This cross-sectional study uses molecular and serologic methods to investigate the 2021 surge in COVID-19 cases among vaccine recipients in Mongolia. This increase led the Mongolian government to mandate a shutdown in March 2021. A nationwide vaccination program was also initiated at that time, and 4 vaccines  were offered. This shutdown was relaxed in early June 2021 because the number of COVID-19 cases had decreased from more than 1400 per day in April to approximately 500 per day by the end of May, which was attributed to vaccination. At the end of June 2021, the number of cases increased rapidly to approximately 3000 per day .1This study was conducted to determine whether the surge of cases in Mongolia was associated with the emergence of the SARS-CoV-2 B.1.617.2 (Delta) variant or another SARS-CoV-2 variant or with breakthrough infections after vaccination.STROBE) reporting guideline.This cross-sectional study was conducted under Onom Foundation institutional review committee protocols and was approved by the Ministry of Health of Mongolia (eAppendix in the NC_045512), and variant analyses were performed using the GISAID database and the NextClade tool  and viral RNA from the anterior nares of patients with a breakthrough infection. Reads were mapped to the SARS-CoV-2 reference sequence . The threshold for statistical significance was set at P < .05.Plasma samples were obtained from 100 healthy asymptomatic participants who had received 1 of the following 4 COVID-19 vaccines: ChAdOX1-S , Gam-COVID-Vac , BBIBP-CorV , or BNT162b2  . The antIn this study, we sequenced 97 anterior nares samples from hospitalized patients with a symptomatic breakthrough SARS-CoV-2 infection. Full-length SARS-CoV-2 sequences were obtained from 92 of 97 patient samples with sufficient RNA. Of the 92 sequences, we identified 87 (94.60%) as the B.1.1.7 (Alpha) variant and 5 (5.40%) as the Delta variant. Of the 87 patients with an Alpha variant breakthrough infection, 54 (62.10%) had received the BBIBP-CorV vaccine, 23 (26.40%) the ChAdOX1-S vaccine, 7 (8.00%) the Gam-COVID-Vac vaccine, and 3 (3.40%) the BNT162b2 vaccine. Of the 5 patients with a breakthrough Delta variant infection, 1 (20.00%) had received the BBIBP-CorV vaccine, 3 (60.00%) the ChAdOX1-S vaccine, and 1 (20.00%) the BNT162b2 vaccine. At the time of sample collection, more than 1.8 million individuals (90.00%) had received the BBIBP-CorV vaccine.Of the 100 healthy asymptomatic participants, more individuals had a reactive spike-ELISA result after they received the BNT162b2 (24 [100.00%]) or ChAdOX1-S (19 [79.17%]) vaccine compared with those who received the Gam-COVID-Vac (12 [50.00%]) or BBIBP-CorV (7 [29.17%]) vaccine. Three BBIBP-CorV vaccine recipients (12.50%) had a reactive NCP-ELISA result. Three ChAdOX1-S vaccine recipients (12.50%) had a positive NCP result; this suggested a past SARS-CoV-2 infection, because currently the ChAdOX1-S vaccine contains the only spike protein. Only BBIBP-CorV, an inactivated whole virus vaccine, contains NCP. Of 4 participants who had received 2 BBIBP-CorV doses followed by 1 BNT162b2 dose, 1 (25.00%) had a weak spike-ELISA result .More participants had 100% end-point neutralizing antibodies to the Washington strain after they received the BNT162b2 (19 [79.17%]) or ChAdOX1-S (12 [50.00%]) vaccine compared with those who received the Gam-COVID-Vac (6 [25.00%]) or BBIBP-CorV (5 [20.83%]) vaccine. In addition, 100% end-point neutralizing antibodies to the Delta variant were present in fewer participants after they received the BNT162b2 (7 [29.17%]), Gam-COVID-Vac (2 [8.33%]), or BBIBP-CorV (3 [12.5%]) vaccine. Nine recipients (37.50%) had 100% end-point neutralizing antibodies to the Delta variant after they received the ChAdOX1-S vaccine. However, 3 of 9 participants (12.50%) had a positive NCP-ELISA result, indicating a previous SARS-CoV-2 exposure. Eliminating these 3 individuals would decrease the percentage of participants with 100% end-point neutralizing antibodies to the Delta variant to 25.00%. Of 4 individuals who received a single dose of BNT162b2 vaccine after 2 doses of BBIBP-CorV vaccine, 3 (75.00%) had 100% end-point neutralizing antibodies to the Washington strain and 1 (25.00%) had neutralizing antibodies to the Delta variant .4 Our data also suggest that the current COVID-19 vaccines, based on the prototypic Washington strain, elicit only minimal levels of 100% end-point neutralizing antibodies to currently circulating strains.6This cross-sectional study was limited in terms of the number of participants and its retrospective collection of specimens. Nonetheless, our findings provide support for previously published literature suggesting a hierarchy of protection among vaccine platforms, with messenger RNA vaccines performing better than adenovirus-vectored or inactivated-virus vaccines against SARS-CoV-2 infection.Our results suggest that the surge in COVID-19 cases in Mongolia in June 2021 was not associated with the introduction of the Delta variant but was more likely attributable to relaxed physical distancing and masking requirements. These findings underscore the importance of continuous surveillance for virus evolution, coordinated modification of vaccines, and a cautious approach to the elimination of classic social controls for mitigating contagion.


Patients with coronavirus disease 2019 (COVID-19) predominantly have a respiratory tract infection with various symptoms and high mortality is associated with respiratory failure second to severe disease. The risk factors leading to severe disease remain unclear. Here, we reanalyzed a published single-cell RNA-Seq (scRNA-Seq) dataset and found that bronchoalveolar lavage fluid (BALF) of patients with severe disease compared to those with mild disease contained decreased TH17-type cells, decreased IFNA1-expressing cells with lower expression of toll-like receptor 7 (TLR7) and TLR8, increased IgA-expressing B cells, and increased hyperactive epithelial cells (and/or macrophages) expressing matrix metalloproteinases (MMPs), hyaluronan synthase 2 (HAS2), and plasminogen activator inhibitor-1 (PAI-1), which may together contribute to the pulmonary pathology in severe COVID-19. We propose IFN-I (and TLR7/TLR8) and PAI-1 as potential biomarkers to predict the susceptibility to severe COVID-19. COVID-19, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)caused infectious disease, manifests various symptoms ranging from asymptomatic to mild to very severe, and leads to multiple organ injury and even death. Poor outcomes are associated with older age  and underlying conditions including diabetes, cardiovascular disease, hypertension, obesity, and chronic obstructive pulmonary disease (COPD) , associaThe published BALF scRNA-Seq dataset (GEO accession number GSE145926), including 3 mild and 6 severe SARS-CoV2 cases, and 3 healthy controls , was dowt test. p ≤ 0.05 was considered as significant.Results were expressed as mean ± SD. Differences between groups were calculated for statistical significance using unpaired Student’s + T cell compartments in severe COIVD-19  cells (+) cells +, but also TRDC−, TBX21−, and RORC−, therefore belonging to TH22 (or NKT), but not TH1 or TH17 cells. Whether IL22 plays a role in the disease severity remains to be determined. Besides the dysregulation in the T cell compartment, severe cases had increased frequencies of IgA1 (IGHA1+)-expressing B cells (and a trend of increasing IgG1 (IGHG1+)) (The scRNA-Seq dataset (GEO accession number GSE145926), including 3 healthy controls, 3 mild cases, and 6 severe cases , was dowre cases A. Intere+] cells A and γδT+) cells B; the la patient . TherefoIGHG1+)) D, in agr+ macrophages and a few other cells, such as keratin 18 (KRT18)+ epithelial cells. Macrophages of severe cases had decreased frequencies of IFNA1, but not IFNA2- and IFNB1-expressing cells relative to those of mild cases , whereas tissue inhibitors of metalloproteinases (TIMPs) inhibit the enzymatic activity of MMPs ,31. Of acentages A. There re cases A. In addre cases B. Macropld cases B. Togeth+, but not tPA+ macrophages (+ to PAI-1+ (tPA/PAI-1) cell ratios were decreased in severe cases relative to mild cases, and there was no difference in urokinase-type plasminogen activator (uPA)/PAI-1 /PAI-1 G. tPA an+), the neutralizing capability of these IgA antibodies is unknown. Third, this study analyzed mRNA expression of a variety of biomarkers in BALF cells, but lacked corresponding protein expression data, although there was some evidence at the protein levels from studies on peripheral blood (see references cited).This study has several limitations. First, the existing dataset only contains small numbers of mild cases and healthy controls. Second, although severe cases had increased frequencies of IgA1  and IFN T cells ,5,10), i


In particular, iPSC-derived NK cells gained interest in adoptive anti-cancer immunotherapies, since they enable generation of homogenous populations of NK cells with and without genetic engineering that can be grown at clinical scale. However, the phenotype of in-vitro generated NK cells is not well characterized. NK cells derive in the bone marrow and mature in secondary lymphoid tissues through distinct stages from CD56brightCD16- to CD56dimCD16+ NK cells that represents the most abandoned population in peripheral blood. In this study, we efficiently generated CD56+CD16+CD3- NK lymphocytes from hiPSC and characterized NK-cell development by surface expression of NK-lineage markers. Hematopoietic priming of hiPSC resulted in 31.9% to 57.4% CD34+CD45+ hematopoietic progenitor cells (HPC) that did not require enrichment for NK lymphocyte propagation. HPC were further differentiated into NK cells on OP9-DL1 feeder cells resulting in high purity of CD56brightCD16- and CD56brightCD16+ NK cells. The output of generated NK cells increased up to 40% when OP9-DL1 feeder cells were inactivated with mitomycine C. CD7 expression could be detected from the first week of differentiation indicating priming towards the lymphoid lineage. CD56brightCD16-/+ NK cells expressed high levels of DNAM-1, CD69, natural killer cell receptors NKG2A and NKG2D, and natural cytotoxicity receptors NKp46, NKp44, NKp30. Expression of NKp80 on 40% of NK cells, and a perforin+ and granzyme B+ phenotype confirmed differentiation up to stage 4b. Killer cell immunoglobulin-like receptor KIR2DL2/DL3 and KIR3DL1 were found on up to 3 and 10% of mature NK cells, respectively. NK cells were functional in terms of cytotoxicity, degranulation and antibody-dependent cell-mediated cytotoxicity.The differentiation of human induced pluripotent stem cells (hiPSCs) into T and natural killer (NK) lymphocytes opens novel possibilities for developmental studies of immune cells and  Natural Killer (NK) cells are an important part of the innate immune system. They play a crucial role in the defense of viral infections and cancer containment, as well as in immune regulation .In recent years, NK cells became favorable candidates for cellular anti-cancer immunotherapies because of their HLA-independent cytotoxic capacity against malignant and virally infected cells . The opt+CD45+ HPCs, embryonic bodies (EB) are generated using different approaches, such as spinning of single cell iPSCs in round-shaped wells (spin EBs), culture on murine stroma cells, or direct induction of iPSC monolayer fragments in media with cytokines inducing differentiation towards the hematopoietic lineage. HPCs are often enriched by cell sorting or cell separation of CD34+ and/or CD45+ cells, and subsequently placed on murine feeder cells  in medium containing IL-3 (during the first week), IL-7, IL-15, SCF, IL-2, and Flt3L. NK-cell differentiation without usage of xenogeneic stromal feeder cells has also been reported -derived NK cells after induction of hematopoietic progenitor cells (HPCs) –13. In oreported . HoweverbrightCD16- into CD56dimCD16+ NK cells takes place in secondary lymphoid tissues (SLTs), whereas CD56brightCD16+ NK cells are considered as functional intermediates  that allow homing to SLTs, liver, skin, and uterus where they interact with MHC molecules, antigens and homeostatic cytokines   is more specific to CD56dimCD16+ (Human NK cells derive from a common lymphoid progenitor cell in the bone marrow and their maturation can be categorized into 5 major stages . The lasmediates . CD56brit tissue . Althougceptors)  consistsceptors) . CD56briof NKp80 . WhereasdimCD16+ .in-vitro are supposed to lack self MHC interactions, although the impact of xenogeneic feeder cells on the maturation processes is not well understood.IPSC-derived NK cells generated In this study, we aimed to establish an efficient protocol to generate mature and functional NK cells from hiPSC with optimized efficiency. Furthermore, we characterized the phenotype of generated NK cells in terms of maturation and activation status by NK-lineage marker expression, as well as functional studies of degranulation and cytotoxicity.+ cell line P815  were used for functional NK cell studies. A human healthy control iPSC line derived from reprogramming of newborn human foreskin fibroblasts (NuFF) C.3 using the CytoTune iPS Sendai Reprogramming kit (ThermoFisher). Karyotype analysis showed no chromosomal abnormalities  and reprogramming-associated genes  was assessed in a quantitative PCR normalized on NHDF fibroblasts  by SSP-PCR  was used as feeder for differentiating NK cells. The human K562 cell line  and the murine mastocytoma FcR SSP-PCR . Materia+/CD45+ HPCs, the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies) was used according to manufacturer’s instructions. EBs from cell suspension were collected using a 40µm cell strainer  and rinsed off with 1ml differentiation medium A (1ml/12 well). 1% Penicillin-Streptomycin (ThermoFisher) was added to the differentiation medium A and B. On day 12, the suspension cells were harvested and passed through a 40μm cell strainer. No positive selection of HPCs was performed prior NK-cell differentiation.IPSCs were placed in one well of a 6-well culture plate coated with Vitronectin XF™ (STEMCELL Technologies) and grown to 60% - 100% confluency. With the help of the StemPro™ EZPassage™ Disposable Stem Cell Passaging Tool (ThermoFisher), colonies were cut into squares, carefully washed and transferred into one well of a 6-well ultra-low attachment plate. During overnight incubation, cell clusters developed into EBs of different sizes. To induce differentiation towards CD345 OP9-DL1 feeder cells/well were prepared in 6-well culture plates. On day 0, 2.5x105 HPCs (CD34+/CD45+) were plated on prepared feeder cells in 2ml NK differentiation medium  ] . All cyt4 - 2.5x105 cells were washed twice with PBS and stained with antibody mixes in 100µl PBS or staining buffer  for 15 to 30 min at room temperature (RT) in the dark. The following antibodies and isotype controls were used at concentrations of 1:100: Anti-Human CD43-PE (STEMCELL Technologies), Anti-Human CD45-FITC (STEMCELL Technologies), APC anti-human CD34 (BioLegend), PE anti-human CD3 (BioLegend), FITC anti-human CD16 (BioLegend), APC anti-human CD56 (NCAM) (BioLegend), FITC anti-human CD94 (BioLegend), PE anti-human CD117 (c-kit) (BioLegend), FITC anti-human CD161 (BioLegend), PE anti-human CD335 (NKp46) (BioLegend), PE anti-human CD7 (BioLegend), PE anti-human CD244 (2B4) (BioLegend), PE anti-human CD57 (BioLegend), PE anti-human CD226 (DNAM-1) (Miltenyi Biotec), PE anti-human CD159c (NKG2C) (Miltenyi Biotec), PE anti-human CD159a (NKG2A) (Miltenyi Biotec), PE anti-human NKp80 (Miltenyi Biotec), APC anti-human CD62L (BioLegend), PerCP/Cyanine5.5 anti-human CD56 (NCAM) (BioLegend), APC/Cyanine7 anti-human CD45 (BD Biosciences), APC anti-human CD69 (Miltenyi Biotec), APC anti-human CD181 (CXCR1) (Miltenyi Biotec), APC anti-human CD336 NKp40 (Miltenyi Biotec), APC anti-human CD337 (NKp30) (Miltenyi Biotec), APC anti-human CD314 (NKG2D), (Miltenyi Biotec), PE anti-human CD158b  (BioLegend), APC anti-human CD158e1  (BioLegend), APC Mouse IgG1, κ Isotype Ctrl (BioLegend), FITC mouse IgG1, κ Isotype Ctrl (BioLegend), PE Mouse IgG1, κ Isotype Ctrl (BioLegend). After two washes, cells were suspended in 50-100µl PBS or staining buffer and analyzed on a BD Accuri C6 Flow Cytometer (Becton Dickinson Biosciences), or a BD FACSAria™ Cell Sorter (BD Biosciences), respectively.2x10NK cells generated on OP9-DL1 cells were harvested after w3, and incubated with K562 cells at effector:target (E:T) ratios of 1.5:1, 3:1, 6:1, 12:1, 25:1, 50:1, and 100:1 at 37°C for 1h. K562 cells were labeled using CellTrace™ far red cell proliferation kit (ThermoFisher) according to manufacturer’s instructions. Lysis of K562 cells was calculated based on the same numbers of K562 cells cultured without NK cells. For degranulation studies, CD107a  was analyzed on surfaces of non-permeabilized NK cells cultured with K562 at the same E:T ratios used for the cytotoxicity assay. Additionally, NK cells were fixed and permeabilized , and subsequently stained for perforin , granzyme B , and interferon-gamma . IFNγ production was further analyzed in NK cells stimulated with phorbol-12-myristat-13-acetat (PMA) (Sigma-Aldrich) (10ng/ml) and Calcium Ionophore A23187 (Sigma-Aldrich) (1µg/ml) at 37°C for 3h, followed by BD GolgiPlug™ (BD Biosciences) (2µl/ml) for 2h. Cells were analyzed on a BD FACSAria™ Cell Sorter.+ cell line P815 was coated with mouse anti-human CD16 antibody (BioLegend) or IgG1 kappa isotype control (BioLegend). Coated and uncoated P815 were subsequently used as target cell line for NK cells in various E:T ratios . P815 were labeled using CellTrace™ (ThermoFisher) according to manufacturer’s instructions, and percentages of lysed P815 cells were analyzed. In addition, CD107a surface expression was studied on NK cells in response to P815 as mentioned above.The redirected antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated based on a protocol reported by Byceson et al. . The murp-values on multiple comparisons were calculated using two-way ANOVA and bonferroni post test; p<0.05 was considered significant. Mean values and standard errors of the mean (SEM) are plotted. NK cell differentiation efficiencies per HPC were calculated using Microsoft Excel.All experiments were performed 3-5 times. GraphPad Prism 5 software was used for statistical analysis; +CD45+ double positive. CD43, a common marker to measure hematopoietic induction efficiency besides CD34  expression could increasingly be detected on NK progenitors from w1-w3 up to 66%. According to the model of NK-cell differentiation suggested by Freud et al. , and on 94,9% of cells obtained in week 3 (w3)  and inhibiting NK cell receptors (NKG2A) was detected, however, expression of NKG2C was much lower , NKp44 , and NKp30  were expressed in high levels on most NK cells , was detin-vitro  from hiPSC –41. OP9-genitors  and perigenitors , it is kgenitors , 44. Of in vitro differentiation process from HPCs to NK lineage committed cells (CD117+ and CD122+), NK precursor cells (CD161+ and CD56+), immature NK cells , and eventually mature NK cells    . The maj in vivo , 45. The in vivo , 51, and6- cells . CD56dimpression . Inhibits (MSCs) , 33.brightCD16-/+ NK cells generated with our protocol displayed a highly activated phenotype shown by upregulation of CD69, and CD62L. However, DNAM-1 (CD226), could be detected on only one third of NK cells in our study. DNAM-1 is expressed early in NK cell development and associated with education  on OP9-DL1 stroma cells and are highly functional in terms of degranulation, cytokine production and cytotoxicity including ADCC. IPSC-derived CD56bright NK cells can acquire KIR expression, although in a limited fashion and most likely dependent on donor source. NK cell yield can be considerably increased through inactivation of feeder cells with MMC without impacting on maturation or functional properties.We report a robust protocol for The original contributions presented in the study are included in the article/KF and KS conceptualized, planned and supervised the study. JE and JS performed the experiments and analyzed the data. TC provided the iPSC line. DF performed the HLA and KIR genotyping of the iPSC line. HS and K-MD supervised the study. JE and KF wrote the manuscript, which was approved by JS, TC, HS, K-MD, and KS. All authors contributed to the article and approved the submitted version.JE and JS are fellows of the International Graduate School in Molecular Medicine, Ulm. KF holds a grant from the German Research Foundation (FE1253/3-1).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.


Diabetes mellitus is a global threat affecting millions of people of different age groups. In recent years, the development of naturally derived anti-diabetic agents has gained popularity. Okra is a common vegetable containing important bioactive components such as abscisic acid (ABA). ABA, a phytohormone, has been shown to elicit potent anti-diabetic effects in mouse models. Keeping its anti-diabetic potential in mind, in silico study was performed to explore its role in inhibiting proteins relevant to diabetes mellitus- 11β-hydroxysteroid dehydrogenase (11β-HSD1), aldose reductase, glucokinase, glutamine-fructose-6-phosphate amidotransferase (GFAT), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and Sirtuin family of NAD(+)-dependent protein deacetylases 6 (SIRT6). A comparative study of the ABA-protein docked complex with already known inhibitors of these proteins relevant to diabetes was compared to explore the inhibitory potential. Calculation of molecular binding energy (ΔG), inhibition constant (pKi), and prediction of pharmacokinetics and pharmacodynamics properties were performed. The molecular docking investigation of ABA with 11-HSD1, GFAT, PPAR-gamma, and SIRT6 revealed considerably low binding energy  and predicted inhibition constant (pKi from 6.01 to 5.21 µM). The ADMET study revealed that ABA is a promising drug candidate without any hazardous effect following all current drug-likeness guidelines such as Lipinski, Ghose, Veber, Egan, and Muegge. Diabetes is one of the most prevalent epidemics, affecting almost 382 million people worldwide. According to the International Diabetes Federation (IDF) report, it is alleged that approximately 1.3 million people die from diabetes every year. IDF suggests that around 629 million people will have diabetes by 2045 worldwide  is a nutritive vegetable known by various names such as lady’s finger, green ginseng, and plant Viagra , aldose reductase (PDB ID-3G5E), glucokinase (PDB ID-4IXC), glycogen synthase kinase-3 [GSK-3 (PDB ID-3F7Z)], glucosamine:fructose-6-phosphate amidotransferase [GFAT (PDB ID-2ZJ4)], pyruvate dehydrogenase kinase (PDB ID-4MP2), peroxisome proliferator-activated receptor-gamma (PDB ID-3DZY), sirtuin family of NAD(+)-dependent protein deacetylases [SIRT6 (PDB ID-3K35)] and Tyrosine kinase (PDB ID-1IR3), shown in ABA was found to have a binding energy of −8.1 kcal/mol, −7.3 kcal/mol, −7.3 kcal/mol, −7.3 kcal/mol, −6.8 kcal/mol, −6.6 kcal/mol, −6.6 kcal/mol, 6.3 kcal/mol, and −6.2 kcal/mol, with 11β-HSD1, GFAT, PPAR-gamma, SIRT6, glucokinase, aldose reductase, glycogen synthase kinase-3, Pyruvate dehydrogenase kinase (PDK), and Tyrosine kinase proteins, respectively . The binIntracellular conversion of metabolically inert cortisone to active cortisol using NADPH as a co-factor is carried out by the 11β-HSD1 enzyme ,13,14,15The X-ray crystallography investigations of the crystal structure of an inhibitor molecule -3-(cyclohexylamino)-4a,5,6,7,8,8a-hexahydrobenzo[e]oxathiazine 1,1-dioxide (C13H22N2O3S) docked with human and murine 11β-HSD1 proteins revealed that its cyclohexyl-NH interacts with the key active site residue Tyr183 . FurtherInterestingly in our in silico analysis, we found that key residues of active site viz., TYR(A)183 and SER(A)169 of the human 11β-HSD1 enzyme interact with the two oxygen atoms of the 3-Methylpenta-2,4-dienoic acid substructure of abscisic acid -3-methylpenta-2,4-dienoic acid)]. Nine other amino acid residues of the human 11β-HSD1 enzyme, LEU(A)126, VAL(A)168, SER(A)170, ALA(A)172, VAL(A)180, LYS(A)187, LEU(A)215, GLY(A)216, and VAL(A)231 formed Van-der-Waals interaction with ABA A–D. ThisGlutamine-fructose-6-phosphate amidotransferase (GFAT) is a rate-limiting enzyme in the hexosamine biosynthetic pathway a key regulator in T2DM ,21,22. IThe C-terminal 40 kDa isomerase domain of GFAT (residues Gln313–Glu680) contains the active site (near the CF helix) and is involved in converting fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P) utilizing ammonia (NH3) as a substrate. The X-ray diffraction pattern shows that the bound ligand AGP (2-deoxy-2-amino glucitol-6-phosphate) with GFAT (PDB ID 2ZJ4) establishes hydrogen bonds with Thr375, Ser376, Ser420, Gln421, Ser422, and Thr425 directly, and other hydrogen-bonds with Val471, His576, and Ala674 from the neighboring subunit pyrimidin-4-yl]oxy}-Our docking and binding analysis exhibit a good binding pattern  of ABA with human glucokinase (PDB ID-4IXC) protein A,B, whicAldose reductase is the rate-limiting enzyme in the polyol pathway. It converts excess D-glucose to D-sorbitol with NADPH as a co-factor . It is cOur docking and binding analysis exhibit a good binding pattern  of ABA with aldose reductase (PDB ID- 3G5E) protein A–D, whicGSK-3, a unique multifunctional serine/threonine kinase, is involved in the glycolysis pathway. GSK-3 is active and capable of synthesizing glycogen in its unphosphorylated state. PKB/AKT phosphorylates GSK-3 on serine 9 in response to insulin binding . As a reHowever, unlike previously mentioned proteins, GSK-3 (PDB ID -3F7Z) did not show a good binding pattern with ABA  A–D. TherPKD negatively regulates the mitochondrial pyruvate dehydrogenase complex (PDC) activity by reversible phosphorylation. PDK isoforms are upregulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases .Our analysis showed a poor binding pattern  of ABA with PKD (PDB ID- 4MP2) protein A–E due tFurther, ABA displayed a weak binding pattern  with the human tyrosine kinase (PDB ID-1IR3) protein A–D due tThe molinspiration bioactivity score (v2018.03) is calculated and presented  for actiThe pharmacokinetic properties and drug-likeness data are summarized in A BOILED-Egg plot predicts the gastrointestinal absorption and brain penetration of small molecules. Here PGP+/− shows the P-glycoprotein substrate positive/negative nature of the molecule under study. The BOILED-Egg’s white (white area) predicts that the molecule located in this area may be passively absorbed by the human intestinal tract (HIA). The ABA molecule (red circle) is located at the extreme periphery of BOILED-Egg’s yolk (yellow area), which predicts that the ABA molecule may passively permeate through the blood-brain barrier (BBB) but have very low chances of −0.047 log BB, contrary to the chances of ABA being absorbed by the human intestinal tract (HIA) is 96.712% .The radar graph shows that various physico-chemical and molecular characteristics of ABA like LogP (2.342), LogS (−2.465), LogD (1.656), molecular weight (264.32 g/mol), nHA (4), nHD (2), number of rotatable bonds (3), number of rings (1), rigid bonds (10), heteroatoms (4), and atoms in the biggest ring number of rings (6), formal charge (0), and topological polar surface area (74.60 Å²) have optimum values within the upper and lower limit .The human proteins related to diabetes mellitus 11-β-hydroxysteroid dehydrogenase (PDB ID-4K1L), aldose reductase (PDB ID-3G5E), glucokinase (PDB ID-4IXC), glycogen synthase kinase-3 (PDB ID-3F7Z), glutamine:fructose-6-phosphate amidotransferase [GFAT (PDB ID-2ZJ4)], pyruvate dehydrogenase kinase (PDB ID-4MP2), peroxisome proliferator-activated receptor-gamma (PDB ID-3DZY), sirtuin family of NAD(+)-dependent protein deacetylases [SIRT6 (PDB ID-3K35)], and Tyrosine kinase (PDB ID-1IR3) were retrieved from the RCSB Protein Data Bank   44]. Th. Th44]. https://pubchem.ncbi.nlm.nih.gov/compound/5280896) (accessed on 15 April 2021) (The ligand molecule ABA -3-methylpenta-2,4-dienoic acid)] in 2D (SDF) format was downloaded from PubChem (il 2021) . The ligBefore docking, the protein structures downloaded from PDB were analyzed in PyMol software , and the already docked ligands or nucleic acid or heteroatoms or water molecules were removed from the X-ray crystallographic protein-ligand complexes. Then the pure proteins as a receptor were prepared in Swiss-Pdb viewer (version 4.1.0) by optimizing bonded atoms, angles, torsions, non-bonded atoms, and improper atoms of the protein backbone and side chains.The docking calculations were performed using the AutoDock Vina version 4.2 (ADT4.2) software suite . The recThe visualization and analysis of protein-ligand complexes were performed using PyMOL software . The receptor’s active sites and interactions with the ligand or drug were determined using the PDBe  and PDBsMoreover, it is concluded that ABA may act as a competitive inhibitor that should compete with the known substrates to the active centers of the protein targets relevant to diabetes mellitus. Therefore, it induces a competitive type of inhibition, that inhibitors could bind to only the free enzyme and formed reversible enzyme-inhibitor (E-I) complexes. An enzyme-inhibitor complex’s inhibition constant (Ki) is traditionally calculated by the basic equations of enzyme kinetics of the Lineweaver–Burk assay extrapolated on 2D plots. If there is inconsistency in the Lineweaver–Burk plots, non-linear regression of the Michaelis–Menten equation is used to validate the related constants obtained.Sophisticated arithmetic and analytical in silico algorithms have been proposed to compute the inhibition constant (Ki) parameter, since the Ki principally depends on the binding (or association) constant (Kb) and dissociation constant (Kd) of an enzyme-inhibitor complex, which occurs in opposite directions (ln Kb = −ln Kd).Therefore, Ki is computationally calculated using the following formula:The binding energy ΔG is in kcal/mol, the universal gas constant R = 1.987 kcal/K/mol, at room temperature (25 °C) T = 273 + 25 = 298 K. Ki is having a unit of mM.https://www.molinspiration.com/cgi-bin/properties), (accessed on 29 May 2021), an online screening server based on sophisticated Bayesian statistics, was implemented to analyze the pharmacodynamics properties of ABA. It compares representative ligands’ structures and determines physico-chemical properties of a particular molecule for being active molecules. There is no need to know about the target’s 3D structure or binding mode. The trained model makes it possible to screen large libraries of hundreds of thousands of molecules in less than an hour to identify molecules with the highest chance of becoming active drugs, pesticides, irritants, or toxic substances. The larger the value of the bioactivity score is, the higher the probability that the particular molecule will be active.The molinspiration  of chemicals have long been recognized as important considerations in the early stages of CADD.The pkCSM , SwissADSwissADME  of SwissADMETlab 2.0 has a greater capacity to assist medicinal chemists in accelerating the drug research and development process. It allows users to calculate and predict 17 physicochemical parameters, 13 medicinal chemistry measures, 23 ADMET endpoints, 27 toxicity endpoints, and eight toxicophore rules (751 substructures) quickly and easily, allowing them to identify interesting lead compounds for further investigation.The major target of the study was to examine if the substance in question inhibited the cytochrome P450 (CYP) family’s CYP1A2 and CYP2D6 isoforms. Pharmacokinetics parameters such as human intestinal absorption, P-glycoprotein, and the BBB and drug-likeness prediction Lipinski, Ghose, and Veber criteria, as well as the bioavailability score, are crucial in judging the molecule ,42,54. AAccording to Lipinski’s “Rule of 5” most “drug-like” compounds have logP ≤ 5, molecular weight (MW) ≤ 500, number of hydrogen bond acceptors (nHA) ≤ 10, and number of hydrogen bond donors (nHD) ≤ 5 . The molTopological Polar Surface Area (TPSA) is calculated based on the methodology published by Ertl et al. (2000) depending on the fragment contributions . TPSA isEffective medications with no cytotoxicity are needed to treat diabetes mellitus, and phytohormones such as ABA are among the best natural extract with no side effects. Molecular docking investigation of ABA with nine different protein targets relevant to diabetes mellitus revealed four potential target proteins perfectly docks with ABA. Docking analysis also revealed that based on binding energy (ΔG) and predicted inhibition constant (pKi), 11β-HSD1 (4K1L) showed best binding with ABA, followed by GFAT (2ZJ4), PPAR-gamma (3DZY), and SIRT6 (3K35), which were equal in inhibition constant. The docking and interaction pattern of ligands were fantastically able to interact with the key residues of the catalytic cavity of the enzyme or located in the very close proximity of the active sites of these proteins. Following all current drug-likeness guidelines such as Lipinski, Ghose, Veber, Egan, and Muegge, the pharmacodynamic and pharmacokinetic features with ADMET study revealed that ABA could be taken best molecule without any hazardous effect. A BOILED-Egg plot and radar graph analysis confirm that all the molecular and physico-chemical properties of ABA are within the upper and lower limit fulfilling all the criteria of an ideal drug. Thus, ABA can be considered a potential candidate for developing a potent anti-diabetic drug and a promising bioactive compound of okra for developing nutraceuticals and functional foods.


Furthermore, [18F]AlF-mNOTA-GZP showed positive associations with changes in tumour-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells, and decreases in suppressive F4/80+ cells. [18F]AlF-mNOTA-GZP tumour uptake was mediated by GZB expressing CD8+ cells and successfully stratifies therapy responders from non-responders, potentially acting as a non-invasive biomarker for ICIs and combination therapy evaluation in a clinical setting.Immune checkpoint inhibitors (ICIs) block checkpoint receptors that tumours use for immune evasion, allowing immune cells to target and destroy cancer cells. Despite rapid advancements in immunotherapy, durable response rates to ICIs remains low. To address this, combination clinical trials are underway assessing whether adjuvants can enhance responsiveness by increasing tumour immunogenicity. CpG-oligodeoxynucleotides (CpG-ODN) are synthetic DNA fragments containing an unmethylated cysteine-guanosine motif that stimulate the innate and adaptive immune systems by engaging Toll-like receptor 9 (TLR9) present on the plasmacytoid dendritic cells (pDCs) and B cells. Here, we have assessed the ability of AlF-mNOTA-GZP, a peptide tracer targeting granzyme B, to serve as a PET imaging biomarker in response to CpG-ODN 1585 in situ vaccine therapy delivered intratumourally (IT) or intraperitoneally (IP) either as monotherapy or in combination with αPD1. [ Immunotherapy has emerged as the fourth pillar of cancer treatment, along with chemotherapy, radiotherapy, and surgery. Immunotherapy exploits the host immune system, activating it to identify and destroy cancer cells . Immune CpG-oligodeoxynucleotides (CpG-ODN) are small synthetic DNA fragments containing unmethylated cysteine-guanosine motif dinucleotides that stimulate the innate and adaptive immune systems. These CpG-ODNs can mimic the immunostimulatory activity of bacterial DNA, activating Toll-like receptor 9 (TLR9) ,4,5,6 pr18F]AlF-mNOTA-GZP) and demonstrated that it can successfully stratify tumours responding to combinations of immune therapeutics from non-responding tumours [18F]AlF-mNOTA-GZP, can also be used for the stratification of response to CpG-ODN in situ vaccine therapy combined with the immune checkpoint inhibitor αPD1 in a syngeneic mouse model of colon cancer. Furthermore, we explored whether different routes of administration of CpG-ODN could influence different tumour-associated immune responses, correlating tumour uptake of [18F]AlF-mNOTA-GZP to differences in tumour-infiltrating immune cells using FACS.Non-invasive assessment of response to these novel combination therapies is complicated. Currently, there are a lack of specific biomarkers capable of providing a readout of in situ immune responses to different treatment strategies, complicating interpretation of clinical trials combining different treatment strategies, such as in situ vaccines and ICIs. Numerous studies have demonstrated that tumour-associated immune infiltrates are required for immune therapy efficacy, and numerous imaging biomarkers have been developed to quantify immune cell infiltrates, such as the T lymphocyte populations CD3 and CD8. However, these biomarkers provide little context and can be negatively affected by immune suppression. Granzyme B is a serine protease released by active NK cells and CD8+ cytotoxic T-cells along with the pore-forming protein perforin to induce apoptosis in tumours providing information on tumouricidal activity. We have previously characterised a PET imaging-based peptide biomarker targeting granzyme B  Accell™ plus QMA carbonate cartridges and Sep-Pak® C18 light cartridges were purchased from Waters Corporation . Saline solution (0.9% w/v) was purchased from Braun Medical Industries (Singapore). All other chemicals and reagents were purchased from Sigma-Aldrich (Singapore), Fisher Scientific  and Tokyo Chemical Industry . Carrier free aqueous [18F] fluoride was produced via the [18O18F] nuclear reaction . Quality control of radiolabelled tracer was performed on a UFLC Shimadzu HPLC  system equipped with a dual-wavelength UV detector and a PMT-radio detector . Radioactivity measurements were made with a CRC-55tPET dose calibrator .All reagents were purchased from commercial suppliers and used without further purification. H-Asp (OtBu)-H NovaSyn TG resin (0.21 mmol/g) was obtained from Merck (Singapore). Fmoc-amino acids, HATU and HOAt, were obtained from Advanced Chemtech . Fmoc-glutamic acid was t-butyl protected. (p-SCN-Bn)-NOTA was purchased from Macrocyclics . Sep-Pak18F]AlF-mNOTA-GZP was prepared as previously described [18F]AlF-mNOTA-GZP was prepared as a colourless solution  with a non-decay corrected radiochemical yield of 19–25% (reaction time ~50 min). The purity of the radiotracer was >98%, and molar activity was 45 ± 20 GBq/µmol (n = 6).The synthesis of NOTA–β-Ala–Gly–Gly–Ile–Glu–Phe–Asp–CHO (mNOTA-GZP) was prepared using standard Fmoc chemistry characterisation was done by using HPLC and mass spectroscopy as previously described . Radioch3 (after 5 days of implantation). The animals were randomised into treatment groups, treated either with rat IgG2a isotype control antibody, CpG-ODN monotherapy (300 μg of CpG-ODN 1585 oligonucleotide (Integrated DNA technology)  delivered either via intratumoural (IT) or intraperitoneal (IP) route on days 5, 8 and 11), αPD1 monotherapy  on days 6, 9 and 12), or a combination therapy groups (αPD1 & CpG-ODN (IT) or αPD1 & CpG-ODN (IP) as per the regimen described above (2) [All animal procedures were carried out according to Institutional Animal Care and Use Committee Singapore (IACUC No. 181399) and NIH guidelines. Five- to seven-week-old female BALB/c mice were purchased from InVivos (Singapore). A CT-26 murine colon model was developed as described previously [ed above . Animalsbove (2) . Tumour 18F]AlF-mNOTA-GZP (~15 MBq) via the lateral tail vein. A 10-min static PET acquisition was performed at 60 min post-injection (p.i.) of the tracer, and a CT scan was acquired for anatomical co-registration. Animals were monitored during imaging using the BioVet physiosuite for their body temperature and respiration rate. The reconstruction and the data analysis were performed as described previously AlF-mNOTA-GZP tumour uptake was correlated across all data sets before post hoc manipulation . When individual treatment groups were assessed prior to stratification, overall %TGI and tumour uptake of [18F]AlF-mNOTA-GZP were found to be positively correlated only in the CpG-ODN (IT)-  and αPD1 + CpG-ODN (IT)-treated groups . The αPD-1 monotherapy cohort did not reach significant difference  due to the variability in tumour responses (<40% of response rate). Similarly, the monotherapy CpG-ODN (IP)-treated cohort  and combination therapy cohort of αPD1 + CpG-ODN (IP)  were not well correlated, perhaps due to low tumour accumulation and rapid clearance of CpG-ODN from the body (Tumour volumes were converted to percentage tumour growth inhibition (%TGI), and AlF-mNOTA-GZP revealed that tumours could be visualised above background, and the radiotracer uptake was found to be heterogeneous across different treatment arms  and TNRs . Significantly higher uptake was observed in all TRs from both monotherapy αPD1  and CpG-ODN (IT)  and combination therapy αPD1 + CpG-ODN (IP)  and αPD1 + CpG-ODN (IT)  cohorts .In-vivo PET imaging with [ent arms A. [18F]Aapy arms B,C. In g cohorts B. Furthep > 0.05) . We observed some uptake in immune-related organs, particularly the spleen; however, no significant differences in spleen uptake were observed between responders and non-responders  .p < 0.05), CpG-ODN (IT) , combination therapy αPD1 + CpG-ODN (IP)  and αPD1 + CpG-ODN (IT) . However, there was no significant increase in the GZB+CD45+T cell population in the CpG-ODN (IP) alone group when compared to TNRs   A.p < 0.05), CpG-ODN (IT)  and in combination therapy αPD1 + CpG-ODN (IP)  and αPD1 + CpG-ODN (IT) . Again, no significant changes were noticed in the GZB+CD8+ cells numbers in the CpG-ODN (IP)-treated mice in contrast to TNRs group  (p < 0.05), CpG-ODN (IT)  and combination therapy αPD1 + CpG-ODN (IP)  and αPD1 + CpG-ODN (IT)  responders. Again, the CpG-ODN (IP) monotherapy group did not show any significant changes in the CD25+CD4+ cell population when compared to the TNR group   B. In add > 0.05) C.p * < 0.05), CpG-ODN (IT)  and combination αPD1 +CpG-ODN (IT) . However, no such reduction was observed in the CpG-ODN (IP)  or combination αPD1 + CpG-ODN (IP) groups  compared to TNRs (21.03 ± 2.56) (p > 0.05). When looking at total lymphocyte cell composition as the % GZB+CD45+, most of the cell type is associated with CD8+ and is significantly higher in TRs when compared to TNRs. We also noticed a majority of NK+ cells, CD4+ and other non-T cells at the tumour site  D. Intere ± 2.56) . Althougour site .CpG-ODN is presented as an effective adjuvant to elicit a stronger anti-tumour response when combined with immune checkpoint inhibitors. CpG-ODN has been shown to modulate the immune system through engagement with TLR9 receptors expressed on plasmacytoid dendritic cells (pDC) or B cells ,24,25. TCpG-ODN is being explored clinically for use as a vaccine adjuvant in combination with ICIs, chemo, or radiotherapy for various cancer types  ,27,28,2918F]AlF-mNOTA-GZP, a probe binding to granzyme B released by activated cytotoxic T cells, to act as a surrogate biomarker of response to CpG-ODN, alone or in combination with αPD1. CpG-ODN elicited a stronger anti-tumoural effect when administered intratumourally compared to intraperitoneally  from non-responders (TNRs). The increased tumour uptake of [18F]AlF-mNOTA-GZP in TRs was associated with a robust increase in tumour-associated activated T cells, especially GZB+ CD8+ cells, GZB+ CD45+ cells and CD25+ CD4 cells  confers selectivity for human granzyme B , potenti18F]AlF-mNOTA-GZP to stratify response to combined αPD1 therapy and in situ vaccination with CpG-ODN in a CT-26 murine colon cancer model. Therapy response was mediated by GZB expressing CD8+ cells and strongly correlated to [18F]AlF-mNOTA-GZP tumour uptake. Potentially, granzyme B imaging agents could be developed as a biomarker for adjuvant in situ vaccine therapy for use in combination with ICIs in a clinical setting.In summary, we have assessed the ability of [


Cell Death and Disease (2019) 10:1–12 10.1038/s41419-019-1940-3, published online 18 September 2019Correction to: The original version of this article unfortunately contained a mistake in Fig.


The expression and biological function of long intergenic noncoding RNA00265 (LINC00265) in gastric cancer (GC) have not yet been explored. This study aimed to detect LINC00265 expression in GC tissues and cell lines, investigate its roles in the proliferation of GC cells in vitro, and elucidate the regulatory mechanisms of LINC00265 action. It was found that LINC00265 expression was significantly upregulated in GC tissue samples and cell lines compared with their normal counterparts. Additionally, LINC00265 knockdown could inhibit GC cell proliferation in vitro. Further investigation revealed that LINC00265 acted as a competing endogenous RNA for microRNA-144-3p (miR-144-3p) and inhibition of miR-144-3p markedly counteracted LINC00265 knockdown-meditated suppression on GC cell proliferation. Additionally, Chromobox 4 (CBX4) was upregulated in GC and silencing CBX4 could reduce GC cell proliferation. Then, CBX4 mRNA was demonstrated to be a direct target of miR-144-3p in GC cells and LINC00265/miR-144-3p axis could regulate CBX4 expression. Taken together, LINC00265 may promote GC cell proliferation via the miR-144-3p/CBX4 axis.  The incidence rate has been dramatically increasing in Eastern Asia during the past decades .Click here for additional data file.


Bax-/-) prolongs RGC body survival in models of glaucoma and optic nerve trauma, axonopathy persists, thus raising the question of whether dendrites and the RGC light response are protected. Here, we used an inducible model of glaucoma in Bax-/- mice to determine if Bax contributes to RGC dendritic degeneration. We performed whole-cell recordings and dye filling in RGCs signaling light onset (αON-Sustained) and offset (αOFF-Sustained). We recovered RGC dendritic morphologies by confocal microscopy and analyzed dendritic arbor complexity and size. Additionally, we assessed RGC axon function by measuring anterograde axon transport of cholera toxin subunit B to the superior colliculus and behavioral spatial frequency threshold . We found 1 month of IOP elevation did not cause significant RGC death in either WT or Bax-/- retinas. However, IOP elevation reduced dendritic arbor complexity of WT αON-Sustained and αOFF-Sustained RGCs. In the absence of Bax, αON- and αOFF-Sustained RGC dendritic arbors remained intact following IOP elevation. In addition to dendrites, neuroprotection by Bax-/- generalized to αON-and αOFF-Sustained RGC light- and current-evoked responses. Both anterograde axon transport and spatial acuity declined during IOP elevation in WT and Bax-/- mice. Collectively, our results indicate Bax contributes to RGC dendritic degeneration and distinguishes the proximal and distal neurodegenerative programs involved during the progression of glaucoma.The BCL-2 (B-cell lymphoma-2) family of proteins contributes to mitochondrial-based apoptosis in models of neurodegeneration, including glaucomatous optic neuropathy (glaucoma), which degrades the retinal ganglion cell (RGC) axonal projection to the visual brain. Glaucoma is commonly associated with increased sensitivity to intraocular pressure (IOP) and involves a proximal program that leads to RGC dendritic pruning and a distal program that underlies axonopathy in the optic projection. While genetic deletion of the Bcl2-associated X protein (The online version contains supplementary material available at 10.1007/s12035-021-02675-5. During ll death , 7.Bax (Bax-/-) prevents cell death in affected tissues in models of neurodegeneration, including Parkinson’s disease [slow Wallerian degeneration (WldS) allele reduces axon dropout, preserves anterograde axon transport, and protects spatial acuity, but somatic and dendritic degeneration persists [Bax-/- proves protective against RGC body death in models of glaucoma and acute focal injury to the optic nerve, yet axons comprising the optic nerve still undergo significant degeneration [Genetic deletion of  disease , Alzheim disease , amyotro disease , and gla disease , 11. Gla disease . Evident disease –20. Neurpersists , 21, 22.neration , 23.Bax and WldS elegantly demonstrate compartmentalized degeneration [Bax-/- on RGC dendrites and signaling prior to significant cell death. Using an inducible microbead model of glaucoma [Bax contributes to dendritic pruning and degradation of evoked responses of alpha RGCs signaling light increments (αON-Sustained) and decrements (αOFF-Sustained). IOP elevation decreased anterograde axonal transport and spatial acuity during glaucoma in both WT and Bax-/- animals. Our results indicate Bax contributes to the reduction in RGC dendritic complexity and signaling during IOP elevation and illustrates the separability of the proximal and distal programs involved in degeneration during glaucoma.Genetic and inducible models of glaucoma combined with genetic dosing of neration , 18, 22.glaucoma , we founBax gene  from Jackson Laboratory , CCG CTT CCA TTG CTC AGC GG (Mutant forward), and GAG CTG ATC AGA ACC ATC ATG (WT forward). We obtained primers from Integrated DNA Technologies . Mice were maintained on a 12-h light-dark cycle with standard rodent chow and water available as desired.All experimental procedures were approved by the Vanderbilt University Institutional Animal Care and Use Committee. We obtained 6- to 8-week-old male C57Bl/6 (WT) mice from Charles River Laboratory . We obtained 4- to 10-week-old male mice with a targeted null mutation in the bor, ME, ), We conWe anesthetized mice with isoflurane (2.5%), and we applied tropicamide (1%), proparacaine (0.5%), and lubricating drops to both eyes. We unilaterally elevated IOP by injecting 1.5 μL of 15-μm polystyrene microbeads  into the anterior chamber of the eye via a borosilicate glass pipette attached to a micromanipulator  driven by a microsyringe pump . For an2, 5% CO2) bicarbonate-buffered Ames’ media supplemented with glucose (20 mM) heated to 32°C . Extracellular solution pH was 7.4 and osmolarity 290 . Whole-cell recordings were performed using borosilicate pipettes filled with (in mM): 125 K-gluconate, 10 KCl, 10 HEPES, 10 EGTA, 4 Mg-ATP, 1 Na-GTP, and 0.1 ALEXA 555 . Intracellular solution pH was 7.35 and osmolarity was 285. When filled with intracellular solution, pipette resistance was 4–8 MΩ. Electrical signals were amplified and sampled at ≥10 kHz .We performed whole-cell current-clamp (0 pA) recordings from RGCs in whole-mount retinas, using previously established protocols from our laboratory –29. WholDuring whole-cell current-clamp recordings, we targeted alpha-type RGCs. For 70% of cells analyzed (256 out of 364), we determined the distance from the optic nerve head. On average, cells were in the mid peripheral retina. We tested RGC responses to light and current injections (0 to 180 pA in 20 pA increments). Light responses were evoked by a light-emitting diode system , and light was focused onto the retina through the microscope objective . After physiology, retinas were fixed overnight in 4% paraformaldehyde (PFA) at −4°C. The following day, the PFA solution was removed and substituted with PBS, and we prepared retinas for immunohistochemistry.Bax-/- whole mount retinas by immunolabeling against mouse-SMI-32  and goat-Brn3a . For SMI-32-positive and Brn3a-positive cell counts, confocal micrographs were taken with an Olympus FV1000 using a 60× objective. Four images were taken within each quadrant  of the retina. Cell counts were performed manually by a masked investigator and then averaged. SMI-32 intensity was determined by creating ROIs around the soma of each SMI-32-positive cell and normalizing to background. We determined neuronal density in SC sections by immunolabeling against rabbit-NeuN . Z-stack images were obtained using a 40× objective with a 2× zoom. Max intensity projections were generated for each z-stack and NeuN-positive cell counts were performed manually by a masked investigator.We performed immunohistochemistry on whole-mount retinas and sections from superior colliculus (SC) as previously described , 29–31. We imaged tissues using confocal microscopy (Olympus FV-1000). RGC dendritic arbors were montaged and manually traced using Adobe Illustrator and Adobe Photoshop, respectively. Skeletonized dendritic arbors were analyzed using Fiji . We determined the dendritic field area using the polygon selection tool in Fiji. We determined dendritic branch points by manually counting the number of dendritic bifurcations , 26. ShoMice were anesthetized with isoflurane (2.5%), and we injected 1 μL of cholera toxin subunit B 488  into the anterior chamber of both eyes , 29, 32.Bax-/- mice with 0.1 M cacodylate buffer followed by 2.5% glutaraldehyde in cacodylate buffer. We carefully removed eyes with the optic nerve attached. We then isolated 3-mm segments of optic nerve proximal to the globe and post-fixed them for 1 h in 2.5% glutaraldehyde in cacodylate buffer. Next, we embedded the optic nerve segments in Epon resin and obtained semi-thin  and ultra-thin  cross-sections. We stained semi-thin cross-sections with 1% paraphenylenediamine  and 1% toluidine blue to identify myelin sheaths and glia, respectively. We imaged sections en montage using a Nikon H600L microscope equipped with a 100× oil-immersion objective, motorized X-Y-Z stage, a digital SLR camera, and differential interference contrast optics. Ultra-thin nerve cross-sections were prepared and photographed at 2700× (10 images per nerve for axon counts) and 240× magnification  using a Philips CM-12, 120-keV transmission electron microscope at Vanderbilt Cell Imaging Shared Resource Core. A naïve observer manually counted total and degenerating axons using Fiji ImageJ. We identified degenerating axons based on multilaminar myelin sheaths and diminishment of cytoskeletal content.One month after IOP elevation, we perfused WT and We measured spatial frequency threshold  using the OptoMotry system  as previously described , 29. We We analyzed data using GraphPad  or SigmaPlot . We determined if data fit a normal distribution using Shapiro-Wilk tests. If data passed normality tests, we performed parametric statistics; otherwise, non-parametric statistics were performed. We identify each statistical test performed in figure captions in the “Bax animal by gel electrophoresis of PCR products. Mice homozygous for the mutant Baxtm1Sjk allele (Bax-/-) showed a single band at 507 bp. Heterozygous mice for the mutant Baxtm1Sjk allele (Bax+/-) produced bands at 507 bp and 307, indicating the WT Bax allele. WT animals produced only a single band at 307 bp  and Bax-/- eyes  compared to respective saline-injected eyes  by blocking the outflow of aqueous fluid by microbead occlusion of the trabecular meshwork, we measured IOP for 2 consecutive days . We did not detect a significant difference in baseline IOP between WT (14.64 ± 0.13 mmHg) and yes Fig. .2, p=0.92) or SMI-32-positive RGCs in WT retinas  and SMI-32-positive RGCs  compared to WTs . However, IOP elevation does not significantly impact either Brn3a-  or SMI-32-positive RGC density in Bax-/- retinas  RGCs in WT and  WT Fig.  and Bax--/- Fig.  αON-S RGpth Fig.  or dendrpth Fig. .Fig. 2Bp≤0.04, Fig. p<0.001, Fig. p=0.98, Fig. R2=0.002, p=0.71) or microbead-injected eyes  and microbead-injected eyes , but RMP of Bax-/- αON-S cells appear unaffected by IOP elevation  RGCs in both WT Fig.  and Bax-nas Fig.  based onp=0.02, Fig. Bax-/- animals  and microbead eyes , but this relationship strengthened following IOP elevation  or microbead-injected eyes  and peak spike rate (p=0.73) in αOFF-S RGCs  and Bax-/- mice  is an early indicator of axonopathy in glaucoma , 39. Antice Fig. . When quBax-/- intrinsically increased optic nerve size and axon density  optic nerves from control eyes , axon density (p≥0.17), and the percentage of degenerating axons (p≥0.76) largely unchanged in both WT and Bax-/- mice  in WT mice . Here, wBax-/- intrinsically improved spatial acuity by 21% compared to WT control eyes  and not significantly different compared to saline-injected eyes of WTs  and Bax-/-  animals compared to respective control eyes  Distance from optic nerve head (ONH) of αON-S RGCs from control and microbead-injected eyes of WT and Bax-/- mice. WT αON-S RGCs from control and microbead-injected eyes were sampled at similar retinal eccentricities (p=0.98). αON-S RGCs from Bax-/- animals were sampled at more distant locations compared to WTs . Bax-/- αON-S RGCs from control and microbead-injected eyes were sampled at similar retinal eccentricities (p=0.36). (B-C) Relationship between branch points and distance from ONH of αON-S RGCs from WT (B) and Bax-/- (C) control and microbead-injected eyes. (D) Distance from ONH of αOFF-S RGCs from control and microbead-injected eyes of WT and Bax-/- mice. WT αOFF-S RGCs from control and microbead-injected eyes were sampled at comparable retinal eccentricities (p=0.98). αOFF-S RGCs from Bax-/- animals were sampled at more distant eccentricities compared to WT cells . Bax-/- αOFF-S RGCs from control and microbead-injected eyes were sampled at similar distances (p=0.61). (E-F) Relationship between branch points and distance from ONH of αOFF-S RGCs from WT (E) and Bax-/- (F) control and microbead-injected eyes. Statistics: One-way ANOVA Tukey Post hoc test , Linear regression . Bar graphs indicate mean, Error bars indicate SEM. Sample number is indicated within bars. (PNG 298 kb)High resolution image (TIF 1670 kb)Figure S2.IOP Elevation Does Not Affect Optic Nerve Area or RGC Axon Density. (A-B) Representative semi-thin cross-sections of optic nerves from WT (A) and Bax-/- (B) control and microbead-injected eyes. Example ultra-thin cross-section of optic nerve from WT (C) and Bax-/- (D) control and microbead-injected eyes. Example degenerating axons exhibiting multilaminar myelin sheaths indicated by arrowheads. (E) IOP elevation does not affect nerve area (p≥0.98), (F) axon density (p≥0.175), or (G) percent of degenerating axons (p≥0.767) in either WT or Bax-/- nerves. However, Bax-/- significantly increases nerve area (p=0.009) and axon density (p=0.0005). Statistics: One-way ANOVA Tukey Post hoc test (C). Bar graphs indicate mean, Error bars indicate SEM. Animal numbers: WT Ctrl n = 5, WT 4Wk n = 4, Bax-/- Ctrl n = 4, Bax-/- 4Wk n = 4. (PNG 2684 kb)High resolution image (TIF 9261 kb)


Systemic sclerosis (SSc) is a connective tissue disease associated with vascular damage and multi organfibrotic changes with unknown pathogenesis. Most SSc patients suffer from defective angiogenesis/vasculogenesisand cardiac conditions leading to high mortality rates. We aimed to investigate the cardiovascular phenotype of SSc bycardiogenic differentiation of SSc induced pluripotent stem cells (iPSC). In this experimental study, we generated iPSC from two diffuse SSc patients, followed bysuccessful differentiation into endothelial cells (ECs) and cardiomyocytes (CMs). SSc-derived EC (SSc-EC) expressed KDR, a nearly EC marker, similar to healthy control-EC (C1-EC). Aftersorting and culturing KDR+ cells, the resulting EC expressed CD31, a late endothelial marker, but vascular endothelial(VE)-cadherin expression markedly dropped resulting in a functional defect as reflected in tube formation failure ofSSc-EC. Interestingly, upregulation of SNAI1  was observed in SSc-EC whichmight underlie VE-cadherin downregulation. Furthermore, SSc-derived CM (SSc-CM) successfully expressed cardiac-specific markers including ion channels, resulting in normal physiological behavior and responsiveness to cardioactivedrugs.This study provides an insight into impaired angiogenesis observed in SSc patients by evaluating in vitrocardiovascular differentiation of SSc iPSC. Systemic sclerosis (SSc) develops as a chronicconnective tissue disease which involves multiple organs;however, its etiology remains unknown. It is characterizedby vascular injury, immune dysregulation and extensivefibrosis of several organs including the skin . Thecomin vitro studies demonstrated an impaired angiogenesis inSSc. Moreover, several studies suggested that ECs might be the origin of a subset ofactivated fibroblasts or myofibroblasts. Furthermore, endothelial-mesenchymal transition(EndoMT) and their differentiation into collagen-producing cells are likely to represent anadditional source of extra collagen . To generate patient-specific iPSC, dermal fibroblasts were reprogrammedby four Yamanaka factors delivered by retrovirus in serum- and feeder-free conditions based on a previouslyreported protocol  supplemented with 20% Knockout serum replacement , 1%non-essential amino acids , 1% penicillin and streptomycin, 2 mM L-glutamine and 0.1 mM β-mercaptoethanol . At day 14-20 of re-plating,embryonic stem cells (ES)- like colonies were observed  and cultured in fibroblast medium  enriched with 10% fetal bovine serum and 1% penicillin and streptomycin , as previously described .Instituprotocol . On tranobserved . For eacobserved  wereacqKaryotyping was performed at cytogenetic laboratoryof the Institute for Human Genetics  according to a standard procedure describedpreviously . BrieflyTo evaluate the spontaneous differentiation capacity ofthe above-noted derived hiPSC lines into three embryonicgerm layers, we generated embryoid body (EB). Briefly,hiPSC colonies were dispersed into single cells usingAccutase  and transferred into non-adhesivebacterial plates  containingDMEM/F12 medium supplemented with 20% KOSR, 1%non-essential amino acids, 2 mM L-glutamine, and 0.1mM β-mercaptoethanol without bFGF. After 12 days, thegenerated EBs were plated on ECM Gel-coated culturedishes for another 8 days. On day 21 of spontaneousdifferentiation, EB samples were collected and ectoderm,mesoderm and endoderm differentiation were evaluatedwith respect to transcriptional expression of each germlayer’s specific genes using quantitative real-timepolymerase chain reaction (qRT-PCR).6) were suspended in phosphate-buffered saline (PBS) andinjected into the subrenal capsule of 8-week-old NOD/SCID mice (BioLASCO) using a 26-gaugesyringe . Eight weeks after injection, tumors were harvested, weighedand fixed in 10% formalin. After fixation, tumors underwent histological analyses forectoderm, mesoderm and endoderm formation.Sc iPSC .Cells were fixed in 4% paraformaldehyde at roomtemperature (RT) for 25 minutes, washed with PBS/0.05%Tween 20 and permeabilized using 0.5% Triton X-100 inPBS for 30 minutes at RT. Thereafter, cells were washedand blocked in blocking solution  for 1 hour atRT. Primary antibodies were diluted in blocking solutionand added to the cells overnight at 4˚C. Cells were thenwashed three times with PBS/0.05% Tween 20, each timefor 5 minutes, and incubated with appropriate secondaryantibodies in blocking solution for 45 minutes at RT. Lastly,cells were washed three times with PBS/0.05% Tween20 and nuclei were counterstained with 4’,6-diamino-2-phenylindole . Images were capturedusing a fluorescent microscope .Antibodies used in the present study are mentioned inWe performed Alkaline phosphatase (ALP) stainingbased on the manufacturer’s instructions .5 cells/ml in hESC medium. Afteraggregate formation, the directed cardiogenic differentiation of hiPSC was performed instatic suspension culture using a cocktail of small molecules (SM) as previously described coated 4-welltissue-culture plate in differentiation medium. After adequate cell attachment, CM werefixed and all steps of immunofluorescence staining were taken as described insupplementary material.hiPSC colonies were dispersed into single cells by 4-5 minutes treatment with Accutase at 37˚C and incubated in non-adhesive bacterial plates  at the density of 2×10escribed. Briefly+ sorted cells were sub-cultured on collagen type I-coatedplates  in EGM-2 medium containing 50 ng/mlVEGF-A until reaching appropriate confluencyFor endothelial differentiation, we used a previously reported protocol with somemodifications . hiPSC aTM RT Reagent Kit   based on the manufacturer’s instructions and qRT-PCR was performedusing a SYBR Premix Ex Taq Kit  and a Rotor Gene Corbett System. Results were analyzed by Rotor-Gene 6000 analysissoftware . All experiments were done intriplicate. The relative gene expression level of the desired genes was calculated by ∆∆CTmethod and normalized against the housekeeping gene, glyceraldehyde 3-phosphatedehydrogenase (GAPDH). All primer sequences used in the present work, arelisted in www.celljournal.org).Total RNA was extracted using TRIzol reagent . Toprevent DNAcontamination, extracted RNA was treated with RNase-free DNase I . cDNAsynthesis was performed using a PrimeScriptDifferentiated hiPSC aggregates were collected atparticular time-points of differentiation and dissociatedinto single cells using Accutase solution and 0.05%trypsin/ ethylenediaminetetracetic acid for hiPSC-CM and hiPSC-EC, respectively. Then, singlecells were fixed by treatment with 4% paraformaldehydefor 20 minutes at 4˚C. After washing with PBS/0.05%Tween 20, the fixed cells were permeabilized by 0.2%Triton X-100 in PBS for 30 minutes at RT, blocked inserum and stained, either 1 hour for surface markers orovernight for cytoplasmic markers, with appropriateprimary antibodies at 4˚C. Cells were then washed and incubated with appropriate secondary antibody for 1 hourat RT. Cells were analyzed using a BD FACS Calibur flowcytometer . Data analysis wasdone by Flowing Software .For sorting, cells were dissociated into single cellsusing 0.05% trypsin/EDTA on day 6 of endothelialdifferentiation. Cells were then washed in PBS containing2% FBS (FACS buffer) and incubated with anti-humanKDR for 1 hour at 4˚C. After washing, KDR-positivecells were sorted using FACS Calibur.ECs were incubated with 10 µg/ml of acetylated low-density lipoprotein  for 4 hours at 37˚C. Then, cells were fixed bytreatment with 4% paraformaldehyde for 10 minutes atRT. After washing with PBS, nuclei were counterstainedwith DAPI  and visualized using afluorescence microscopy .4 cells/well were seeded onto the matrix in 150 μl of EGM-2medium and incubated for 2 hours at 37˚C with 5% CO2 . Tube formation wasassessed using Olympus CKX41 inverted microscope and analyzed using “Image J”software.ECMatrix™  was aliquoted into all wellsof a 96-well plate (50 µl/well) and incubated for 1-2 hour at 37˚C to polymerize.Thereafter, 103 √ ). Drugsused in pharmacological studies are listed in www.celljournal.org).A multielectrode array (MEA) data acquisition system  was used to record the extracellular field potential (FP) of hiPSC-CM. The MEAplate is composed of 60 titanium nitride electrodes with an inter-electrode space of 200µm. Beating spheroids were plated on ECM Gel-coated MEA plates and allowed to attach for48-72 hours before recording. Baseline FP recording and drug testing were performed 30 ± 5days post-differentiation. On the day of the experiment, the MEA plates were connected toa head stage amplifier. FP was acquired at 2 kHz, and all recordings were performed at37˚C. Signals were recorded for 60 seconds at baseline and 5 minutes after drugapplication. All drugs were purchased from Sigma-Aldrich, otherwise stated. Stocksolutions were prepared daily in appropriate solvent and used at desired concentrationsmade in RPMI/B27 medium. Data were analyzed by Cardio2D software . FP durations (FPD) was normalized to beating rate using theBazett correction formula  by a P-97 horizontal puller  to a tip resistance of 3-6 MΩ.The pipette solution contained 135 mM KCl, 10 mM NaCl, 1 mM EGTA, 10 mM HEPES, and 5 mMMgATP, and the pH was adjusted to 7.2 using KOH.Action potential (AP) was recorded from spontaneously beating hiPSC-CM using thecurrent-clamp mode of the whole cell patch-clamp configuration. On differentiation day 30,beating spheroids were dissociated into single cells by Accutase accompanied by gentlepipetting. Single beating CM were plated onto ECM-Gel coated glass coverslips andincubated at 37˚C overnight. Then, the cover slips were transferred to a recording chambermounted on the stage of an Olympus inverted microscope . The bath solutionwithin the chamber contained 135 mM NaCl, 5.4 mM KCl, 10 mM HEPES, 10 mM D-glucose, 1 mMMgCl, and 1.8 mM CaClData were acquired using a multiclamp 700B amplifier, a Digidata 1440 analog-to-digital board and pClamp10 software (Axon Instruments), at a sampling frequencyof 10 kHz and low-pass filtered at 2 kHz. Data analysis wasperformed using Clampfit 10 (Axon Instruments) and Prism6  software.+2 transients in hiPSC-CM, beating spheroids wereincubated with 1 µM Fura-2 AM  for 30 minutes at 37˚C, 30-daypost-differentiation. Then, beating spheroids were washed with RPMI/B27 medium for 15minutes at 37˚C. Afterwards, Ca+2 imaging was performed using a fluorescentmicroscope  equipped with a DP72 digital camera  andanalyzed in a custom- made Matlab macro. Calcium transient amplitude and calcium transientduration at 80% decay (CTD80) were calculated. In order to study the Ca+2content of sarcoplasmic reticulum (SR), rapid puffs of 10 mM caffeine were applied andCa+2 release was imaged. Fractional Ca+2 release (FCR) from thestores was calculated as the ratio of the amplitude of Ca+2 transient to theamplitude of the caffeine-induced Ca+2 release. In order to record CaAll data are presented as mean ± SEM from at least threeindependent biological replicates for each hiPSC line.Comparisons were made by analysis of variance  or unpaired t test when appropriate,using GraphPad Prism version 6.01  and considered significant whenP<0.05.The datasets generated during and/or analysed duringthe current study are available from the correspondingauthor on reasonable request.OCT4and NANOG was evaluated which revealed that both S1-iPS2 and S2-iPS3expressed endogenous OCT4 and NANOG  in derived S1-iPS2 and S2-iPS3 . Differentiation potency ofS1-iPS2 and S2-iPS3 into embryonic germ layers was evaluated by EB formation. To achievethat, iPSC were cultured in suspension for 12 days to form EB in the absence of bFGF andthen transferred to ECM Gel-coated dishes for another 8 days. RT-PCR analyses demonstratedthat ectoderm (PAX6 and TAU), mesoderm(Brachyury) and endoderm (ALB andFOXA2) specific markers were only expressed in differentiated S1-iPS2and S2-iPS3but not in undifferentiated state . Moreover, SSci PSCwere transplanted into the subrenal capsule of 5-week-old NOD mice. After about 7 weeks,teratoma was formed, harvested and subjected to immunohistochemistry analyses. Our resultsdemonstrated the presence of cartilage (mesoderm), gut-like epithelium (endoderm) andneural rosette (ectoderm) tissues in teratoma which further confirmed stemness of iPSC. Flow cytometry analyses indicated that almost30% of cells were KDR-positive .hiPSC lines were differentiated to ECs by a cocktail of growth factors and SMs. Cells won day 1 .The exppositive  implicatCD31(PECAM-1) as a late endothelial marker was substantially upregulated ondifferentiation day 8  which was followed by afairly similar protein expression pattern  . In contrast, whileVE-cadherin (CD144) expression in C1-EC peaked ondifferentiation day 8, neither S1-EC nor S2-EC showed upregulation of this endothelialspecific marker  in a similar manner (KDR-positive cells were sorted on day 6 and cultured in VEGF-A supplemented media oncollagen I-coated plates to confluency . Expressctively) . Relativc marker . C1-EC er manner . Toevalr manner . Howeverr manner . While Cr manner . This chr manner . These rin vitro differentiated SSc-ECs, while the expression ofMMP1 was 2.4-fold upregulated in S1- EC, MMP9expression showed 12-fold decrease compared to C1-EC, suggesting a dysregulation ofMMP1 and MMP9 expression in S1-EC (RGS5 was similarin C1-and S1-EC characterized in this study (endothelin 1 (EDN1), which is involved in vascularremodeling of SSc  in ECs during angiogenesis. The impin S1-EC .Sclerodin S1-EC . Interesis study . Moreoveg of SSc , showed g of SSc .MTOR andPI3KCA in S1-EC compared to C1-EC. Furthermore, the expression ofSNAI1, a transcriptional repressor of VE-cadherin, was markedlyincreased in SSc-EC, indicating the possible role of SNAI1 indownregulation of VE-cadherin  and phosphoinositide-3 kinase (PI3K) signaling . In ordecadherin .TNNT2(cardiac type of troponin), MYL2 (myosin light chain 2) andMYH (myosin heavy chain), encoding calcium handling proteins;SERCA (sarco/endoplasmic reticulum Ca2+-ATPase),SLC8A1 (solut carrier family 8 of member A1), CACNA1C, RYR2 (ryanodine receptor),TRDN (Triadin) and CASQ , and encodingion channels; KCNH2 (potassium voltage-gated channel). WhileMYL2, MYH6 and MYH7 were upregulated in S1-CM, relativeexpression of these contractile apparatus-related genes were similar in S2-CM and C2-CM were similar in allthree hiPSC-derived CM. Protein expression of cTNT showed a similar pattern to that of itsgene expression as reflected in immunostaining images; also, both SSc and C2 iPSC linesgenerated similar abundance of cTNT+ CM (iPSC lines were subjected to cardiomyocyte differentiation in static suspension cultureusing a SM-based protocol  which rend C2-CM. FurthercTNT+ CM . Altogetmax) and AP duration at different time-points ofrepolarization (APDx) as well as APD90/ APD50 ratio wereused for AP classification .To evaluate the functional properties of differentiated CM, two components ofexcitation-contraction coupling were studied. FPs generated by spontaneously beatingspheroids were recorded to study the electrophysiological properties of SSc and C2-CM. All diffication . Analysification . Electro2+ transients as another component of excitation-contraction coupling wascharacterized in C2-CM, S1-CM and S2-CM. Ca2+ transient amplitude  and CTD80 did notsignificantly differ between patient-specific and healthy CM . Furthermore, fractional Ca2+release which represents the ratio of active intracellular Ca2+ bulk to wholeintracellular Ca2+, did not vary betweenC1-CM, S1-CM and S2-CM . Fractional Ca2+release is basically defined as the ratio of the excitation-induced Ca2+release to caffeine-induced Ca2+ release which depletes all intracellularCa2+ stores. Ca1 -adrenergic stimulation by Iso substantiallyincreased the spontaneous beating frequencies of iPSC-derived CM  which were antagonized byPro as reflected by substantial reduction of beating rates to values approaching baselinefrequencies. Furthermore, Iso administration caused significant shortening of FPD and cFPDwhich were reversed by Pro treatment  . ImportaThe role of MTOR in vascular development waspreviously reported . Bieri eMultiple mechanisms are involved in VE-cadherinregulation including Twist/Slug/Snail family which aretranscriptional repressors of VE-cadherin gene .Lopez eAbsence of VE-cadherin expression and subsequentdefective angiogenic activity of SSc-EC motivated usto assess the effect of VE-cadherin signaling on MMPsexpression, which play multiple roles in angiogenesis .Kiran eAs majority of SSc patients suffer from cardiacinvolvements , we alsoThe present study reports the successful differentiationof SSc iPSC into endothelial and functional cardiac cellsthat would provide a unique opportunity for mechanisticstudies of scleroderma pathogenesis and possible targeteddrug discovery.


Scientific reports 10.1038/s41598-021-02636-x, published online 01 December 2021Correction to: The original version of this Article contained an error in the Abstract.“The best cut-off index was determined, and clinical performance was tested using 115 serum samples obtained from 46 patients with coronavirus disease 2019 (COVID-19) and 69 individuals who tested negative for COVID-19 through reverse transcription quantitative polymerase chain reaction (RT-qPCR).”now reads:“The best cut-off index was determined, and clinical performance was tested using 115 nasopharyngeal swab samples obtained from 46 patients with coronavirus disease 2019 (COVID-19) and 69 individuals who tested negative for COVID-19 through reverse transcription quantitative polymerase chain reaction (RT-qPCR).”The original Article has been corrected


Enterobacterales (CPE) strains recovered from rectal screening swab samples obtained from children at a tertiary-care pediatric hospital in Qatar during a 3-year period. A total of 72 CPE isolates recovered from 61 fecal carriers were characterized. Escherichia coli (47 isolates [65.3%]) and Klebsiella pneumoniae (22 isolates [30.6%]) were the most common species identified. High levels of genetic diversity were observed for both species. These 72 isolates produced 78 carbapenemases, characterized as either NDM-type (41 enzymes [52.6%]) or OXA-48-type (37 enzymes [47.4%]). NDM-5 (24 enzymes [30.8%]), NDM-1 (15 enzymes [19.2%]), and OXA-181 (15 enzymes [19.2%]) were the most common variants detected within each type. Twenty-three NDM producers exhibited difficult-to-treat resistance, compared with only 2 of the OXA-48 producers. Multiple comorbidities were identified in 88.5% of the patients, whereas recent travel history to countries in which CPE are endemic was documented for 57.4% of the patients. All 9 blaOXA-48-type-gene-containing E. coli sequence type 38 (ST38) strains were isolated from patients without international travel history. The mean quarterly incidence of fecal carriage decreased more than 6-fold after the implementation of coronavirus disease 2019 (COVID-19)-related international travel restrictions in Qatar in mid-March 2020. Our data suggest that NDM-type and OXA-48-type carbapenemases expressed by a large diversity of E. coli and K. pneumoniae genotypes are largely dominant in the pediatric population of Qatar. Although our data indicate successful local expansion of E. coli ST38 strains harboring blaOXA-244 genes, at least within health care settings, blaOXA-48-type and blaNDM-type genes appear to have been mainly introduced sporadically by asymptomatic carriers who visited or received health care in some nearby countries in which the genes are endemic.Whole-genome sequencing was used to characterize carbapenemase-producing IMPORTANCE To the best of our knowledge, this is the first study addressing the molecular characteristics of CPE in a pediatric population in Qatar using whole-genome sequencing. Since several countries in the Arabian Peninsula share relatively similar demographic patterns and international links, it is plausible that the molecular characteristics of CPE in children, at least in the middle and eastern parts of the region, are similar to those observed in our study. Enterobacterales (CPE) is growing at an alarming pace in many regions of the world, including the Middle East  and AmpC β-lactamases  technology to study the molecular characteristics of CPE detected through our proactive surveillance program from pediatric patients over a 3-year period. We also aimed to determine the extent to which the phenotypic and genomic antimicrobial resistance data generated by our WGS data analysis could facilitate optimization of antimicrobial stewardship in children with CPE infections in our institution and to assess whether our infection control procedures are effective in preventing the spread of these microorganisms.Escherichia coli strains (65.3%), 22 Klebsiella pneumoniae strains (30.6%), 2 Citrobacter sp. strains (2.8%), and 1 Klebsiella oxytoca strain (1.4%). High clonal diversity was observed among the E. coli and K. pneumoniae isolates . Twenty-one different sequence types (STs) were detected in E. coli, including ST38 (9 isolates [19.1%]), ST410 (8 isolates [17%]), and ST405 (4 isolates [8.5%]) as the most common clones. Seven of 9 E. coli ST38 isolates were highly similar genetically, but they were different in terms of acquired blaCTX-M genes  and ST14 and ST17 (2 isolates [9.1% each]) as the only STs with more than 1 representative.During the study period, 72 unique CPE strains producing 78 carbapenemases were detected and characterized . CPE strains of four species were found, including 47 -M genes . In K. pE. coli isolates, 26 (55.3%) produced OXA-48-type enzymes and 23 (48.9%) NDM-type enzymes. Among K. pneumoniae isolates, 17 (77.2%) produced NDM-type enzymes and 9 (40.9%) OXA-48-type enzymes. Four (18.2%) K. pneumoniae isolates and 2 (4.2%) E. coli isolates harbored blaNDM-type and blaOXA-48-type genes simultaneously (see Data Set S1 and Tables S1 and S2).Among 78 carbapenemases characterized, only NDM-type (41 enzymes [52.6%]) and OXA-48-type (37 enzymes [47.4%]) carbapenemases were detected. The variants identified within each type were NDM-5 (24 enzymes [30.8%]), NDM-1 and OXA-181 (15 enzymes each [19.2%]), OXA-244 (13 enzymes [16.7%]), OXA-48 (7 enzymes [9%]), and NDM-7 and OXA-232 (2 enzymes each [2.6%]). Among E. coli isolates tested against fosfomycin were susceptible to this antibiotic, according to the breakpoint defined for susceptibility (MIC of ≤64 μg/ml) by the CLSI.Susceptibility patterns for 35 single NDM producers and 31 single OXA-48 producers are shown in blaCTX-M-15 genes, 10 isolates (11.9%) harboring blaCTX-M-14b genes, and 5 isolates (6%) harboring blaCTX-M-27 genes. Cocarriage of blaCTX-M-15 genes was higher among NDM producers (27 isolates [77.1%]) than among OXA-48 producers (10 isolates [32.3%]) ; blaCTX-M-27 genes were detected only in 5 genetically related E. coli ST38 isolates carrying blaOXA-244 genes (gyrA), S80I (parcC), and S458A (parE) were detected in 21 isolates (29.2%), including all E. coli ST410 isolates and all but 1 E. coli ST405 isolates. Nine isolates (12.5%) harbored 16S rRNA methylase  genes. All rmtB and rmtC genes were carried by isolates bearing blaNDM-type genes, particularly blaNDM-5 (6/7 isolates [85.7%]). One blaNDM-1-containing E. coli ST540 isolate with a colistin MIC of 4 μg/ml harbored the mcr-1 gene (Genes encoding CTX-M-type ESBL were coharbored by 54 isolates 75%), including 39 isolates (54.2%) harboring 44 genes . Genes e5%, inclublaOXA-48-type genes (14 blaOXA-181 and 2 blaOXA-232), whereas 1 blaNDM-1 gene was located on an IncA/C2 replicon and 1 blaNDM-5 gene on an IncFII replicon (see Data Set S1).Although a wide range of plasmid replicons was found in 69 isolates (95.8%), including IncFIA (35 isolates [48.6%]), IncFIB(AP0001918) (32 isolates [44.4%]), ColKP3 (16 isolates [22.2%]), IncFIB(K) (15 isolates [20.8%]), and IncX3 (14 isolates [19.4%]), a plasmid location of carbapenemase-encoding genes was identified in 19 isolates (26.4%). All ColKP3 replicons contained E. coli ST38 isolates were recovered from patients without travel history.The isolates in our collection were carried by 61 patients. Thirty-five (57.4%) had visited or come from a foreign country within 12 months before CPE detection (see Table S1). Sixteen (45.7%) patients visited Indian subcontinent countries (India and Pakistan), 15 (42.9%) Northeast Africa countries , and 2 (5.7% each) Middle East countries (Kuwait and Iraq) and Turkey. Thirty-two patients (91.4%) with a history of staying in a foreign country had ≥1 underlying medical condition, and 28 (80%) were hospitalized or received medical care in their home or visited countries. Twenty-six patients (42.6%) lacked travel history (see Table S2). Of those, 22 (84.6%) had ≥1 underlying medical condition and 24 (92.3%) had a history of previous hospitalizations and/or residency in long-term-care facilities locally. All Similarly, whereas the quarterly mean of rectal swab samples processed by our laboratory and pediatric admissions increased slightly, the CPE screening positivity rate decreased almost 7 -fold after the implementation of the coronavirus disease 2019 (COVID-19)-related travel restriction (see Table S3). The mean quarterly carriage incidence decreased from 18.3 cases per 10,000 admissions in the pre-COVID-19-related-travel-restriction period to 3.1 cases per 10,000 admissions in the post-COVID-19-related-travel-restriction period  . In our blaOXA-48-type and blaNDM-type genes distributed mostly among a great variety of E. coli and K. pneumoniae strains carried by a cohort of children with underlying medical conditions with health care encounters locally or abroad. Our findings concur with those of studies performed in the adult population in Gulf Cooperation Council (GCC) countries , pointing out that the molecular epidemiology of CPE in our region is dominated by far by NDM-type and OXA-48-type enzymes produced by multiple clonal linages of both species   . Second,E. coli ST38 strains harboring blaOXA-48-type genes in several countries  and determine the precise plasmid location of carbapenemase-encoding genes in the majority of NDM producers . Short-rountries .blaNDM-type genes and blaOXA-48-type genes carried by multiple E. coli and K. pneumoniae clones are largely dominant among CPE in the pediatric population in Qatar. The predominance of E. coli ST38 strains harboring the blaOXA-244 gene among children with multiple comorbidities and without overseas exposure suggests a clonal spread of this high-risk clone locally. Since several countries in the Arabian Peninsula share relatively similar demographic patterns and international links, it is plausible that the molecular characteristics of CPE in children, at least in the middle and eastern parts of the region, would be similar to those observed in our study. Finally, with CPE established as a major public health threat worldwide, our study emphasizes that WGS is an emerging tool that can be used in clinical practice to optimize antimicrobial stewardship programs targeting these pathogens and to assess the impact of infection control strategies to prevent their spread.In summary, This retrospective observational study was conducted at Sidra Medicine, a 400-bed tertiary-care women’s and children’s hospital with approximately 2,000 pediatric admissions and 75,000 emergency visits per year. The hospital serves as a national referral center for all pediatric medical subspecialties, including oncology, neonatal and pediatric intensive care, and surgical specialties, including cardiac surgery and neurosurgery. Sidra Medicine is a new hospital that started providing outpatient care in January 2016, opened inpatient wards in January 2018, and became fully operational in July 2018, when the emergency department opened.All nonduplicate CPE strains recovered from surveillance rectal swab samples from children younger than 18 years of age during a 3-year period, between January 2018 and December 2020, were analyzed. In our hospital, risk assessment for CPE colonization is based on a three-question survey at emergency and elective admissions. These questions inquire about the history of colonization with MDROs and the history of having undergone either medical procedures or hospital admission in local or overseas health care facilities in the past 12 months. Patients who answer affirmatively to any of the questions and those who are unsure about the answer to any of the questions are deemed to be at high risk for CPE carriage, and surveillance rectal swab samples are obtained. Patients admitted to the NICU or PICU have routine surveillance swab samples collected on admission and once a week thereafter. Patients hospitalized in other wards for more than 2 weeks are also surveyed weekly. Isolates recovered from the same patient during different hospital admissions were counted as different carriage episodes when the strains were different. Patients’ electronic medical records were reviewed for underlying medical conditions, history of travel abroad, history of overseas medical care, and history of hospitalization or institutionalization in local facilities in the 12 months prior to CPE detection.Escherichia coli, the susceptibility to this agent was studied only in this species. In addition, MICs for colistin were determined by broth microdilution , and isolates were separated into wild-type and non-wild-type groups based on the epidemiological cutoff values provided by the same CLSI guidelines  and incubated aerobically in the dark at 35 ± 2°C for 18 to 24 h. Carbapenemase genes in presumptive CPE colonies on chromogenic agar plates were confirmed by the multiplex PCR Xpert Carba-R assay . All isolates were identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)  and tested for antimicrobial susceptibility using the BD Phoenix automated susceptibility testing system . MICs and breakpoints were interpreted according to CLSI 2019 guidelines . Isolateidelines . We defiidelines . Isolateexcluded .http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and quality trimmed by Trim Galore  to eliminate low-quality sequences. Trimmed reads were assembled using SPAdes v3.9.0 (https://github.com/tseemann/mlst) and the ResFinder v3.2 database (https://github.com/tseemann/abricate). Mutations in the QRDRs were detected using AMRFinderPlus v3.8 . Isolates were sequenced (paired-end reads with a read length of 300 bp) on the Illumina MiSeq platform with MiSeq reagent kit v3. Raw reads were assessed by FastQC .PRJNA667192.Raw sequence data are available in GenBank under BioProject accession number


Coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a serious infectious disease that has led to a global pandemic with high morbidity and mortality. High-affinity neutralizing antibody is important for controlling infection, which is closely regulated by follicular helper T (Tfh) cells. Tfh cells play a central role in promoting germinal center reactions and driving cognate B cell differentiation for antibody secretion. Available studies indicate a close relationship between virus-specific Tfh cell-mediated immunity and SARS-CoV-2 infection progression. Although several lines of evidence have suggested that Tfh cells contribute to the control of SARS-CoV-2 infection by eliciting neutralizing antibody productions, further studies are needed to elucidate Tfh-mediated effector mechanisms in anti-SARS-CoV-2 immunity. Here, we summarize the functional features and roles of virus-specific Tfh cells in the immunopathogenesis of SARS-CoV-2 infection and in COVID-19 vaccines, and highlight the potential of targeting Tfh cells as therapeutic strategy against SARS-CoV-2 infection. Globallorldwide . SARS-CoHumans who are immune-naive to SARS-CoV-2 are considered to be a major factor for the COVID-19 pandemic worldwide, and high-affinity neutralizing antibodies are especially essential for the control and clearance of SARS-CoV-2 infection –10. Seve+T cell subsets that play important roles in the control of viral infections, including T helper (Th) 1 (Th1), Th2, and Th17 cells and follicular helper T (Tfh) cells , inducible T cell costimulator (ICOS), programmed cell death protein 1 (PD-1), B-cell lymphoma 6 (Bcl-6), and interleukin-21 (IL-21) in both mice and humans and can usually initiate B cells to differentiate into plasma cells that produce high-affinity antibodies to neutralize the virus, such as lymphocytic choriomeningitis virus (LCMV), influenza virus and hepatitis B virus (STAT3 deficiency and common variable immunodeficiency (Antibody responses are closely correlated with CD4h) cells , 19. Amoesponses . Tfh cel B virus –25. Lossficiency . Howeverficiency . Here, w+T cells are differentiated into Tfh cells with the upregulation of CXCR5 and downregulation of CC-chemokine receptor 7 (CCR7), which are mediated by antigen-specific conventional dendritic cells (DCs) or monocyte-derived DCs  cells characterized as PD-1+ICOS+Bcl-6lowCCR7lowCXCR5+CD4+T cells, and memory Tfh cells similar to Pre-Tfh cells in lymphoid tissue  and central memory Tfh cells (PD-1-ICOS-CCR7highBCL-6-CXCR5+CD4+T cells) (+CCR6-), Tfh2 (CXCR3-CCR6-), Tfh17 (CXCR3-CCR6+), and Tfh1/17 (CXCR3+CCR6+) cells, which share the signature transcription factors and cytokines of Th1 (T-bet and IFN-γ), Th2 , Th17  cells, respectively (+PD-1highCCR7lowcTfh1 cells effectively regulate B cell differentiation and induce antibody responses (+Treg cells in the GC that are initiated from Foxp3+/- precursors but not from Tfh cells (Circulating Tfh (cTfh) cells in the peripheral blood are usually composed of two distinctive phenotypes: effector memory Tfh cells (PD-1T cells) , 57, 58.ectively , 58, 59.esponses . These sesponses , 66–71. esponses –74. Currfh cells –78. Tfr fh cells –82. Impofh cells .+T cells are primed by binding their T cell receptors with peptide-loaded major histocompatibility complex (MHC) class II (pMHC-II) on professional antigen-presenting cells (APCs), such as DCs and monocytes. Strong TCR signaling and continuous antigenic stimulation play critical roles in favoring Tfh cell differentiation by upregulating BATF to promote Bcl-6 expression (+CXCR5+Pre-Tfh cells are attracted by the chemokine CXCL13 (CXCR5 ligand) produced within the B cell follicle zones toward the T-B border  are consistently elicited after S peptide stimulation in convalescent COVID-19 cases and exhibit a clear phenotypic bias to aCCR6+CXCR3-cTfh17 cell phenotype; however, neutralizing activity is inversely correlated with S-specific cTfh17 cell frequencies but positively correlated with S-specific cTfh, cTfh1 (CCR6-CXCR3+) and cTfh2 (CCR6-CXCR3-) cell frequencies , cTfh1 and cTfh2 cells in CXCR5+CD45RA-CD25-CD4+T cells are significantly increased, as well as high IL-1β and TNF-α, and that the frequencies of cTfh1 cells are associated with SARS-CoV-2-specific IgG/IgM antibodies, although CCR7highPD-1-cTfh-central memory (cm) and cTfh17 cells in CXCR5+CD45RA-CD25-CD4+T cells are decreased, as well as cTfr cells in Treg cells in convalescent patients compared to healthy subjects. Moreover, the frequencies of high cTfh-em, low cTfh-cm and cTfr cells are positively correlated with disease severity  were observed in acute and convalescent COVID-19 cases, and the frequencies of both SARS-CoV-2-specific cTfh cells and S-specific CCR6+CXCR3-cTfh17 cells were closely associated with low disease severity  and aberrant TNF-α production in COVID-19 lymph nodes in COVID-19 patients, as well as loss of GCs in lymph nodes and spleens from acute and dead COVID-19 patients  cells were positively correlated with anti-SARS-CoV-2 IgM and IgG titers in hospitalized COVID-19 patients  cohort (LCR) than in those in the short-time clinically recovered (4∼9 weeks) cohort (SCR). However, the frequencies of cTfh cells in both the LCR and SCR cohorts were lower than those in the healthy donor cohort (HD). Moreover, three cTfh subsets were similar between the LCR and HD cohorts; cTfh1 cell frequencies in the SCR cohort were shown to be significantly low, but cTfh2 and cTfh17 subsets were found to be high compared with the LCR and HD cohorts  specific for the SARS-CoV-2 spike and nucleocapsid proteins and produce high titers of antiviral serum IgG and IgM antibodies againstSARS-CoV-2  cells include CD69+OX40+ or CD69+CD40L+ orCD69+4-1BB+ or OX40+4-1BB+ or CD40L+4-1BB+ or CD40L+OX40+cells, and the frequency of AIM+cTfh cells is positively correlated with anti-Spike-specific IgA and IgG antibody titers , Funding for Chongqing International Institute for Immunology (2020YJC10), Hong Kong Research Grants Council General Research Fund (17113319) and Theme-Based Research Scheme (T12-703/19R), Zhejiang Provincial Key Research and Development Program (Grant No. 2020C03032).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.