The mechanism(s) involved in H2S-gastroprotection against ischemia/reperfusion (I/R) lesions and their time-dependent progression into deeper gastric ulcerations have been little studied. We determined the effect of l-cysteine, H2S-releasing NaHS or slow H2S releasing compound GYY4137 on gastric blood flow (GBF) and gastric lesions induced by 30 min of I followed by 3, 6, 24 and 48 h of R. Role of endogenous prostaglandins (PGs), afferent sensory nerves releasing calcitonin gene-related peptide (CGRP), the gastric expression of hypoxia inducible factor (HIF)-1α and anti-oxidative enzymes were examined. Rats with or without capsaicin deactivation of sensory nerves were pretreated i.g. with vehicle, NaHS (18–180 μmol/kg) GYY4137 (90 μmol/kg) or l-cysteine (0.8–80 μmol/kg) alone or in combination with (1) indomethacin (14 μmol/kg i.p.), SC-560 (14 μmol/kg), celecoxib (26 μmol/kg); (2) capsazepine (13 μmol/kg i.p.); and (3) CGRP (2.5 nmol/kg i.p.). The area of I/R-induced gastric lesions and GBF were measured by planimetry and H2-gas clearance, respectively. Expression of mRNA for CSE, CBS, 3-MST, HIF-1α, glutathione peroxidase (GPx)-1, superoxide dismutase (SOD)-2 and sulfide production in gastric mucosa compromised by I/R were determined by real-time PCR and methylene blue method, respectively. NaHS and l-cysteine dose-dependently attenuated I/R-induced lesions while increasing the GBF, similarly to GYY4137 (90 μmol/kg). Capsaicin denervation and capsazepine but not COX-1 and COX-2 inhibitors reduced NaHS- and l-cysteine-induced protection and hyperemia. NaHS increased mRNA expression for SOD-2 and GPx-1 but not that for HIF-1α. NaHS which increased gastric mucosal sulfide release, prevented further progression of acute I/R injury into deeper gastric ulcers at 6, 24 and 48 h of R. We conclude that H2S-induced gastroprotection against I/R-injury is due to increase in gastric microcirculation, anti-oxidative properties and afferent sensory nerves activity but independent on endogenous prostaglandins.Hydrogen sulfide (H On the other hand, recent data has shown that H2S may be involved in human colon cancer cell proliferation and this mechanism is accompanied by a decrease of NO production [2S donor, dose-dependently inhibited HT29 human colon cancer cells proliferation indicating that these discrepancies are dose related [2S inhibited the lipid peroxidation and formation of water immersion and restraint stress-induced gastric damage. Wallace et al. [l-cysteine and H2S donors accelerated the healing of experimental chronic gastric ulcers. Furthermore, H2S has been shown to prevent gastric mucosal damage induced by intragastric application of ethanol, alendronate or non-steroidal anti-inflammatory drugs [Hntenance . Physiolat colon . In anotat colon  have demoduction . In cont related . Lou et  related  have shoe et al.  observedry drugs ,18,19.l-cysteine attenuated gastric injury induced by I/R. Despite these reports concerning the effect of NaHS in the upper and lower GI-tract, the potential role of this gaseous molecule in protection against gastric I/R injury and the mechanism of its protective action has not been fully investigated. Little is known whether this endogenously produced H2S can affect gastric lesions caused by I/R with the respect to their progression into deeper gastric erosions caused by extending the time of reperfusion up to 12, 24 or 48 h. Moreover, the potential gastroprotective effect of slowly H2S-releasing compound, GYY4137 against I/R-induced gastric damage has not been so far investigated [2S-mediated gastroprotection against this type of gastric mucosa injury involves regulation of oxidation and hypoxia, activity of prostaglandins (PGs)/cyclooxygenases (COX) system, afferent sensory nerves releasing calcitonin gene related peptide (CGRP) and vanilloid receptors (TRPV)-1 activity.NaHS abrogated the gastric epithelial cells against I/R injury via mitogen-activated protein kinase-dependent anti-apoptotic action and NF-κB-dependent anti-inflammatory pathway . Moreovestigated . Further2S precursor, l-cysteine  dose-dependently attenuated the area of gastric lesion and significantly increased the GBF in rats exposed to I followed by 3 h of R . Pretreatment with GYY4137 administered i.g. in a dose of 90 μmol/kg, equimolar with the dose of NaHS inhibiting I/R-induced lesions by 50%, significantly decreased the area of I/R-induced gastric damage and significantly raised the GBF as compared with vehicle-control group (p < 0.05) (Pretreatment with NaHS (18–180 μmol/kg i.g.) and 3 h of R . The dos < 0.05) .l-cysteine (80 μmol/kg i.g.) with 1) non-selective COX inhibitor, indomethacin (14 μmol/kg i.p.), 2) selective COX-1 inhibitor, SC-560 (14 μmol/kg i.g.) or 3) selective COX-2 inhibitor, celecoxib (26 μmol/kg i.g.) (l-cysteine-induced decrease in the area of I/R-induced gastric lesions and the accompanying increase in the GBF as compared with the vehicle-pretreated control group (p < 0.05).l-cysteine as compared with the groups treated with this H2S donor or H2S precursor alone (p < 0.05) (Pretreatment with capsazepine (13 μmol/kg i.p.) which did not significantly increase the area of I/R-induced gastric damage and failed to significantly affect GBF, significantly increased the area of I/R-induced gastric lesions and significantly decreased the GBF evoked by NaHS or  < 0.05) .l-cysteine (80 μmol/kg i.g.) as compared with rats with intact-sensory nerves pretreated with this H2S donor or its precursor (p < 0.05)  or  < 0.05) . l-cysteine with CGRP (2.5 nmol/kg i.p.) which by itself significantly reduced the I/R injury and significantly increased the GBF in rats with capsaicin denervation, restored the values of area of I/R-induced gastric lesions and the GBF to those observed in groups of rats pretreated with NaHS or l-cysteine alone without capsaicin denervation (The combination of NaHS or ervation .p < 0.05) (p < 0.05). NaHS (90 μmol/kg i.g.) administered to intact rats failed to affect mRNA expression for CSE ( < 0.05) A. In the < 0.05) B and 3-M < 0.05) C was obs for CSE A, CBS (F for CSE B and 3-M for CSE C as compp < 0.05) (p < 0.05). < 0.05) A–C. In g < 0.05) B and SOD < 0.05) C but not < 0.05) A mRNA exp < 0.05).p < 0.05). Pretreatment with NaHS accelerated the healing of I/R-induced gastric damage as demonstrated by a significant decrease in the area of gastric lesions in groups subjected to 24 and 48 h of R compared with the NaHS -pretreated groups exposed to 3 and 6 h of R (p < 0.05)  .p < 0.05). In groups pretreated with vehicle or NaHS and exposed to I followed by 48 h of R, sulfide release reached the values similar to that obtained in intact rats.l-cysteine (80 μmol/kg i.g.) or NaHS (90 μmol/kg i.g.) significantly increased sulfide release by the activity of CSE/CBS pathway in gastric mucosa exposed to I and followed by 3 of R as compared with vehicle-control group (p < 0.05) ( < 0.05) A.l-cysteine (80 μmol/kg i.g.) or NaHS (90 μmol/kg i.g.) did not affect sulfide release by the 3-MST activity pathway in gastric mucosa exposed to I and followed by 3 of R as compared with intact or vehicle-control group (p < 0.05) (Pretreatment with  < 0.05) B.Exposure of gastric mucosa to I/R is known to induce gastric mucosal hemorrhagic lesions due to increased generation of reactive oxygen metabolites, microvascular dysfunction and adhesion of neutrophils to the vascular endothelium and their infiltration of gastric mucosal structure ,25,26. A2S, exogenously delivered by the administration of H2S donor, NaHS or slow H2S-releasing compound, GYY4137 [l-cysteine, in the mechanism of gastroprotection against the formation of I/R-induced damage. Our second goal was to examine an effect of NaHS administration on further progression of these acute I/R lesions into deeper ulcerations by extension of the time of R from initial 3 h up to 6 h, 1 day and 2 days, respectively. We determined the possible role of H2S in restoration of gastric microcirculation and involvement of classic protective factors such as endogenous prostaglandins and afferent sensory nerves releasing vasodilatory mediators such as CGRP in H2S-mediated gastroprotection against I/R injury. We also attempted to assess if H2S released from NaHS can affect changes of mRNA expression for anti-oxidative HIF-1α, GPx-1 and SOD-2 induced by exposure of gastric mucosa to I/R.In this study, we investigated the involvement of H GYY4137  and that2S donor or l-cysteine, which acts as a H2S precursor, dose-dependently reduced I/R-induced gastric damage and this effect was accompanied by restoration of gastric microcirculation. Similar effect was observed when animals were pretreated with GYY4137 which has been considered as a slow releaser of H2S. These results are in pair with previous data that the vasodilatory activity of H2S is involved in the mechanism of gastroprotection against stress-induced lesions [2S exerted protective activity against I/R-induced gastric damage [2S can mainly derive from the activity of CSE biosynthetic pathway. Moreover, pretreatment with NaHS or l-cysteine increased endogenous H2S production, possibly due to CSE activation since sulfide release from gastric mucosa exposed to I/R and pretreated with NaHS or l-cysteine was increased via CSE/CBS but not 3-MST activity pathway. Similarly, Bos et al. [We observed that pretreatment with NaHS, H lesions . Our resc damage ,22, hepac damage  and myocc damage ,32. In ac damage  reporteds et al.  demonstr2S gastroprotection against I/R-induced injury does not depend on endogenous PGs biosynthesis by COX-1 or COX-2 activity since the pharmacological inhibition of the activity of these enzymes failed to affect beneficial effect of NaHS or l-cysteine observed in our present study. This seems to be contrary to the previous observation in another experimental model of gastric damage because NaHS increased COX-1, COX-2 and PGE2 content and prevented gastric mucosa against stress-induced lesions [2S biosynthesis in rodent colon is associated with downregulation of COX-2 mRNA expression. We assume that these discrepancies can be explained by the evident differences in animal models and experimental conditions used in our and previous studies. Based on the evidence presented in this study, we propose that H lesions . Wallace lesions  demonstr2S-induced protection against I/R-injury involves the anti-oxidative properties of this gaseous molecule since in our study the pretreatment with NaHS increased mRNA expression for SOD-2 and GPx-1. On the other hand, in gastric mucosa compromised by I followed by 3 h of R, HIF-1α mRNA expression was also decreased. Indeed, previous studies reported that mRNA expression of the gene for this particular protein was not affected in gastric mucosa when stomach was subjected to standard I/R injury with an extension of reperfusion even up to 1 or 6 days [2S-mediated gastroprotection against this type of injury does not involve regulation by HIF-1α.The mechanism of Hr 6 days . In contr 6 days  reportedl-cysteine was attenuated in rats with capsaicin-induced sensory nerves ablation. The NaHS- and l-cysteine-mediated gastroprotection in capsaicin-treated rats was then restored when CGRP was co-administered with H2S donor or its metabolic precursor. Similarly to capsaicin-denervation, inhibition of TRPV1 activity by capsazepine [l-cysteine. Therefore, we assume that the mechanism of H2S-mediated regulation of gastric microcirculation and gastroprotection against I/R-induced damage may depend upon the activation of TRPV1 receptors and the activity of afferent sensory nerves releasing vasodilatory CGRP. Based on these results obtained in rats with capsaicin denervation and those with inhibited TRPV1 receptors by capsazepine, we conclude that the potent vasodilatory and gastroprotective activity of H2S against I/R gastric lesions may depend, at least in part, on activation by this gaseous molecule of sensory nerves and vanilloid receptors. Since some degree of H2S protection was still observed in rats pretreated with capsazepine, the possibility is not excluded that other factors i.e., nitric oxide (NO), potassium channels, guanylyl cyclase/cGMP system, or yet unidentified factors can also mediate this protection and an increase in the GBF observed in rats pretreated with H2S donor. Our notion that H2S protection involves sensory nerves activation matches with data published recently that H2S-induced protection against gastric oxidative bleeding erosions caused by another ulcerogen such as stress has been ameliorated in animals with capsaicin-denervation [2S modulates intestinal motility and excites duodenal contraction by the mechanism involving activation of TRPV1 in afferent nerves endings and opening of KATP channels.On the other hand, we observed that, the gastroprotective effect of NaHS and sazepine ,39, althervation . Indeed,ervation  provided2S in gastric mucosa has been increased in rats pretreated with donor of this gaseous mediator. We assume that elevated H2S bioavailability caused by the administration of NaHS releasing H2S enhanced gastric mucosal resistance to oxidative I/R-induced gastric mucosal damage. Moreover, H2S production in gastric tissue was maintained after 6 and 24 h of R. Thus, we conclude that H2S can strengthen the mucosal barrier since this gaseous mediator not only reduced I/R-induced gastric damage formation but also accelerated healing of these acute erosions progressing with time into gastric ulceration. This supports the observation by Wallace et al. [2S donors accelerated the healing of pre-existing gastric ulcers.Interestingly, we observed that pretreatment with NaHS not only protected gastric mucosa against the formation of I/R-induced lesions but also reduced development of gastric damage during their progression to deeper gastric ulcerations and restored the decrease in GBF as observed after 6 h of R in vehicle-pretreated animals exposed to I/R. Moreover, in groups pretreated with NaHS, almost no gastric damage after 24 and 48 h of R has been observed. Furthermore, the exposure to I/R resulted in upregulation of mRNA expression for CSE but downregulated CBS and 3-MST mRNA expression in gastric mucosa and this effect was not affected by NaHS. Additionally, endogenous production of He et al. , who repn= 150, average weight about 250 g) were used in the study. Rats were fasted for 24 h with free access to drinking water before the exposure to I/R. The study was approved by the Institutional Animal Care and Use Committee of Jagiellonian University Medical College in Cracow and run in accordance with the statements of the Helsinki Declaration regarding handling of experimental animals.Male Wistar rats ; (4) GYY4137 (90 μmol/kg i.g.). The doses of 90 and 80 μmol/kg i.g. for NaHS and l-cysteine, respectively, have been selected based on ability of these compounds to reduce the area of I/R gastric damage by 50%. Separate groups of rats were treated with saline or NaHS (90 μmol/kg i.g.) and were not exposed to I/R. Rats with intact sensory nerves (series A) were randomized to the groups (4–5 rats each) and were administered 30 min before exposure to I/R with: (1) saline (vehicle-control group); (2) NaHS (18–180 μmol/kg i.g.); (3) l-cysteine (80 μmol/kg i.g.) [Rats of series B were pretreated with capsazepine (5 mg/kg i.p.), TRPV1 blocker ,39, indokg i.g.) ,43.l-cysteine (80 μmol/kg i.g.) alone or in combination with CGRP (2.5 nmol/kg i.p.) and exposed to I/R.Two weeks before experiment, in separate group of rats (series C), capsaicin was administered for three days at a dose of 82.5, 165 and 165 μmol/kg  to induce functional ablation of afferent sensory nerves as described previously ,44. Next2S production in gastric mucosa. All tested compounds were of analytical grade and were purchased from Sigma-Aldrich .In series D, rats were pretreated with vehicle or NaHS (90 μmol/kg i.g.) and were exposed to I followed by 3, 6, 24 and 48 h of R to investigate the progression of I/R injury and changes in H2 gas clearance technique as described previously [After the end of I/R, the animals of series A–D were anesthetized with pentobarbital (60 mg/kg i.p.), the abdomen was opened, and the stomach was exposed to measure GBF by Heviously . The GBFl-cysteine (10 mM) and pyridoxal 5′-phosphate  or α-ketoglutarate  were added. H2S biosynthesis via CSE and CBS required the presence of P5P, while that via 3-MST required α-KG. Tube containing a piece of filter paper soaked with zinc acetate  was placed inside the larger vial. The vials were then flushed with nitrogen gas and capped with an airtight serum cap. The vials were then transferred to water bath (37 °C) and after 90 min, trichloroacetic acid  in 7.2 M HCl and FeCl3  in 1.2 M HCl were added. After 20 min, absorbance at 670 nm was measured with a microplate reader . The calibration curve of absorbance vs. H2S concentration was obtained by using NaHS solution of varying concentrations. Results were expressed as total sulfide pool released from 1 g of gastric mucosa per one hour.Measurement of sulfide production in gastric mucosa was determined by modified methylene blue-method, as described previously ,19,29. BGastric mucosa from rats exposed to I/R and pretreated with vehicle or NaHS (5 mg/kg i.g.) was collected, snap-frozen in liquid nitrogen and stored at −80°C for further determination of mRNA expression for CSE, CBS, MST, HIF-1α, GPx-1, SOD-2 by real time PCR, as described previously ,19,46.Total RNA was isolated using commercially available kit with spin-columns  according to manufacturer protocol. Reversed transcription was performed using High-Capacity cDNA Reverse Transcription Kit with RNAse inhibitor  and random primers. −ΔCt method [Expression for CSE, CBS, MST, HIF-1α, GPx-1, SOD-2 and β-actin as reference gene was determined using specific primers ,19. The t method ,48.post-hoc test where appropriate. p < 0.05 was considered as statistically significant.Results of the experiment were expressed as mean ± S.E.M and the statistical analysis was performed with t-Student test or ANOVA test and Tukey 2S released form NaHS or produced endogenously form l-cysteine mainly due to CSE activity acts as an important, protective factor in gastric mucosa against I/R-induced gastric damage which can also accelerate the healing of these erosions. The mechanism of this H2S-mediated gastroprotection does not involve the endogenous PGs biosynthesis and regulation of HIF-1α expression but may depend upon the vasodilatory properties of this gaseous molecule mediated by the activity of CGRP-releasing afferent sensory nerves, finally leading to increase in gastric microcirculation under conditions associated with the generation of reactive oxygen metabolites and the enhanced oxidative metabolism. We conclude that H


IL-22 is another cytokine implicated in AS pathophysiology and is produced by Th17 and Th22 cells. In this study, we aimed to analyze conventional and non-conventional T cell subsets producing IL-17A and IL-22 in patients with AS. We thus evaluated the intracellular staining for IL-17A, IL-22, and IFN-γ in peripheral blood mononuclear cells of 36 patients with AS and 55 age- and sex-matched healthy controls (HC). Conventional CD4+ and CD8+ T cells, γδ T cells, and mucosal-associated invariant T (MAIT) cells were evaluated. In patients with AS, we found a decreased frequency and number of γδ T cells, of MAIT cells and of IFN-γ+ CD4+ and CD8+ T cells. Th17-related IL-17A+/IFN-γ− CD4+ T cells were decreased in AS. The number of IL-22+ MAIT cells was higher in AS compared with HC, as well as the number of IFN-γ+/IL-17A+ MAIT cells. The number of IFN-γ−/IL-17A+ MAIT cells was higher only in female patients with AS compared with female HC. The cellular source of IL-17A was thus not restricted to conventional Th17 CD4+ T cells and might involve innate-like T cells, such as MAIT cells. Circulating MAIT cells producing IL-22 were increased in AS. These results strengthen the importance of innate and innate-like sources of IL-17A and/or IL-22.The IL-23/T helper 17 (Th17) axis plays an important role in joint inflammation in ankylosing spondylitis (AS). Conventional CD4 IL-23R) gene  is the prototypical and most common form of spondyloarthritis (SpA). The IL-23/T helper 17 (Th17) axis has been suggested to be a key player in joint inflammation in AS (3R) gene . Circulad IL-17A . Moreovend IL-22 . FinallyIt is estimated that around 50% of patients with AS exhibit microscopic signs of ileum or colon inflammation and an average of 6.5% of SpA patients develop clinically established inflammatory bowel disease, highlighting the link between gastrointestinal tract inflammation and SpA . New celComité de Protection des Personnes CPP-EST-II, Besançon, France), and the sample collection was approved by the French Ministry of Higher Education and Research (agreement number #DC-2008-713).Thirty-six patients with AS and 55 sex-matched and age-matched HC were included in the study. All the AS patients met the modified New York criteria and were all under nonsteroidal anti-inflammatory drugs and/or conventional synthetic disease-modifying anti-rheumatic drugs . None of the patients had corticosteroids or had previously received a TNF inhibitor (most of them were enrolled before initiating such agent). The study was approved by the local Ethics Committee  according to the manufacturer’s recommendations, as previously described .Erythrocyte sedimentation rate (ESR) test and C-reactive protein (CRP) levels were used as laboratory parameters to assess inflammation.+ lymphocyte number. These values were obtained by extrapolation of CD4+, CD8+, and MAIT T cells by using the absolute numbers of T cells which were obtained on fresh blood samples and the percentages of the specific subpopulations among CD3+ T cells obtained in thawed samples. The differences in the number of the evaluated cellular subsets, cytokine-secreting T cell subsets, and MAIT cells between patients with AS and HC were analyzed using the non-parametric Mann–Whitney test. Spearman’s correlation coefficient was used to test the correlation between disease activity and the percentage of cytokine-secreting MAIT cells and between IL-22-positive cells and circulating IL-22.Results are expressed as mean ± SEM. Absolute number of the different T cell subsets, cytokine-secreting T cell subsets, and MAIT cells was obtained by multiplying the specific cell subset percentages by the total CD3p = 0.008). The absolute number and percentage of CD4+ T cells, CD8+ T cells, and CD3+ T cells did not differ between patients and HC  . Then, we found that the following CD8+ T cell subsets: IL-17A+/IFN-γ+, IL-17A+/IFN-γ−, IL-22+/IFN-γ+, and IL-22+/IFN-γ− were in equal proportions in both groups. Thus, no difference exists for CD8+ T cells, except a decrease in IFN-γ+ Tc1 cells in AS patients.We then analyzed the intracellular expression of IL-17A, IFN-γ, and IL-22 in different T cell subpopulations , while the other cytokine-secreting CD4+ T cell subsets did not differ between patients and HC   compared with HC . For the MAIT cells that were only positive for IL-17A , a significant higher frequency was observed only in female AS (p = 0.037)  Table A,B, as wC Figure , we alsop = 0.7). Other circulating inflammatory cytokines  were significantly higher in the AS group , but their levels were very low .Finally, serum levels of IL-22 were found marginally elevated in patients with AS, albeit without reaching a statistical significance  cells. In a previous study, the proportion of IL-23R-expressing T cells  in the periphery was twice as high in AS patients than in HC, but this increased frequency was only observed in the γδ T cell subset that represented the main source of IL-17A secretion  coding for factors that regulate bone formation and promote ligamentous ossification, a hallmark of AS disease , indicating that these effects on MSC may represent a pathway for new bone formation in AS , but this was also found in previous studies analyzing IL-17A-secreting cells –18. Anal+ Th17 cells, suggesting that the type-17 axis may be broader in AS than initially thought. Besides the IL-23 and IL-17A cytokines, IL-22 also seems to be implicated in the pathogenesis of AS. MAIT cells are deregulated in AS, strengthening the importance of innate and innate-like sources of IL-17A and IL-22 and emphasizing the link between the gut and the joint in AS. Future studies are needed to better understand the importance of this cellular population and IL-22-producing cells in AS.In conclusion, our results indicate that the cellular source of IL-17A is not restricted to conventional CD4Ethics committee: Comité de Protection des Personnes CPP-EST-II, Besançon, France. Sample collection was approved by the French Ministry of Higher Education and Research (agreement number #DC-2008-713).ET, BG, and PS conceived and designed the study. ET recruited the patients and healthy controls. BG and CL designed the laboratory assessments and performed all the analysis. ET and BG analyzed the results. ET wrote the manuscript together with BG and PS. DG designed the figures. All the authors contributed substantially to editing the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli ST131, a global, high-risk clone, comprises fluoroquinolone resistance (FQ-R) mutations and CTX-M extended-spectrum beta-lactamases associated with the fimH30-encoding clades, C1 and C2. Further carbapenem resistance development in ST131 is a public health concern.E. coli (CP E. coli) ST131 across England.This observational study aimed to probe the diversity of carbapenemase-producing E. coli from human samples submitted to the national reference laboratory from January 2014 to June 2016. Antimicrobial resistance (AMR) gene content and single nucleotide polymorphism (SNP) data were compared against a published ST131 phylogeny and analysed alongside patient metadata.ST131 isolates were identified using whole-genome sequencing (WGS) data generated for all non-duplicate CP E. coli, from eight of nine regions, represented 10% of CP E. coli isolates sequenced. Ten and eight isolates were from the FQ-susceptible (FQ-S) clades A and B, while eight and 15 isolates belonged to the FQ-R clades C1 or C2, respectively. Seven distinct carbapenemases were identified: KPC-2  frequently occurred among clade C2 isolates (n = 10). OXA-48-producers  were often from clade A (n = 5). NDM-1 (n = 4), NDM-5 (n = 1), VIM-1 (n = 1), VIM-4 (n = 1) and OXA-181 (n = 1) were also identified. Clade C2 isolates encoded more AMR genes than those from clades A (p = 0.02), B (p = 9.6 x 10−3) or C1 (p = 0.03).Thirty-nine genetically diverse ST131 CP E. coli, ST131 represented a fraction of the CP E. coli received, belonging to diverse clades and encoding diverse carbapenemases. The greater accumulation of resistance genes in clade C2 isolates highlights the need for ongoing monitoring of this high-risk lineage.When compared with its global predominance among ESBL- Escherichia coli with horizontally acquired CTX-M extended-spectrum beta-lactamases (ESBLs) and, more recently, carbapenemases, have heralded the advent of resistance to last-resort treatment options for many serious infections because of E. coli and other Enterobacterales.The increasing incidence of antimicrobial resistance worldwide and a paucity of new drugs in development presents a major threat to the treatment of bacterial infections ,2. SinceE. coli has been associated particularly with the uropathogenic E. coli lineage ST131. The population structure of the ST131 lineage was shaped by the acquisition of virulence and resistance elements which has resulted in three dominant clades; A, B and C [gyrA (coding for gyrase) and parC (coding for topoisomerase) which impart elevated fluoroquinolone resistance (FQ-R) [fimH gene variants into the C1 (fimH30-R) and C2 (fimH30-Rx) clades in 1987 [fimH encodes for the type 1 fimbrial adhesin which binds to, and facilitates colonisation of, the bladder epithelium [E. coli carrying CTX-M-15 ESBL [The pandemic of resistant  B and C . Around e (FQ-R) . This clithelium ,5 and ha-15 ESBL  and multkpc gene occurring in ST131 globally [E. coli have also recently been recorded in Spanish rivers [E. coli globally across many environmental niches demonstrates the breadth of reservoirs that contain CP E. coli.Recent evidence has demonstrated ongoing dynamics and diversification of clade C2, with the emergence and spread of new resistant forms. An example of further acquisition and proliferation in the ST131 clades has been observed with CTX-M-27 ESBL-encoding isolates being reported first in Japan, followed by India and then northern Europe -10. Concglobally ,15. Impoh rivers  and longh rivers . This obE. coli from community- and hospital-onset infections alike [E. coli (CP E. coli) in England.In the United Kingdom (UK), ST131 has been widely described in ns alike . HoweverE. coli were isolated from human samples by clinical diagnostic laboratories and submitted to Public Health England’s (PHE) Antimicrobial Resistance and Health Care Associated Infections (AMRHAI) Reference Unit for carbapenemase gene detection and/or antimicrobial susceptibility testing. The referring laboratories were located in the nine English Regions . Between January 2014 and June 2016, AMRHAI undertook whole genome sequencing (WGS) of every initial CP E. coli isolate received per patient. DNA was extracted from RNase-treated lysates via a QIAsymphony DSP DNA Midi Kit . DNA libraries were prepared using the Nextera XT sample preparation method and sequenced with a standard 2 x 101 base protocol on a HiSeq 2500 ).CP E. coli from different patients were identified and extracted for analysis.Data for carbapenemase-producing ST131 E. coli ST131 isolates were submitted to the European Bioinformatic Institute’s European Nucleotide Archive as project PRJEB32306.Ethical approval was not required as no personal identifiable information was used in this study. Short-read sequence data for the 39 CP E. coli samples.Minimum inhibitory concentrations (MICs) of polymyxins, beta-lactams, aminoglycosides, carbapenems, tetracyclines and fluoroquinolones were determined at the time of receipt (January 2014– June 2016) by BSAC agar dilution methodology and were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines version N.0, 2018) for 26 of the CP WGS data were deplexed via Casava, and nucleotides with a Phred score less than Q30 at the ends of the reads were removed with Trimmomatic . SpeciesE. coli isolates from England, additional raw read sequences for 188 global non-carbapenemase producing ST131 isolates identified and validated previously [To provide a contextual framework for the 39 ST131 CP eviously  and trimSingle nucleotide polymorphisms (SNPs) were determined through read mapping using the PHEnix pipeline . Briefly−6; correlation coefficient = 0.5059; R2 = 0.256). BEAST 2 version 2.4.8 [E. coli using the 3,779-bp non-recombinant SNPs conserved across all 227 ST131 isolates. As only SNP sites were used in the alignment a Gamma site model (Gamma Category count 0) was used, which assumes a gamma distribution for site-to-site rate heterogeneity, and the proportion of invariant sites was fixed at 0%. The Tamura-Nei 1993 (TN93) model, which weights transitions and transversion mutations according to their likelihood, was found to be the best model to represent the phylogeny (path sampling maximum likelihood estimates are described in Supplementary Table S1A and effective sample size scores (ESS) in Supplementary Table S1B). A strict molecular clock, constant population size and uniform clock rate was found to be the most appropriate model as all isolates were from the same E. coli sequence type. The suitability of this model was reflected in the ESS scores when compared against GTR, HKY and JC69 site models under like-for-like parameters. To ensure convergence, Markov Chain Monte Carlo (MCMC) generations for each analysis were performed in triplicate for 30 million steps  sampling every 1,000 steps, producing ESS scores equal to or greater than 200 (Supplementary Table S1B). Replicate analyses were then combined using the BEAST program LogCombiner with 10% burn-in.TempEST was used to assess the variance between the time of sampling and the root-to-tip divergence in maximum likelihood trees  using heon 2.4.8  was used2 and ANOVA statistical tests were all performed using standard libraries contained within the R statistics package [T-test, X package .E. coli isolates were referred to and sequenced at PHE’s AMRHAI reference unit between January 2014 to June 2016 (30 months) from 20 laboratories that represented eight of the nine English regions. These accounted for 10% of the total number of CP E. coli isolates submitted. Temporal analysis showed that the average number of CP E. coli ST131 isolates submitted to PHE on a monthly basis rose from 0.67 in 2014 (ST131 CP E. coli = 8), to 1.67 in 2015 (ST131 CP E. coli = 20), and to 1.83 in 2016 . As a percentage of the total CP E. coli isolates across England, ST131 represented 4.5% in 2014 (CP E. coli = 177), 6.2% in 2015 (CP E. coli = 324) and 4.9% in the first half of 2016 (CP E. coli = 223). This increase in CP E. coli was linked to the number of KPC-2 producing isolates  and OXA-48 producing isolates .Thirty-nine ST131 CP E. coli ST131 isolates in this study for the occurrence of regional or local clusters, irrespective of time, showed that the majority were isolated from London (n = 13) or the North West region (n = 11) (E. coli and was found most often in the North West region (n = 6). The remaining carbapenemase alleles occurred sporadically across London , the North East (NDM-1), West Midlands (NDM-5) and the South East (VIM-1). None of the isolates in this study were associated with known outbreaks of CP E. coli. (Supplementary Table S2).Analysis of all PHE CP (n = 11) . Seven dE. coli ST131 with a previously published WGS-based population structure for ST131 identified that 23 of 39 ST131 CP E. coli from across England were from the FQ-R clades C1 (n = 8) and C2 (n = 15). Clade C2 was the most widely distributed clade of ST131 CP E. coli (isolated from 7 regions) and was the most highly represented clade in three regions (Supplementary Table S2).Comparison of WGS data from the CP  regions . The FQ-E. coli clade, C2, were most frequently associated with KPC-2 (n = 13) and predominated among ST131 CP E. coli isolated in London (n = 7). In contrast, the second most frequent carbapenemase, OXA-48 (n = 10), occurred most often in clade A isolates from the North West region (n = 5), demonstrating the contrasting phylogeographical patterns in ST131 CP E. coli for the two most frequent carbapenemases.The combined data for ST131 clades and carbapenemase alleles indicated that isolates from the largest CP E. coli isolates were incorporated into and visualised within the context of a previously validated phylogenetic BEAST tree [E. coli isolates were widely distributed across the diversity present in each of the clades with little evidence for clear expansions of sub-clades or genetic clusters of carbapenemase producers emerging, although there was a close relative cluster in clade C2 . Those 12 most resistant isolates remained susceptible to colistin and tigecycline. They originated from seven English regions, but were most often isolated in London (n = 6). We observed that 10 of the 12 isolates were from clade C2 and originated from six regions. Seven of the 10 isolates from clade 2 encoded KPC-2, two encoded OXA-48, and the remaining isolate encoded VIM-4 (Supplementary Table S3).MIC analyses for 26 of 39 isolates highlighted 12 isolates of ST131 CP −3, mean = 6.3, median = 5.0; clade C1, p = 0.03, mean = 8.25, median = 6.5). This was attributable to mobile genes that encoded resistance to aminoglycosides ; fluoroquinolones  and sulfonamides . Further comparisons revealed that clade C2 CP E. coli also had more AMR genes than their non-CP E. coli C2 counterparts , despite almost half (7/15) lacking the CTX-M-15 gene that has previously been strongly associated with the multi-resistant status of clade C2. Supplementary Table S4 describes the most common resistance genes in CP E. coli and non-CP E. coli isolates.To better explore the suggestion that isolates from the C2 clade had a propensity to have a greater number of antimicrobial resistances, we examined the number of AMR genes per isolate. The number of AMR genes did not differ significantly between isolates expressing different carbapenemase genes (ANOVA: p = 0.826), but AMR gene counts were affected by the clade that an isolate belonged to (ANOVA: p = 0.0214). Clade C2 isolates were associated with significantly higher numbers of acquired AMR genes   (t-test:E. coli ST131 isolates most often indicated the presence of replicons from the FIA (n = 23), FII (n = 35) and FIB (n = 35) plasmid incompatibility groups (Supplementary Table S5). Non F-type replicons were less evenly distributed across clades as indicated by the higher numbers in isolates from clades A (mean = 2.2) and C2 (mean = 2.13) compared with isolates in clades B (mean = 0.67) and C1 (mean = 0.88) . For isolates in clade A, particular carbapenemase types did show unique associations with replicons, with VIM-1/-4 associated with A/C replicons, KPC-2 to N-type replicons and OXA-48 association with IncL/M replicons. Isolates in the other clades  showed no clear relationships between unique plasmid replicons and particular carbapenemase genes (Supplementary Table S5).The plasmid replicon sequences found in the 39 CP E. coli represented only 10% of CP E. coli isolates that were submitted to the reference laboratory over the study period contrasted the current narrative that ST131 shows a global predominance among ESBL-producing E. coli. Nevertheless, the wide distribution of ST131 CP E. coli across all but one of the nine English regions indicates the ongoing success of ST131 E. coli and highlights its progression beyond being an ESBL encoding lineage in England. The diversity of clades and carbapenemase alleles also greatly differed from the emergence of a single lineage akin to the CTX-M-15 expressing C2 isolates responsible for the pandemic established in the mid-2000s. While no evidence was found for distinct newly emerged phylogenetic clusters reaching predominance, the relative success of clade C2 in particular highlights the potential to repeat the clonal success of the CTX-M-15 positive clade [The observation that ST131 CP E. coli in the region at the time. The principal mechanism of antibiotic gene acquisition in bacteria is through horizontal gene transfer, which is primarily mediated by plasmids. It is not possible to ascertain from short-read data whether these OXA-48 positive clade A isolates were part of a wider circulation of isolates and/or plasmids in the area at the time. However, nine of 10 OXA-48-positive isolates had reads mapping to the origin of replication for IncL/M-type plasmids, which have previously been identified as the main vehicles encoding OXA-48 in many Enterobacterales species [The contrast between the two most successful carbapenemases were marked. The predominance of KPC-2 among the FQ-R C2 isolates from London occurred contemporaneously to a large outbreak of KPC-2 in multiple species of the Enterobacterales in the North West region . In that species -36. For E. coli clade is concerning. The example of Klebsiella pneumoniae has shown that some high-risk clones spread in successive forms, first encoding ESBLs (CTX-M-15), followed by carbapenemases and subsequently developing resistance to colistin (an agent of last resort) [E. coli and especially ST131 would be further heightened by the emergence and wide geographic dissemination of mobile colistin resistance (mcr) genes in the species, not least as these have already been reported on IncHI2-based multi-resistance plasmids that are mobile and can replicate in E. coli and other members of the Enterobacterales [The marked differences in the multi-resistance gene profiles, evidenced by the higher number of AMR genes noted in clade C2 when compared against their ESBL-positive counterparts, suggests that clade C2 may have been differentially affected, as compared with clades A-C1, over time by a pressure to accumulate multiple resistances. This may be because of clade C2 isolates being exposed more regularly to antimicrobial pressure in healthcare settings. We speculate that clade C2 isolates possess a mechanism(s) or are otherwise predisposed towards the increased acquisition of resistance genes; and we are investigating this further. The detection of repeated accumulation of resistance genes in this epidemic  resort) . This prcterales .E. coli within England, the emergence of multiple new resistance profiles across ST131 and a propensity for these to be newly-emerging, increasingly-resistant forms of clade C2 in particular. Detecting genomic signals associated with the repeated acquisition and evolution of resistance profiles in the most successful E. coli clades provides a platform on which to base healthcare strategies and interventions that can assist in the early detection and mitigation of high-risk lineages in the future. It also underlines the importance of continued monitoring of this high-risk lineage with known pandemic potential, given the current lack of good alternatives to carbapenems for the treatment of Gram-negative antimicrobial-resistant infections.The data presented highlight the ongoing diversification and success of the clade C ST131 CP


C. necator H16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression in C. necator H16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and two negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme.This report provides tools for robust and predictable control of gene expression in the model lithoautotroph  Cupriavidus necator H16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within a >700-fold dynamic range was compared to the native PphaC, with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were evaluated. By supplying different concentrations of inducers, a >1,000-fold range of gene expression levels was achieved. Application of inducible systems for controlling expression of the isoprene synthase gene ispS led to isoprene yields that exhibited a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was also evaluated. A second-order polynomial relationship was observed between the RBS activities and isoprene yields. This report presents quantitative data on regulatory genetic elements and expands the genetic toolbox of C. necator.A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements, such as constitutive and inducible promoters as well as ribosome binding sites (RBSs), are required. In this study, we designed, built, and tested promoters and RBSs for controlling gene expression in the model lithoautotroph IMPORTANCE This report provides tools for robust and predictable control of gene expression in the model lithoautotroph C. necator H16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression in C. necator H16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and two negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme. Cupriavidus necator H16 is a Gram-negative betaproteobacterium, formerly known as Ralstonia eutropha, Alcaligenes eutrophus, Wautersia eutropha, and Hydrogenomonas eutropha. It has been extensively studied for its capacity to store large amounts of organic carbon in the form of poly-3-hydroxybutyrate (PHB) that can be utilized as a source of bioplastics (2) as sources of energy, utilizing them to power metabolic processes and fix carbon dioxide (CO2) (2), C. necator H16 can switch to anaerobic respiration-denitrification, exploiting alternative electron acceptors such as nitrite (NO2−) or nitrate (NO3−)  . Such flinterest –8.C. necator H16 has gained prominence as a chassis for the production of fuels, chemicals, and proteins , PxylS (m-toluic acid), Plac (lactose), PhpdH (3-hydroxypropionic acid), and PrhaBAD (l-rhamnose)  hydratases, allowing modulation of the proportion of the (R)-3-hydroxyhexanoate monomer in poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate]  –29, the amnose) –, 31, andamnose) – have beexanoate] . A few cter Ptac . In addiC. necator. Inducible control systems involving either positive or negative regulators are also required for highly controllable circuits in synthetic biology and biotechnology applications. Importantly, the quantitative and comparative evaluation of existing and new functional genetic elements is needed to facilitate genetic and metabolic engineering. The aim of this study was to design, build, and quantitatively characterize sets of constitutive promoters and RBSs, which would provide a wide range of strengths for gene expression control. Four inducible systems, known to have practical application in other microbial chassis, were assembled and characterized in a comparative manner. Inducible systems and a set of RBSs are applied for controlling expression of the ispS gene, leading to variable isoprene production.A strong need to expand the synthetic biology toolbox remains, aiming to broaden the range of functional genetic elements for controlling gene expression in 10 (T7g10) -based engineering and cloning method . Using p (T7g10) , was pla4 and T5 , 36–38 wnd T5 36–. Such innd T5 36–. The corC. necator H16 by electroporation. A promoterless construct (P0) was included as negative control. Resulting C. necator strains were cultured in minimal medium (MM) and the single-time-point fluorescence of the logarithmically growing cells was determined as a measure of eYFP protein concentration normalized to the cell density (phaC (P0_1+SD) or any other C. necator promoters characterized so far, with promoters P22 and P24 showing 9-fold higher activity. Strengths of respective promoters were comparable in the pBBR1MCS-2 and pMTL71101 vector backbones  synthesis rate at any time point during the time course of experiment. The resulting maximum synthesis rate was fitted to the corresponding inducer concentration using a Hill function , providing key parameters such as the maximum possible rate of RFP synthesis, the Hill coefficient, or the inducer concentration mediating half-maximal RFP synthesis (Table S3). According to the fitted data, the l-rhamnose-inducible system demonstrated the highest induction cooperativity (h = 3.88). It requires a minimum concentration of 0.156 mM l-rhamnose to be activated and achieves about 85% of maximum expression at 2.5 mM. The acrylate- and cumate-inducible systems generally require lower inducer levels to initiate gene expression. Moreover, the range of inducer concentration mediating a linear fluorescence output spans more than 1 order of magnitude  and therefore can be fine-tuned more easily.Plasmids harboring inducible systems were transformed into trations . The restrations . For exaacuRI activity. This behavior is also reflected in its dose-response curve . Four hours after supplementation with 5 mM acrylate, 49% of the initial amount was consumed. Upon depletion of inducer, gene expression was maintained at the level of basal promoter activity. Neither of the other three inducers, l-arabinose, l-rhamnose, or cumate, appeared to be consumed. C. necator H16 was not able to grow in MM supplemented with these three inducers as the sole carbon source (data not shown).Regardless of inducer concentration, we noticed that the absolute fluorescence, corrected by fluorescence that derives from basal promoter activity, generally increased during the time course of experiment. This was not the case for the acrylate-inducible system. Six hours after inducer addition, the increase in absolute fluorescence output facilitated by acrylate concentrations of 1.25 mM and less was at the same level as mediated by basal Pse curve . Normalil-arabinose-, l-rhamnose-, acrylate-, and cumate-inducible systems, respectively. The induction factors that were achieved by AcuR/PacuRI and CymR/Pcmt were much lower than for the two positively regulated systems due to high background levels of reporter gene expression in the absence of inducer . All of the systems can be considered to activate reporter gene transcription immediately after inducer addition. Fluorescence above background levels could be detected within 30 min, representing the time which is required for RFP production and maturation . A linear fluorescence output was achieved by addition of l-arabinose from 0.313 to 2.5 mM. The induction factor was considerably higher , mainly due to an 8-fold lower background level of RFP synthesis. However, removing the stem-loop also decreased absolute normalized fluorescence levels 3.6-fold at an inducer concentration of 10 mM  showed a significantly (P < 0.05) lower fluorescence output in the pBBR1MCS-2 vector backbone than that in pMTL71101.In this study, we used an RBS calculator  to develpromoter . Resultith phase . A major2), medium (RBS8), and strong (RBS1)—were evaluated using constructs containing combinations of upstream (promoter) and downstream (reporter) genetic elements, including eyfp, driven by the PphaC, P1, or PrhaBAD promoter, and rfp, driven by PphaC. As expected, the change from medium-strength PphaC to the strong P1 promoter contributed to the overall increase in gene expression but had little effect on the relative strength of the three cognate RBSs, displaying unchanged hierarchy of strengths RBS1 > RBS8 > RBS2 . Strikingly, the downstream change of genetic element (rfp gene) had a more pronounced effect on the relative strength of RBSs, significantly increasing it for RBS1 and RBS8 but not for RBS2 . These findings suggest that variable-strength RBSs can aid in achieving differential levels of protein synthesis. However, depending on the genetic context, the absolute levels of gene expression should be fine-tuned by testing a range of RBS alternatives.It should be noted that the activity of an RBS can vary extensively depending on the sequence context . To asseE. coli  pathway. Notably, due to the poor catalytic properties of the enzyme (high mK and low kcat), isoprene synthase is considered one of key bottlenecks in isoprene biosynthesis  emerged as the most significant. IspS has been demonstrated previously to catalyze production of isoprene from dimethylallyl pyrophosphate (DMAPP) , 48, whiynthesis .C. necator H16, and to investigate how gene expression of the enzyme (isoprene synthase) translates into the product of enzymatic reaction (isoprene), Populus alba ispS under the control of two positively (AraC/ParaBAD and RhaRS/PrhaBAD) and two negatively (AcuR/PacuRI and CymR/Pcmt) regulated inducible systems was introduced into C. necator on a plasmid. Induction of ispS expression with l-arabinose, l-rhamnose, acrylate, or cumate confirmed that isoprene was biosynthesized to different levels, resulting in a yield up to 7 μg/g of cells (dry weight) .First, to establish whether production of isoprene can be achieved in  weight) . MoreoveispS was cloned under the control of the PphaC promoter and different RBS variants. Isoprene production was measured after 24 h of plasmid-transformed C. necator cell growth as described in Materials and Methods. By applying a range of RBS variants to control translation, we were able to explore the relationship between IspS abundance and isoprene production efficiency  and two negatively (acrylate and cumate) regulated inducible systems using standardized experimental conditions. In our experimental design and using MM, the positively regulated inducible systems exhibited a tighter control of gene expression than the negatively regulated ones. The highest induction level was achieved using the l-arabinose-inducible system, whereas the l-rhamnose-inducible system exhibited the highest induction factor. The acrylate- and cumate-inducible systems showed significantly lower induction and higher background levels. The application of the acrylate-inducible system in C. necator is limited due to consumption of acrylate by this bacterium. l-Arabinose-, l-rhamnose-, and cumate-inducible systems are suitable for continuous activation of gene expression as well as biosensors. Notably, the cumate-inducible system appeared to be most sensitive, responding to the nanomolar to micromolar range of inducer concentrations. Overall, the systems' dose-response curves are consistent with data from previous induction experiments with C. necator and E. coli , encoding protein S1 with 67% identity (94% coverage) to the E. coli K-12 homologue, and the genes cafA1 and cafA2 (locus tags H16_A0909 and H16_A2580), encoding potential RNase E/G superfamily members with 38% identity (42% coverage) and 52% identity (67% coverage), respectively, to the E. coli K-12 homologues. These results underline existing similarities of gene expression control between members of gammaproteobacteria (E. coli) and betaproteobacteria (C. necator).The mRNA secondary structure as well as nucleotide sequence composition, such as the A/U-rich sequence, can also contribute significantly to gene expression regulation. The introduction of A/U-rich sequences into the RBS, upstream of the SD sequence, increases translational efficiency and the stability of mRNA in ribosome . On the ribosome . These oC. necator H16 cells transformed with plasmids harboring the ispS gene under the control of medium-strength RBS yielded the highest isoprene production, while further increases in gene expression resulted in no further increase and even decreases in levels of the product. This was somewhat surprising, since the gene expression data using fluorescence reporter showed that the increase in strength of RBS translated to higher yields of protein. The observed limitation in the isoprene production can be explained by several lines of reasoning: (i) if the overproduction of isoprene synthase is toxic, cells can potentially reduce this effect by either turning down expression or increasing degradation of IspS protein; (ii) due to the inherently high mK of IspS, the reduced availability of substrate can become a limiting factor in the biosynthesis of isoprene  asisoprene , proteinC. necator. In this study, we have quantitatively evaluated and compared an array of new genetic elements, including constitutive and inducible promoters, RBSs, mRNA stem-loop structure, and A/U-rich sequences, expanding the choice of tools. Our quantitative results will inform design and engineering of synthetic pathways and genetic circuitry in C. necator H16 and other related bacterial species.To conclude, the previous work has delivered a limited toolbox of functional genetic elements suitable for application in the metabolic engineering of C. necator H16  was purchased from DSMZ  and used in all experiments, including fluorescence assays, RNA isolation, and isoprene production, as described in the following sections. E. coli TOP10  was used for cloning and plasmid propagation.l-(+)-Arabinose , l-rhamnose monohydrate , magnesium acrylate , 4-isopropylbenzoic acid , and d-(−)-fructose  were used as inducers for assaying inducible systems or substrates in metabolite consumption assays. Isoprene  was used as a standard for isoprene yield quantification. Restriction enzymes were purchased from New England BioLabs (USA).Oligonucleotide primers were synthesized by Eurofins Genomics  and are listed in Table S1 in the supplemental material. 15, GCTGAGGGAAAGTACCCAAAAATTCATCCTTCTCGCCTATGCTCTGGGGCCTCGGCAGATGCGAGCGCTGCATACCGTCCGGTAGGTCGGGAAGCGTGCAGTGCCGAGGCGGATTAATCGATttgacagctagctcagtcctaggtactgtgctagcaacctGAATTCACTAGTTTAACTTTAAGAA. The insulations preceding (122 bases) and following (25 bases) the core promoter are both shown in uppercase and are partially derived from the upstream region of the phaC promoter and upstream nucleotide sequence of the T7 gene 10 RBS, respectively. For each insulated promoter, only the core sequence (in lowercase), including the −35 and −10 hexamers (underlined), up to 12 bases upstream of the −35 box, the 17-base spacer between the −35 and −10 boxes, and up to 20 bases downstream of the −10 box, vary between library members (The core promoter sequences used to construct the library of insulated constitutive promoters are listed in  members . The explacZα (for blue-white color screening of colonies) flanked by inversely oriented nicking endonuclease Nt.BbvCI sites and restriction endonuclease XbaI sites was synthesized by PCR using oligonucleotide primers P001 and P002 and pGEM-T plasmid (Promega) as a DNA template. To exclude any possible transcriptional readthrough, rrnB terminator T2 (5′-AAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTT-3′) and truncated version of terminator T1 (5′-ATCAAATAAAACGAAAGGCCTTCGGGCCTTTCGTTTTATCTGTTGTTT-3′) (Plasmids with insulated constitutive promoters and RBS variants  were conGTTT-3′)  were insGTTT-3′) . The reseyfp gene sequences, were generated by PCR with two pairs of oligonucleotide primers, P003 with P005_pX_r  and P006_pX_f (or P008_rbsX_f) with P004 using plasmid pBBR1MCS-2-PphaC-eyfp-c1 as a template. The overlapping primers, P005_pX_r (or P007_rbsX_r) and P006_pX_f (or P008_rbsX_f), were designed in such a way so that the desired promoter (or RBS sequence) is integrated into the primer sequence. The resulting plasmids with promoter and RBS variants are referred to as pBBR1MCS-2-P0 to pBBR1MCS-2-P25 and pBBR1MCS-2-RBS0 to pBBR1MCS-2-RBS32 in the pBBR1MCS-2 vector backbone and as pMTL71107-P0 to pMTL71107-P25 and pMTL71107-RBS0 to pMTL71107-RBS32 in the pMTL71107 vector backbone.All insulated constitutive promoters and RBSs were assembled in vectors pBBR1MCS-2-USER and pMTL71107 by employing the USER method. For this purpose, two overlapping DNA fragments, including promoter, RBS and 0 and SD sequence-free construct RBS0 (including the complement SD sequence) were generated. P0 was assembled from the DNA fragment that was PCR amplified using oligonucleotide primers P006_p0_f and P004 from pBBR1MCS-2-PphaC-eyfp-c1 and was cloned into either the pBBR1MCS-2-USER or pMTL71107 vector. The assembly of RBS0 involved two overlapping DNA fragments generated by PCR with two pairs of primers, P003-P007_rbs0_r and P008_rbs0_f-P004, using same template as described above.For use as negative controls, the promoterless construct P1, pBBR1MCS-2-SL/RBS2, and pBBR1MCS-2-SL/RBS8, containing RBS variants with upstream T7g10 mRNA stem-loop 9/RBS1, pBBR1MCS-2-(A/U)6/RBS1, and pBBR1MCS-2-(A/U)4/RBS1, bearing RBS variants with A/U-rich (A/U) sequences, were generated using corresponding primers P007_(A/U)12/rbs1_r, P007_(A/U)9/rbs1_r, P007_(A/U)6/rbs1_r and P007_(A/U)4/rbs1_r, which all overlapped with primer P008_rbs1_f. The use of primers P007_(A/U)12/rbs2_r and P007_(A/U)12/rbs8_r overlapping with primers P008_rbs2_f and P008_rbs8_f allowed construction of plasmids pBBR1MCS-2-(A/U)12/RBS2, and pBBR1MCS-2-(A/U)12/RBS8, respectively.To construct plasmids pBBR1MCS-2-SL/RBStem-loop  (SL), prC. necator-E. coli shuttle vector pEH006 (eyfp or rfp) fusion. The utilization of restriction sites for rare-cutter enzymes SbfI, PmeI, FseI, and AscI made these functional modules interchangeable  anangeable  for pMTLl-rhamnose-inducible system RhaRS/PrhaBAD was amplified with oligonucleotide primers EH007_f and EH008_r from pJOE7784.1  under the control of either different RBS variants or inducible systems were used for isoprene production. To construct pBBR1MCS-2-RBSx-ispS series with RBS variants (“x” is the corresponding number of RBSs), eyfp was replaced with ispS gene through restriction sites NdeI and BamHI or by the USER method. To construct plasmids for isoprene production using l-rhamnose-, l-arabinose-, acrylate-, and cumate-inducible systems, rfp was replaced with ispS through restriction sites NdeI and BamHI for plasmids pEH002, pEH006, and pEH020 and through NdeI and AflII for plasmid pEH040, resulting in pEH002-ispS, pEH006-ispS, pEH020-ispS, and pEH040-ispS, respectively. The DNA fragment containing ispS was prepared by either restriction digestion of plasmid pBBR1MCS-2-RBS1-ispS with NdeI and BamHI or by PCR amplification using plasmid pBBR1MCS-2-RBS1-ispS as a template and oligonucleotide primers P009_ispS_f and P010_ispS_r, followed by restriction digestion with NdeI and AflII.Plasmids harboring codon-optimized All plasmids constructed in this work were verified by Sanger sequencing and listed in Table S2.C. necator H16 cells were grown in minimal medium (MM)   with 4 mium (MM) .Relative normalized fluorescence values were calculated to display the fluorescence output of the four evaluated inducible systems on a common axis. This was performed by dividing absolute normalized fluorescence values by 110% of the highest value achieved during the time course of experiment.The normalized absolute fluorescence values were used to calculate the RFP synthesis rate for each inducer concentration at any time as reported previously . The rest), the maximum RFP synthesis rate (vmax), the inducer concentration (I), the Hill coefficient (h), the inducer concentration that results in half-maximal RFP synthesis (mK), and the basal rate of RFP synthesis (vmin). The fitted data for each system are illustrated in Fig. S2. The resulting parameters are summarized in Table S3.The parameters correspond to the time  of 1 for cells in exponential growth phase was pelleted by centrifugation at 13,000 × g for 1 min. The pellet was resuspended in 1 ml of TRI reagent (Sigma) and stored at −80°C. RNA was extracted using TRI reagent (Sigma) according to the manufacturer's recommendations. The samples were treated with 2 units of RQ1 RNase-free DNase (Promega), and 2 μg of total RNA was used for cDNA synthesis in a 20-μl reaction volume using the ProtoScript II first-strand cDNA synthesis kit (New England BioLabs). A real-time PCR analysis was performed in a 20-μl reaction volume using LuminoCt SYBR green qPCR ReadyMix (Sigma) and a Light Cycler 480 II (Roche). The relative quantification of mRNA levels was performed using the threshold cycle (ΔΔTC) method (eyfp gene).The wild-type and plasmid-transformed ) method . All bioC. necator H16 strains were inoculated to an OD600 of 0.1 into 10 ml of MM containing 4 mg/ml of fructose and 50 μg/ml of chloramphenicol and incubated at 30°C with vigorous shaking in sealed 60-ml serum bottles. For constructs with the ispS gene under the control of inducible promoters, the medium was supplemented with either 1.25 mM l-rhamnose, 1.25 mM l-arabinose, 0.5 mM acrylate, or 3.13 μM cumate. Gas samples from the headspace were taken for isoprene analysis 18 h after induction. Cultures of C. necator H16 strains containing plasmids with different RBS variants and ispS under the control of the constitutive promoter PphaC were sampled after 24 h of growth in sealed 60-ml serum bottles.Freshly grown overnight cultures of plasmid-transformed The cell absorbance was measured in a 1-cm-path-length cuvette using BioMate 3S UV-visible (UV-Vis) spectrophotometer at 600 nm .For isoprene quantification, gas samples were collected from the headspace of sealed 60-ml serum bottles containing 10 ml of culture. Isoprene was detected by gas chromatography (GC) using the instrument Focus GC  equipped with a flame ionization detector and an HP-AL/S column . Nitrogen gas was used as the carrier gas at a flow rate of 2 ml/min. The injector, oven, and detector temperatures were maintained at 220°C, 120°C, and 250°C, respectively. The injection volume was 1 ml. The yields of isoprene per gram of cells (dry weight) were estimated from standard curves generated by analyzing known quantities of isoprene. Dry weight was determined by washing cells from a 20-ml culture in distilled water and separation by centrifugation, followed by vacuum-freeze-drying and weighing cell pellet with microbalance.600 of 0.5 in MM supplemented with 20 mM fructose and 5 mM acrylate as carbon sources and allowed to grow for 48 h. Fructose and acrylate concentrations were measured by taking supernatant samples at 0, 2, 4, 8, 24, and 48 h and subjecting them to high-performance liquid chromatography (HPLC) in combination with UV spectroscopy using a Thermo Scientific Ultimate 3000 HPLC system equipped with a Phenomenex Rezex ROA-organic acid H+ (8%) column and a diode array detector with the wavelength set at 210 nm. The concentrations of metabolites in the supernatant were estimated from standard curves generated by analyzing known concentrations of magnesium acrylate and D-(−)-fructose.The metabolite consumption assay was performed as described previously . Cells wt test was applied to identify statistically significant differences in the normalized absolute fluorescence and relative mRNA levels. The correction for multiple comparisons was performed using Holm-Sidak method, and alpha level was set at 0.05. The Pearson correlation coefficient r was calculated to establish the degree of linear correlation between two variables . The t test and correlation analysis were performed using GraphPad Prism 7.01 built-in equations.The unpaired


Several inactivated enterovirus-A71 (EV-A71) vaccines are currently licensed in China; however, the development of additional EV-A71 vaccines is ongoing, necessitating extensive analysis of the molecular epidemiology of the virus worldwide. Until 2012, laboratory confirmation of EV-A71 for hand, foot, and mouth disease (HFMD) and other associated diseases had not occurred in the Philippines. Because EV-A71 has been linked with cases of acute flaccid paralysis (AFP), AFP surveillance is one strategy for documenting its possible circulation in the country. To expand current knowledge on EV-A71, molecular epidemiologic analysis and genetic characterization of EV-A71 isolates were performed in this study.A retrospective study was performed to identify and characterize nonpolio enteroviruses (NPEVs) associated with AFP in the Philippines, and nine samples were found to be EV-A71–positive. Following characterization of these EV-A71 isolates, the complete viral protein 1 (VP1) gene was targeted for phylogenetic analysis.n = 2), 2002 (n = 4), 2005 (n = 2), and 2010 (n = 1) were characterized using molecular methods. Genomic regions spanning the complete VP1 region were amplified and sequenced using specific primers. Phylogenetic analysis of the full-length VP1 region identified all nine EV-A71 Philippine isolates as belonging to the genogroup C lineage, specifically the C2 cluster. The result indicated a genetic linkage with several strains isolated in Japan and Taiwan, suggesting that strains in the C2 cluster identified in the Asia-Pacific region were circulating in the Philippines.Nine EV-A71 isolates detected in 2000 (The study presents the genetic analysis of EV-A71 in the Philippines. Despite some limitations, the study provides additional genetic data on the circulating EV-A71 strains in the Asia-Pacific region, in which information on EV-A71 molecular epidemiology is incomplete. Considering that EV-A71 has a significant public health impact in the region, knowledge of its circulation in each country is important, especially for formulating vaccines covering a wide variety of strains. Picornaviridae, genus Enterovirus, and species EV-A. Using the complete VP1 sequence for genetic classification, EV-A71 is divided into seven genogroups (A–G). Genogroup A contains only one prototype strain (BrCr), and genogroups B and C both have five subgenogroups . D belongs to an Indian genogroup, E and F are of African origin, and G was found in India [Enterovirus-A71 (EV-A71) is an RNA virus belonging to the family in India , 6–9. Siin India , 10–12.Although EV-A71 is implicated in HFMD, which is usually a mild, self-limiting disease, the infection is also associated with severe neurologic diseases including aseptic meningitis, acute encephalitis, polio-like paralysis, acute brainstem encephalitis, cerebellar ataxia, and fulminant neurogenic pulmonary edema –15. MuchIn total, 18,346 stool samples were collected from children presenting with AFP in the Philippines from 1992 to 2016, and 1332 NPEVs were isolated from these samples. Of these NPEVs, nine EV-A71 isolates were characterized in this study, including eight isolates identified as EV-A71 in a report published in 2012 using partial viral protein 1 (VP1) sequences  and an iThe purified PCR products were labeled directly using a cycle sequencing reaction version 3.1 kit and analyzed using a 3100 Avant Genetic Analyzer (Applied Biosystems). Sequencher version 4.10.1 was used to assemble and generate consensus sequences determined from two strands and trimmed to obtain the full-length VP1 sequences using BioEdit v 4.7. MEGA 7.0 was then used to translate nucleotide sequences to amino acid sequences and construct phylogenetic trees using the maximum likelihood method with a bootstrap value of 1000 pseudoreplicates. The substitution model was employed using the Tamura-Nei method. The nucleotide sequences were deposited in GenBank under the following accession numbers: JQ621835 (C2/EV-A71/182/PHL/2005), JQ621836 (C2/EV-A71/241/PHL/2002), JQ621837 (C2/EV-A71/255/PHL/2002), JQ621838 (C2/EV-A71/26/PHL/2000), JQ621839 (C2/EV-A71/263/PHL/2002), JQ621840 (C2/EV-A71/27/PHL/2000), JQ621841 (C2/EV-A71/323/PHL/2002), JQ621842 (C2/EV-A71/80/PHL/2005), and MF465901(C2/EV-A71/217/PHL/2010).EV-A71 was detected in 9 of 1332 NPEV-positive stool samples (0.70%) collected between 1992 and 2016 from seven children with AFP  –26. HoweBased on the available complete VP1 sequences detected between 1970 and 2008, genogroup C is more prevalent than genogroup B; however, the pattern of outbreaks in several Asian countries such as Taiwan indicated the alternating predominance of genogroups C2, B4, C4, B5, and C5 . The cirDuring this 25-year period, we found only EV-A71 genogroup C, in particular, subgenogroup C2 in 2002 and subgenogroup C2-like in 2000, 2002, 2005, and 2010 in different geographic locations in the Philippines (Fig. Generally, enteroviruses use two evolutionary mechanisms, namely mutation and recombination. Studies have demonstrated the presence of both intra- and intertypic EV-A71 recombinants –37. In bAlthough the present study only examined samples from patients with AFP opposed to a diverse group of individuals presenting with other clinical conditions, this study on EV-A71–associated AFP provides a focal premise that Philippine EV-A71 strains may have neurovirulent potential similarly as strains found in the Asia-Pacific region. Given the partial extent of EV-A71 detection in this study, a significant observation is that subgenogroup C2 might have a role in a broad spectrum of diseases. Many of the subgenogroup C2 strains detected previously were associated with fatal HFMD, aseptic meningitis, myelitis, encephalitis, bronchiolitis, and herpangina, and samples in this study were comprehensively obtained via 25 years of virologic surveillance of AFP-associated illness.This study reports the existence of only EV-A71 C2 subgenogroup in the Philippines, indicating that this subgenogroup is indigenously circulating in the country because it was isolated from different geographic areas. Although large outbreaks of EV-A71 infection have been described in the Asia-Pacific region, no outbreak has occurred in the Philippines. The present work could be used for future genetic detection studies on EV-A71 on a much larger scale to determine the overall epidemiology in the country. Given the potential health threat of the virus, EV-A71 should be monitored in the Philippines. Although limited in scope, the present study provides further information on the genetic pool of circulating EV-A71 strains in the Asia-Pacific region.


Blocking antibodies to NKG2A unleashed the reactivity of these effector cells resulting in tumor control in multiple mouse models and an early clinical trial. Monalizumab is inhibiting this checkpoint in human beings and future clinical trials will have to reveal its potency in combination with other cancer treatment options.The implementation of immune checkpoint inhibitors to the oncology clinic signified a new era in cancer treatment. After the first indication of melanoma, an increasing list of additional cancer types are now treated with immune system targeting antibodies to PD-1, PD-L1 and CTLA-4, alleviating inhibition signals on T cells. Recently, we published proof-of-concept results on a novel checkpoint inhibitor, NKG2A. This receptor is expressed on cytotoxic lymphocytes, including NK cells and subsets of activated CD8 HLA-A, −B and -C loci contributes to improved overall survival following ICI therapy compared to patients that were homozygous at one HLA class I locus with the largest effects at HLA-B and -C [+ T lymphocytes. Loss of HLA class I expression on tumors is a well-established and common phenotype associated with many tumor types and has been linked to poor outcomes [+ T cells mediate the strongest anti-tumor response and that maximal heterozygosity is, by design, necessary to achieve optimal presentation of neoantigens, this narrative potentially underestimates the antitumor roles mediated by NK cells in response to ‘immuno-edited’ tumors. We recently reported that blockade of the immune checkpoint NKG2A recruits CD8+ T cell- as well as NK cell-reactivity to the stage [Immuno-oncology has emerged as a revolution in cancer treatment. Unprecedented improvements in tumor control have been achieved with therapeutic blocking antibodies that release immune inhibitory ‘checkpoints’ . In particular, therapeutic monoclonal antibodies (mAbs) directed against the PD-1 (programmed-cell death protein 1)/PD-L1 (programmed-cell death ligand 1) axis have been approved for use in monotherapy or combinations for several cancer indications . Such trB and -C . PrimaryB and -C , making B and -C ), inhibiB and -C , 13 and B and -C ), interfB and -C  and downB and -C , which aoutcomes –25. Whilhe stage , 27. NKGhe stage , 29.www.proteinatlas.org) shows that HLA-E expression is, in general, ubiquitous but low. Exceptions are trophoblast cells in the placenta and ductal epithelial cells in the testis and epididymis, which display high levels of expression, suggesting a role for HLA-E in immune tolerance. Key factors of stabilization of the HLA-E protein at the cell surface are the availability of peptide ligands and proper function of the antigen processing machinery [HLA-A, −B and -C alleles on tumors promotes higher HLA-E cell-surface expression through provision of VML9 peptides [HLA-A encode a suitable HLA-E binding peptide, but polymorphisms across alleles drive differences in HLA-A expression [HLA-B is uniformly transcribed but has a dimorphism in its leader sequence at residue − 21 encoding either a good binding methionine (− 21 M) or a poor binding threonine (− 21 T) and thus varies whether or not it promotes HLA-E expression [A view at the comprehensive tissue slide collection of the human protein atlas  expressed PD-1 and importantly, half of them expressed NKG2A [+ T cells that protected the hosts upon re-challenge with the same tumor.Using a Qa-1ed NKG2A . A major+ TILs in cancer patients. Monalizumab, a humanized anti-NKG2A blocking mAb, increased degranulation and IFN-γ production by NKG2A+ NK cell against HLA-E+ target cells, thereby promoting NK cell effector functions [+ Flu-specific-CD8 T cells upon restimulation with Flu-specific-peptide in vitro. Importantly, when used in combination with durvalumab, an anti-PD-L1 blocking mAb, monalizumab exhibited additive effects promoting both NKG2A+ PD-1+ NK and CD8+ T-cell effector functions. Also, when combined with cetuximab, an anti-epidermal growth factor receptor (EGF-R) mAb which promotes antibody-dependent cell-mediated cytotoxicity (ADCC), monalizumab enhanced the NK cell-mediated ADCC [In human cancer samples, HLA-E was demonstrated widely expressed on the surfaces of several tumor types. Therefore, NKG2A blockade, either alone or in combination with other checkpoint inhibitors, might improve the anti-tumor efficacy of NK and CD8unctions . It modeted ADCC , suggest+ T cells.Following this rationale, evaluations of the efficacy and safety of monalizumab in cancer patients was conducted in phase II clinical trial using monalizumab in combination with cetuximab in patients with SCCHN (NCT026435509). In this interim report, an overall response rate (ORR) of 27.5% (95% CI 16–41%) was reported in 40 evaluable patients as compared to historical ORR of 13% observed for cetuximab monotherapy reported in earlier studies [Recently, dose escalation of first-in-human combination of monalizumab plus durvalumab in cohort of patients with metastatic microsatellite-stable colorectal cancer (MSS-CRC) has been completed (NCT02671435). Preliminary data demonstrate a manageable toxicity profile and indicate that the combination has encouraging activity in patients with MSS-CRC, a population historically nonresponsive to PD-1/PD-L1 blockade.+ αβ T cells. The frequencies of NKG2A expressing CD8+ T cells in blood of SCCHN patients was very low, in the range of 2–10%, whereas up to 50% of NK cells expressed CD94/NKG2A [+ T cells, indicating that NKG2A was induced in the tumor environment or that NKG2A-positive cells were selectively recruited there [+ T cells recognizing tumor antigens indeed are more likely to display this inhibitory receptor [+ TILs in cervical carcinoma samples interestingly suggested a preferential expression of NKG2A on T cells positive for the E-cadherin binding αEβ7 integrin. This CD103+ subset is associated with tissue residency, which is an epigenetically imprinted program mediating localization of lymphocytes to the tissues where they persist and patrol to protect organs for reoccurrence of pathogens [+ T cells and its higher expression on tissue resident cells versus other differentiation statuses of CD8+ T cells suggests an tissue-protective function for NKG2A on activated, antigen-specific lymphocytes [+ CD8+ T cells [In contrast to the rich literature of CD94/NKG2A receptors for NK cell biology, expression and function of NKG2A on adaptive immune cells is covered in paucity. In addition to NK cells, CD94/NKG2A is observed on subsets of innate lymphocytes, NKT cells, γδ T cells and CD894/NKG2A . Interesed there , 39, 46.ed there , 55. CD8receptor . CyTOF aathogens . The higphocytes –59. Howe T cells . To what T cells . NKG2A e+ T cell immunity to some viruses in that virus-driven immunopathology was limited and antiviral T cell responses were sustained by triggering NKG2A [+ T cell responses. In multiple cancer mouse models, NKG2A on CD8+ T cells functions as an immune checkpoint and blockade of the NKG2A/Qa-1 axis release the inhibitory signals  antigen comprising synthetic long peptide, RNA or DNA vaccines.NKG2A has been reported to regulate CD8ng NKG2A , 45, 61.+ T cells for immunotherapy with checkpoint blockers is well recognized [The importance of intratumoral CD8cognized , 8, but cognized . The NK cognized . Indepencognized , 63. Thicognized , 63. Impcognized , 65. Intcognized , 67. Tog+ and CD8+ anti-tumor T cell immunity, increase immune infiltration of human cancers and eradicate pre-malignant lesions [Although interest in cancer vaccines waned long ago due to a sheer lack of objective clinical responses in hundreds of trials, they recently regained attention since novel platforms demonstrated efficacy to induce broad CD4 lesions . Recent  lesions –71. More lesions . The add+ T cells while concurrently modulating NK cell inhibition and functional sensitization to tumors. Perhaps, the dual ability of HLA class I to regulate both NK cells and CD8+ T cells reflects differences in windows of immune activity, where NK cells lack the need for prior antigen-specific sensitization and can rapidly amplify the initial immune reaction [HLA-C locus compared to tumor regions without HLA-C LOH [The human immune system relies on HLA class I to present antigens to CD8reaction , 72–76. LA-C LOH .HLA-A, −B and -C genes indicate that human populations are divided into groups that are stratified by HLA-E expression (higher threshold for NK cell activation) and the presence or absence of KIR ligands (degree of NK cell education) that define whether NKG2A-expressing or KIR-expressing NK cells are dominantly activated in response to cytokines, Fc-receptor-mediated signaling, and to loss of HLA-E or KIR ligands on tumors and HIV-infected CD4+ T cells [HLA-B alleles had significantly better leukemia-free and overall survival compared to patients that were homozygous for -21 T HLA-B alleles and found correlations with diminished expression of HLA-E on primary AML blasts [Analyses of genetic variation in  T cells , 78, 79.L blasts .Future studies should consider a comprehensive analysis of HLA class I expression and immunoediting of HLA genes in the germline and matched tumor tissues when considering alleles of HLA class I that are specifically lost (or even duplicated) and whether they promote high HLA-E expression and encode KIR ligands. The level of HLA-E expression and presence or absence of KIR ligands in germline tissue will determine the educational environment and subsets of NK cells that are trained to react to perturbed expression of HLA on tumors, which has been shown to vary extensively across cancers .Understanding the effects of cytomegalovirus (CMV) infection (and reactivation) is also important in settings of cancer immunotherapy for its ability to imprint NK cell phenotypes and functions and promote expansion of adaptive or “memory-like” NK cell subsets  . Such ex+ NK cells represent over 50% of peripheral blood NK cells and is also expressed on a subset of activated CD8+ T cells during chronic viral infections, such as human immunodeficiency virus (HIV) [+ T cells remain NKG2A negative. André and colleagues showed that monalizumab can potentiate other ICI in a combination therapy, such as anti-PD-1/PD-L1 [combinations’ and future clinical trials will need to carefully determine which combination therapy provides the best results in the interest of our patients.NKG2Aus (HIV)  and hepaus (HIV) , and in us (HIV) . It is u-1/PD-L1  and Van -1/PD-L1 . A centr


Mitoflashes are bioenergetic mitochondrial events that control mitochondrial metabolism and signalling in both healthy and disease states. We discuss how Ca2+ fluctuations in pro- and pre-B cells may translate into mitoflashes in early B cells and speculate about the consequences of these changes.The most important feature of humoral immunity is the adaptation of the diversity of newly generated B cell receptors, that is, the antigen receptor repertoire, to the body’s own and foreign structures. This includes the transient propagation of B progenitor cells and B cells, which possess receptors that are positively selected via anabolic signalling pathways under highly competitive conditions. The metabolic regulation of early B-cell development thus has important consequences for the expansion of normal or malignant pre-B cell clones. In addition, cellular senescence programs based on the expression of B cell identity factors, such as Pax5, act to prevent excessive proliferation and cellular deviation. Here, we review the basic mechanisms underlying the regulation of glycolysis and oxidative phosphorylation during early B cell development in bone marrow. We focus on the regulation of glycolysis and mitochondrial oxidative phosphorylation at the transition from non-transformed pro- to pre-B cells and discuss some ongoing issues. We introduce Swiprosin-2/EFhd1 as a potential regulator of glycolysis in pro-B cells that has also been linked to Ca They are derived from pluripotent stem cells and develop through the following stages: hematopoietic stem cells (HSCs); common lymphoid progenitors (CLPs); B cell-biased lymphoid progenitors (BLPs); and pre-pro-, pro- and pre-B cells. B cell precursors require cell contact and specific niches in the BM for their survival and growth . Prolife+ signal . The fir+ signal ). Pre-pr+ signal . Another+ signal . B lymph+ signal  , enhancing survival and proliferation ,15. In v-B cells . The exp-B cells ) and pro-B cells ,13,14). -B cells ,20. −/− background in mice led to the development of phenotypic pre-B cells, while the introduction of µHC and lambda (λ)-LC led to the production of peripheral, monoclonal and immunoglobulin-secreting B cells [2+ concentration [Ectopic expression of the µHC on a Rag2 B cells . The Prentration ,24,25,26ntration . Hence, ntration . This dentration ,30. The ntration ,32. Exprntration . After tntration . Successntration . As outlntration )  across the IMM is established by the electron transport chain (ETC) by mitochondrial respiratory chain complexes I, III and IV, which pump protons from the matrix into the mitochondrial intermembrane space [p) that drives the synthesis of ATP via the ATP synthase complex (complex V) (∆p = ∆pHm + ∆ψm) [The ultimate downstream biochemical events that supply cells with adenosine triphosphate (ATP) are glycolysis and mitochondrial oxidative phosphorylation (OxPhos) (reviewed in detail in the context of lymphocytes ). The oxne space . ∆pH andm + ∆ψm) . The conm + ∆ψm) ,38,39. N-glycosylation  at 10 or 50 nM did not affect the appearance of-IgM positive WT B cells, while 10 nM oligomycin did alter the appearance of these cells in Fnip1−/− cultures. Extracellular flux analyses performed with a Seahorse analyser showed that pro-/pre-B cells responded to IL-7 by increasing their oxygen consumption rate   [−/− pro/pre-B cells responded better. To determine which substrates fuel the observed increase in OCR, sorted pro-/pre-B cells were treated with 2-DG and Etomoxir. Both treatments reduced the OCR in WT cells but it was reduced even more in Fnip1−/− pro-/pre-B cells. These data suggested that pro-/pre-B cells utilize glucose and FA for OxPhos and that Fnip1 renders pro-/pre-B cells resilient to inhibition of glycolysis and of FA oxidation. Further experiments connected this metabolic checkpoint to the Fnip1:AMPK complex and the pro-/pre-B cell transition (−/− pro-/pre-B cells is likely mediated by an increase in rps6ka1 expression [-/+ and PTEN−/− pre-B acute lymphoblastic leukaemia (ALL) cells [−/− mice [−/−;33.C9μHCki) [−/−;33.C9μHCki mice are mainly small. Extracellular flux analyses performed with sorted primary pro- and pre-B cells obtained from this system revealed that in general, under normoxic conditions, OCR and ECAR were lower in Rag1−/−;33.C9μHCki pre-B cells than pro-B cells. These data were confirmed by Zeng et al., who also analysed immature B cells, which have an OCR similar to that of small pre-B cells [Inhibition experiments performed with 2-deoxyglucose (2-DG), a non-hydrolysable glucose analogue that blocks glycolysis, have shown that pro-/early/pre-B cells depend on the glycolytic pathway, whereas late  pre-B cells do not . In contetail in ). Hence,etail in  and in Ftabolism . In WT Blication , that melication ) and exttracked) . Howeveransition A. The anpression . A similL) cells ,44. WhilL) cells . MitochoL) cells . Pro-B cL) cells . To meas−/− mice  (Rag1−/−C9μHCki) . Pre-B c-B cells . In cont-B cells ,46 and t-B cells  do indee-B cells ,47  revealed that plasma cells in the BM rely on this mechanism [It appears that the reduction of metabolism observed in small pre-B cells is maintained in immature B cells . Additiopyruvate . Interespyruvate . Glycolypyruvate . Glucosepyruvate . No infopyruvate  or BLNK/pyruvate , which fpyruvate , in addiechanism . −/− blastocyst complementation chimeras by reducing the number of proliferating CD43-HSA+B220+ cells [fl/fl or HIF2αfl/fl mice crossed to Mb1-Cre mice is normal [-HSA+B220+ cells affected by HIF1α deficiency are likely proliferating pre B cells, there is the possibility that HIF1α mediated metabolic adaptations influence B cell development already before the mb-1 promotor is active. HIF-1α controls glycolysis in BM precursor B cells in a developmental stage-specific manner by regulating the genes that encode glucose transporters and the key glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bishosphatase 3 [highCD5+ B cells via glycolysis and production of anti-inflammatory IL-10 by those B cells [A caveat of all the studies mentioned so far is that ex vivo experiments are generally performed under normoxic conditions, while many parts of the BM and several of its niches are hypoxic . Immune 0+ cells . On the s normal . Althoughatase 3 . Intereshatase 3 . These dhatase 3 . In acco B cells . Further B cells . Studyin B cells . The genes that control mitochondria and glycolysis are targets of both pre-BCR and BCR ,60. ThismTORC1-deficient pro- and perhaps early pre-B cells (Hardy fractions C and C’) developed in these mice, small pre-B cells (fraction D) were strongly reduced and later stages were absent. Although VDJ and VJ recombination were normal in the remaining C/C’ and D fraction cells, the expression of the IgM (μ) heavy chain was severely impaired, even when expressed as anti-hen egg lysozyme (HEL) transgenic BCR. Hence, B cell development was not rescued by the expression of anti-HEL BCR. It is unclear why mTORC1 activity is required for IgM expression. It is possible that mTORC1 controls IgM stability downstream of glycolysis and OxPhos, or it may support glycosylation via the hexosamine biosynthetic pathway and thereby stabilize the membrane expression of μHC within the pre-BCR complex A.2+-binding protein that localizes to the IMM and consists of an N-terminal disordered region, two central Ca2+-binding EF-hands and a C-terminal coiled-coil domain [efhd1 expression [2+ buffer parvalbumin [Swiprosin-2/EFhd1 (EFhd1) is a Cal domain ,69,70,71l domain ,73. PTENpression   to metabolic fitness in pre-B cells. Along these lines, the Crispr-Cas9-mediated knock-out of EFhd1 as well as the its shRNA-mediated knock-down in the transformed pro-B cell line 38B9 resulted in increased glycolysis and a higher glycolytic rate and glycolytic spare capacity  Figure A,B. We ppatients . sod2) [sod2, observed in pre-B cells [In parallel with decreasing IL-7 responsiveness, the pre-BCR initiates the expression of SLP-65/BLNK, allowing large pre-B cells to differentiate into small pre-B cells, by inhibiting the PI3K/Akt pathway and inducing a concomitant increase in FOXO1 activity ,76,77 FB. In smasod2) . The inc-B cells  is compa-B cells  and migh-B cells   has revealed that pHmito is in dynamic equilibrium with cytosolic pH . Similaiewed in ) and Ca2iewed in ). The spflashes” ,82. Mitohes” [2+  but ther2+ [mito ,82. It t2+ [mito ,84 shouloduction . Experimonomycin . This fuonomycin  in each handling . These dchondria  and that coupled . There i coupled  or succepled [2+ ,24,25,26 signals . In the 2+-binding protein that localizes on the IMM, Swiprosin-2/EFhd1 (EFhd1), might be involved in this phenomenon because it is expressed in pro-B cells and controls Ca2+-dependent mitoflashes. We have shown that EFhd1 becomes downregulated by cell surface expression of pre-BCR and that EFhd1 limits glycolysis in pro-B cells. We propose that EFhd1 might integrate Ca2+ signals with gene regulation and metabolic activity in pro- and early B cells and EFhd1 may thereby serve as a sensor for optimal pre-BCR signalling strength. The interplay between pre-BCR signalling and metabolism was clearly revealed in mice lacking mTORC1 activity in B cells. mTORC1 is required to maintain the stability of the IgM heavy chain and it therefore controls pre-BCR signalling at this level. The mechanism behind this function remains unknown but it may involve the generation of anaplerotic intermediate factors produced during glycolysis. In summary, we suggest that more extensively examining the regulation of metabolism in pro- and pre-B cells would increase our understanding of growth control and the generation of a healthy BCR repertoire. The metabolic regulation of pro- and pre-B cell development has important consequences for the expansion of normal and malignant pre-B cell clones. Under healthy conditions, it affects the normal BCR repertoire and contributes directly to adaptive immunity. To understand how the growth of normal or transformed pro- and pre-B cells is regulated, we have reviewed recent data on the regulation of glycolysis and oxidative phosphorylation in early B cells. It has become clear that pre-BCR ultimately induces metabolic quiescence because it leads to a reduction in glycolysis in late pre-B cells. The current data also suggest that large pre-B cells utilize glycolysis to sustain ATP but previous extracellular flux analyses of primary pro- and pre-B cells have missed large pre-B cells for technical reasons. Large pre-B cells may in the future be enriched from Irf4/8 double or BLNK/SLP-65 knock-out mice to clarify this issue. It remains unclear whether large pre-B cells (a) perform more glycolysis than is found in pro-B cells; (b) depend solely on glycolysis for their growth or (c) require pyruvate generated by glycolysis for ATP production via OxPhos. Hence, it remains completely unknown whether pro-B cells and pre-B cells depend in vivo on the mitochondrial respiratory chain. It will also be important to study metabolism in early B cells under hypoxic conditions, which occur naturally in BM. It is clear that HIF1α plays a role in early B cell development by promoting glycolysis in B cell progenitors. Here, we introduce the idea that recently described bioenergetic events in mitochondria (“mitoflashes”) that have been shown to maintain the proton motive force required to generate mitochondrial ATP may also occur in pro- and early pre-B cells. A Ca