon in E. coli is triggered in response to growth rate-dependent threshold flux-signal of adenosine triphosphate (ATP). Flux analysis revealed that the shortfall in ATP supply observed as the growth rate (⁠|$\mu $|⁠) diminishes from µmax to ≤ 0.43h−1 (⁠|$ \pm 0.02;n4)\ $| is partially redressed by up-regulating flux through succinyl CoA synthetase. Unlike glycerol and glucose, pyruvate cannot feed directly into the two glycolytic ATP-generating reactions catalyzed by phosphoglycerokinase and pyruvate kinase. On the other hand, glycerol, which upon its conversion to D-glyceraldehyde, feeds into the phosphorylation and dephosphorylation parts of glycolysis including the substrate-level phosphorylation-ATP generating reactions, thus preventing ATP flux from dropping to the critical threshold signal required to trigger the acetate-diauxic switch until glycerol is fully consumed. The mRNA transcriptional patterns of key gluconeogenic enzymes, namely, ackA , acetate kinase; pta , phosphotransacetylase; acs , acetyl CoA synthetase and aceA , ICL, suggest that the pyruvate phenotype is better equipped than the glycerol phenotype for the switch from acetogenic to gluconeogenic metabolism. [ABSTRACT FROM AUTHOR]

To evaluate the expression of immune checkpoint genes, their concordance with expression of IFNγ, and to identify potential novel ICP related genes (ICPRG) in colorectal cancer (CRC), the biological connectivity of six well documented ("classical") ICPs (CTLA4, PD1, PDL1, Tim3, IDO1, and LAG3) with IFNγ and its co-expressed genes was examined by NGS in 79 CRC/healthy colon tissue pairs. Identification of novel IFNγ- induced molecules with potential ICP activity was also sought. In our study, the six classical ICPs were statistically upregulated and correlated with IFNγ, CD8A, CD8B, CD4, and 180 additional immunologically related genes in IFNγ positive (FPKM > 1) tumors. By ICP co-expression analysis, we also identified three IFNγ-induced genes [(IFNγ-inducible lysosomal thiol reductase (IFI30), guanylate binding protein1 (GBP1), and guanylate binding protein 4 (GBP4)] as potential novel ICPRGs. These three genes were upregulated in tumor compared to normal tissues in IFNγ positive tumors, co-expressed with CD8A and had relatively high abundance (average FPKM = 362, 51, and 25, respectively), compared to the abundance of the 5 well-defined ICPs (Tim3, LAG3, PDL1, CTLA4, PD1; average FPKM = 10, 9, 6, 6, and 2, respectively), although IDO1 is expressed at comparably high levels (FPKM = 39). We extended our evaluation by querying the TCGA database which revealed the commonality of IFNγ dependent expression of the three potential ICPRGs in 638 CRCs, 103 skin cutaneous melanomas (SKCM), 1105 breast cancers (BC), 184 esophageal cancers (ESC), 416 stomach cancers (STC), and 501 lung squamous carcinomas (LUSC). In terms of prognosis, based on Pathology Atlas data, correlation of GBP1 and GBP4, but not IFI30, with 5-year survival rate was favorable in CRC, BC, SKCM, and STC. Thus, further studies defining the role of IFI30, GBP1, and GBP4 in CRC are warranted. [ABSTRACT FROM AUTHOR]

Naturally-occurring chalcones and synthetic chalcone analogues have been demonstrated to have many biological effects, including anti-inflammatory, anti-malarial, anti-fungal, and anti-oxidant/anti-cancerous activities. Compared to other chalcones, trans-chalcone exhibits superior inhibitory activity in cancer cell growth as shown via in vitro assays, and exerts anti-cancerous effects via the activation of the p53 tumor suppressor protein. Thus, characterization of the specific mechanisms, by which trans-chalcone activates p53, can aid development of new chemotherapeutic drugs that can be used individually or synergistically with other drugs. In this report, we found that trans-chalcone modulates many p53 target genes, HSP40 being the most induced gene in the RNA-Seq data using trans-chalcone-treated cells. CRM1 is also inhibited by trans-chalcone, resulting in the accumulation of p53 and other tumor suppressor proteins in the nucleus. Similar effects were seen using trans-chalcone derivatives. Overall, trans-chalcone could provide a strong foundation for the development of chalcone-based anti-cancer drugs. [ABSTRACT FROM AUTHOR]

ADAMTS13 is a hemostatic enzyme that breaks down pro-thrombotic ultra-large multimers of von Willebrand factor (VWF). The deficiency of ADAMTS13 increases VWF-mediated thrombogenic potential and may lead to thrombotic thrombocytopenic purpura (TTP). Recently, clinical studies have shown the development of acquired TTP after COVID-19 infection and a correlation between low ADAMTS13 plasma levels and increased mortality. As a result, investigating ADAMTS13 as a potential recombinant therapeutic is of broad interest in the field of hematology. ADAMTS13 is considered challenging to purify in its biologically active state. Current purification methods utilize immobilized metal ions, which can interfere with ADAMTS13 metalloprotease activity. For this reason, we optimized an alternative strategy to isolate milligram quantities of highly active recombinant ADAMTS13 (rADAMTS13) from conditioned media after exogenous expression in human cell line, HEK293. HEK293 cells stably expressing C-terminal V5-His-tagged ADAMTS13 were grown in two parallel systems, culture bottles and flasks, for identifying an optimal cultivation strategy. Subsequently, we employed anion exchange followed by anti-V5-tag affinity chromatography to purify rADAMTS13, and extracted rADAMTS13 of high specific activity while preserving its native post-translational modifications. In addition, this process has been optimized and scaled up to produce active rADAMTS13 at levels sufficient for laboratory-scale structural, enzymatic, and biochemical studies. [ABSTRACT FROM AUTHOR]

Very little is known about disease transmission via the gut microbiome. We hypothesized that certain inflammatory features could be transmitted via the gut microbiome and tested this hypothesis using an animal model of inflammatory diseases. Twelve-week-old healthy C57 Bl/6 and Germ-Free (GF) female and male mice were fecal matter transplanted (FMT) under anaerobic conditions with TNFΔARE−/+ donors exhibiting spontaneous Rheumatoid Arthritis (RA) and Inflammatory Bowel Disease (IBD) or with conventional healthy mice control donors. The gut microbiome analysis was performed using 16S rRNA sequencing amplification and bioinformatics analysis with the HIVE bioinformatics platform. Histology, immunohistochemistry, ELISA Multiplex analysis, and flow cytometry were conducted to confirm the inflammatory transmission status. We observed RA and IBD features transmitted in the GF mice cohort, with gut tissue disruption, cartilage alteration, elevated inflammatory mediators in the tissues, activation of CD4/CD8+ T cells, and colonization and transmission of the gut microbiome similar to the donors' profile. We did not observe a change or transmission when conventional healthy mice were FMT with TNFΔARE−/+ donors, suggesting that a healthy microbiome might withstand an unhealthy transplant. These findings show the potential involvement of the gut microbiome in inflammatory diseases. We identified a cluster of bacteria playing a role in this mechanism. [ABSTRACT FROM AUTHOR]

Abstract: Escherichia coli K (JM109) and E. coli B (BL21) are strains used routinely for recombinant protein production. These two strains grow and respond differently to environmental factors such as glucose and oxygen concentration. The differences have been attributed to differential expression of individual genes that constitute certain metabolic pathways that are part of the central carbon metabolism. By implementing a semiparametric algorithm, which is based on a density ratio model, it was possible to compare and quantify the expression patterns of groups of genes involved in several central carbon metabolic pathways. The groups comprising the glyoxylate shunt, TCA cycle, fatty acid, and gluconeogenesis and anaplerotic pathways were expressed differently between the two strains, whereas no differences were apparent for the groups comprising either glycolysis or the pentose phosphate pathway. These results further characterized differences between the two E. coli strains and illustrated the potency of the semiparametric algorithm. [Copyright &y& Elsevier]

Successful adaptation of Escherichia coli to constant environmental challenges demands the operation of a wide range of regulatory control mechanisms, some of which are global, while others are specific. Here, we show that the ability of acetate-negative phenotype strains of E. coli devoid of acetate kinase (AK) and phosphotransacetylase (PTA) to assimilate acetate when challenged at the end of growth on acetogenic substrates is explicable by the co-expression of acetyl CoA-synthetase (AcCoA-S) and acetate permease (AP). Furthermore, mRNA transcript measurements for acs and aceA , together with the enzymatic activities of their corresponding enzymes, acetyl CoA synthetase (AcCoA-S) and isocitrate lyase (ICL), clearly demonstrate that the expression of the two enzymes is inextricably linked and triggered in response to growth rate threshold signal (0.4 h -1 ± 0.03: n4). Interestingly, further restriction of carbon supply to the level of starvation led to the repression of acs (AcCoA-S), ackA (AK) and pta (PTA). Further, we provide evidence that the reaction sequence catalysed by PTA, AK and AcCoA-S is not in operation at low growth rates and that the reaction catalysed by AcCoA-S is not merely an ATP-dissipating reaction but rather advantageous, as it elevates the available free energy (Δ G °) in central metabolism. Moreover, the transcriptomic data reinforce the view that the expression of PEP carboxykinase is essential in gluconeogenic phenotypes.

Very little is known about disease transmission via the gut microbiome. We hypothesized that certain inflammatory features could be transmitted via the gut microbiome and tested this hypothesis using an animal model of inflammatory diseases. Twelve-week-old healthy C57 Bl/6 and Germ-Free (GF) female and male mice were fecal matter transplanted (FMT) under anaerobic conditions with TNF ΔARE-/+ donors exhibiting spontaneous Rheumatoid Arthritis (RA) and Inflammatory Bowel Disease (IBD) or with conventional healthy mice control donors. The gut microbiome analysis was performed using 16S rRNA sequencing amplification and bioinformatics analysis with the HIVE bioinformatics platform. Histology, immunohistochemistry, ELISA Multiplex analysis, and flow cytometry were conducted to confirm the inflammatory transmission status. We observed RA and IBD features transmitted in the GF mice cohort, with gut tissue disruption, cartilage alteration, elevated inflammatory mediators in the tissues, activation of CD4/CD8+ T cells, and colonization and transmission of the gut microbiome similar to the donors' profile. We did not observe a change or transmission when conventional healthy mice were FMT with TNF ΔARE-/+ donors, suggesting that a healthy microbiome might withstand an unhealthy transplant. These findings show the potential involvement of the gut microbiome in inflammatory diseases. We identified a cluster of bacteria playing a role in this mechanism.

Supplementing influenza vaccines with recombinant neuraminidase (rNA) antigens remains a promising approach for improving suboptimal vaccine efficacy. However, correlations among rNA designs, properties, and protection have not been systematically investigated. Here, we performed a comparative analysis of several rNAs produced by the baculovirus/insect cell system. The rNAs were designed with different tetramerization motifs and NA domains from a recent H1N1 vaccine strain (A/Brisbane/02/2018) and compared for enzymatic properties, antigenicity, stability, and protection in mice. We found that the enzymatic properties differ between rNAs containing the NA head domain versus the full ectodomain, the formation of higher-order rNA oligomers is tetramerization domain dependent, whereas the protective efficacy is more contingent on the combination of the tetramerization and NA domains. Following single-dose immunizations, an rNA possessing the full ectodomain and the tetramerization motif from the human vasodilator-stimulated phosphoprotein provided much better protection than an rNA with ∼10-fold more enzymatically active molecules that is comprised of the head domain and the same tetramerization motif. In contrast, these two rNA designs provided comparable protection when the tetramerization motif from the tetrabrachion protein was used instead. These findings demonstrate that individual rNAs should be thoroughly evaluated for vaccine development, as the heterologous domain combination can result in rNAs with similar key attributes that vastly differ in protection. IMPORTANCE For several decades, it has been proposed that influenza vaccines could be supplemented with recombinant neuraminidase (rNA) to improve efficacy. However, some key questions for manufacturing stable and immunogenic rNAs remain to be answered. We show here that the tetramerization motifs and NA domains included in the rNA construct design can have a profound impact on the biochemical, immunogenic, and protective properties. We also show that the single-dose immunization regimen is more informative for assessing the rNA immune response and protective efficacy, which is surprisingly more dependent on the specific combination of NA and tetramerization domains than common attributes for evaluating NA. Our findings may help to optimize the design of rNAs that can be used to improve or develop influenza vaccines.

Background: E. coli B (BL21), unlike E.coli K-12 (JM109) is insensitive to glucose concentration and, therefore, grows faster and produces less acetate than E. coli K-12, especially when growing to high cell densities at high glucose concentration. By performing genomic analysis, it was demonstrated that the cause of this difference in sensitivity to the glucose concentration is the result of the differences in the central carbon metabolism activity. We hypothesized that the global transcription regulator Cra (FruR) is constitutively expressed in E. coli B and may be responsible for the different behaviour of the two strains. To investigate this possibility and better understand the function of Cra in the two strains, cra - negative E. coli B (BL21) and E. coli K-12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and real-time PCR., Results: The deletion of the cra gene in E. coli B (BL21) minimally affected the growth and maximal acetate accumulation, while the deletion of the same gene in E.coli K-12 (JM109) caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. ppsA (gluconeogenesis gene), aceBA (the glyoxylate shunt genes) and poxB (the acetate producing gene) were down-regulated in both strains, while acs (acetate uptake gene) was down-regulated only in E.coli B (BL21). These transcriptional differences had little effect on acetate and pyruvate production. Additionally, it was found that the lower growth of E. coli K-12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producing bet operon (betABT)., Conclusions: The transcriptional changes caused by the deletion of cra gene did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone; rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that cra deletion caused transcription inhibition of the bet operon in E. coli K-12 (JM109) but did not affect this operon transcription in E. coli B (BL21). This property, together with the insensitivity to high glucose concentrations, makes this the E. coli B (BL21) strain more resistant to environmental changes.

Plasmid DNA (pDNA) is an emerging experimental vaccine, produced in E. coli, initially targeted for viral diseases. Unlike traditional protein vaccines whose average dose is micrograms, the average dose of pDNA is on the scale of milligrams. Production yields are, therefore, important for the future development of this vaccine. The E. coli strains currently used for pDNA production, JM109 and DH5alpha, are both suitable for production of stable pDNA due to the deletion of recA and endA, however, these two E. coli K strains are sensitive to growth conditions such as high glucose concentration. On the other hand E. coli BL21 is less sensitive to growth conditions than E. coli JM109 or DH5alpha, this strain grows to higher densities and due to its active glyoxylate shunt and anaplerotic pathways is not sensitive to high glucose concentration. This strain is used for recombinant protein production but not for pDNA production because of its inability to produce stable pDNA. To adapt E. coli BL21 for stable pDNA production, the strain was mutated by deleting both recA and endA, and a proper growth and production strategy was developed. Production values, reaching 2 g/L were obtained using glucose as a carbon source. The produced plasmid, which was constructed for HIV clinical study, was found to have identical properties to the plasmid currently produced by E. coli DH5alpha.