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Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with limited treatment options. Pyruvate kinase, especially the M2 isoform (PKM2), is highly expressed in PDAC cells, but its role in pancreatic cancer remains controversial. To investigate the role of pyruvate kinase in pancreatic cancer, we knocked down PKM2 individually as well as both PKM1 and PKM2 concurrently (PKM1/2) in cell lines derived from a Kras G12D/- ; p53 -/- pancreatic mouse model. Methods We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine metabolic profiles of wildtype and PKM1/2 knockdown PDAC cells. We further used stable isotope-labeled metabolic precursors and LC-MS/MS to determine metabolic pathways upregulated in PKM1/2 knockdown cells. We then targeted metabolic pathways upregulated in PKM1/2 knockdown cells using CRISPR/Cas9 gene editing technology. Results PDAC cells are able to proliferate and continue to produce pyruvate despite PKM1/2 knockdown. The serine biosynthesis pathway partially contributed to pyruvate production during PKM1/2 knockdown: knockout of phosphoglycerate dehydrogenase in this pathway decreased pyruvate production from glucose. In addition, cysteine catabolism generated ~ 20% of intracellular pyruvate in PDAC cells. Other potential sources of pyruvate include the sialic acid pathway and catabolism of glutamine, serine, tryptophan, and threonine. However, these sources did not provide significant levels of pyruvate in PKM1/2 knockdown cells. Conclusion PKM1/2 knockdown does not impact the proliferation of pancreatic cancer cells. The serine biosynthesis pathway supports conversion of glucose to pyruvate during pyruvate kinase knockdown. However, direct conversion of serine to pyruvate was not observed during PKM1/2 knockdown. Investigating several alternative sources of pyruvate identified cysteine catabolism for pyruvate production during PKM1/2 knockdown. Surprisingly, we find that a large percentage of intracellular pyruvate comes from cysteine. Our results highlight the ability of PDAC cells to adaptively rewire their metabolic pathways during knockdown of a key metabolic enzyme.

A large percentage of infants develop viral bronchiolitis needing medical intervention and often develop further airway disease such as asthma. To characterize metabolic perturbations in acute respiratory syncytial viral (RSV) bronchiolitis, we compared metabolomic profiles of moderate and severe RSV patients versus sedation controls. RSV patients were classified as moderate or severe based on the need for invasive mechanical ventilation. Whole blood and urine samples were collected at two time points (baseline and 72 h). Plasma and urinary metabolites were extracted in cold methanol and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), and data from the two biofluids were combined for multivariate data analysis. Metabolite profiles were clustered according to severity, characterized by unique metabolic changes in both plasma and urine. Plasma metabolites that correlated with severity included intermediates in the sialic acid biosynthesis, while urinary metabolites included citrate as well as multiple nucleotides. Furthermore, metabolomic profiles were predictive of future development of asthma, with urinary metabolites exhibiting higher predictive power than plasma. These metabolites may offer unique insights into the pathology of RSV bronchiolitis and may be useful in identifying patients at risk for developing asthma.

The feasibility of gastrointestinal (GI) microbiome work in a pediatric intensive care unit (PICU) to determine the GI microbiota composition of infants as compared to control infants from the same hospital was investigated. In a single-site observational study at an urban quaternary care children’s hospital in Western Michigan, subjects less than 6 months of age, admitted to the PICU with severe respiratory syncytial virus (RSV) bronchiolitis, were compared to similarly aged control subjects undergoing procedural sedation in the outpatient department. GI microbiome samples were collected at admission (n = 20) and 72 h (n = 19) or at time of sedation (n = 10). GI bacteria were analyzed by sequencing the V4 region of the 16S rRNA gene. Alpha and beta diversity were calculated. Mechanical ventilation was required for the majority (n = 14) of study patients, and antibiotics were given at baseline (n = 8) and 72 h (n = 9). Control subjects’ bacterial communities contained more Porphyromonas, and Prevotella (p = 0.004) than those of PICU infants. The ratio of Prevotella to Bacteroides was greater in the control than the RSV infants (mean ± SD—1.27 ± 0.85 vs. 0.61 ± 0.75: p = 0.03). Bacterial communities of PICU infants were less diverse than those of controls with a loss of potentially protective populations.

Metastatic breast cancer is currently incurable. It has recently emerged that different metabolic pathways support metastatic breast cancer. To further uncover metabolic pathways enabling breast cancer metastasis, we investigated metabolic differences in mouse tumors of differing metastatic propensities using mass spectrometry-based metabolomics. We found that sialic acid metabolism is upregulated in highly metastatic breast tumors. Knocking out a key gene in sialic acid metabolism, Cmas, inhibits synthesis of the activated form of sialic acid, cytidine monophosphate-sialic acid and decreases the formation of lung metastases in vivo. Thus, the sialic acid pathway may be a new target against metastatic breast cancer.

Recently, unconventional antiferromagnets that enable the spin splitting (SS) of electronic states have been theoretically proposed and experimentally realized, where the magnetic sublattices containing moments pointing at different directions are connected by a novel set of symmetries. Such SS is substantial, k-dependent, and independent of the spin-orbit coupling (SOC) strength, making these magnets promising materials for antiferromagnetic spintronics. Here, combined with angle-resolved photoemission spectroscopy (ARPES) and density functional theory (DFT) calculations, a systematic study on CrSb, a metallic spin-split antiferromagnet candidate with Néel temperature T N = 703 K, is conducted. The data reveal the electronic structure of CrSb along both out-of-plane and in-plane momentum directions, rendering an anisotropic k-dependent SS that agrees well with the calculational results. The magnitude of such SS reaches up to at least 0.8 eV at non-high-symmetry momentum points, which is significantly higher than the largest known SOC-induced SS. This compound expands the choice of materials in the field of antiferromagnetic spintronics and is likely to stimulate subsequent investigations of high-efficiency spintronic devices that are functional at room temperature., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Abstract: Objective: In this study, our aim was to examine the effects of levosimendan on diaphragmatic dysfunction in patients with sepsis, as well as assess its impact on respiratory muscle contractility and the outcome of weaning. Methods: This was a single-blind, randomized, controlled trial. Patients with diaphragmatic dysfunction and failure of spontaneous breathing trials (SBTs) were randomly and equally assigned to the experimental and control groups. The experimental group received levosimendan at a loading dose of 6 μg/kg for 10 min, followed by a continuous infusion at 0.2 μg/kg/min. The control group received an equivalent dose of a placebo. The preadministration and postadministration respiratory mechanics parameters of the patients were recorded. Evaluation of the effect of levosimendan on patients with sepsis-induced diaphragm dysfunction comprised arterial blood gas analysis as well as ultrasound measurements of diaphragm excursion (DE), diaphragm thickness (DT), diaphragm thickening fraction (TFdi), and diaphragm-rapid shallow breathing index (D-RSBI). Results: Forty-four patients were enrolled in the study. We found that postadministration of levosimendan, the patients' tidal volume (GCSMV) increased, whereas the D-RSBI decreased, and the partial pressure of carbon dioxide (PACO 2 ) decreased when compared to the preadministration levels. Additionally, following levosimendan administration, patients showed increased DE and pressure support (PS) when compared to before administration (1.14 ± 0.177 vs. 1.22 ± 0.170 cm and 0.248 ± 0.03 vs. 0.284 ± 0.06, respectively) and decreased D-RSBI (22.76 ± 6.14 vs. 20.06 ± 6.04, respectively), all of which were statistically significant ( P < 0.05). In contrast, in the control group of patients, there were no statistically significant differences in the postadministration levels of DE, TFdi, and D-RSBI as compared to the preadministration period ( P > 0.05). Furthermore, in terms of weaning outcomes, we did not find any statistically significant difference in the number of patients in the two groups who eventually underwent weaning ( P = 0.545). Conclusion: In this study, we found that levosimendan enhanced diaphragm contractile function. However, further investigations are required to explore its effect on weaning outcomes in patients undergoing mechanical ventilation., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 by the Shock Society.)

Objectives: To investigate the differentiation of regulatory T cells (Tregs) induced by methylprednisolone (MP) pulse therapy in patients with Systemic Lupus Erythematosus (SLE)., Methods: We enrolled 30 patients with SLE and analyzed peripheral blood mononuclear cells (PBMCs) before and after MP pulse therapy. Peripheral Tregs, apoptosis of PBMCs subsets, and TGFβ production by monocytes was quantified by flow cytometry. Proliferation and IFN-γ production of CD4 + T cells were measured. Furthermore, TGFβ1 production by human monocyte-derived macrophages (HMDM) stimulated with MP-treated CD4 + T cells were quantified by ELISA., Results: Peripheral Tregs was significantly increased after MP pulse therapy (6.76 ± 1.46% vs. 3.82 ± 1.02%, p < 0.01), with an expansion of Nrp1 - induced Tregs (4.54 ± 0.46% vs. 1.75 ± 0.38%, p < 0.01). Proliferation and IFN-γ production of CD4 + T cells were significantly decreased after MP pulse therapy. MP pulse therapy induced CD4 + T cell apoptosis (early apoptosis, 26.34 ± 3.54% vs. 14.81 ± 2.89%, p < 0.01) and TGFβ expression on monocytes (6.02% vs. 2.45%, p < 0.01). Furthermore, MP induced CD4 + T cell apoptosis in vitro, which stimulated HMDM to produce TGFβ. Moreover, elevated TGFβ level in supernatant from HMDM stimulated with MP-treated CD4 + T cells promoted Tregs differentiation., Conclusions: MP pulse therapy induces CD4 + T cell apoptosis, which promotes monocytes to produce TGFβ and further facilitates Tregs differentiation. Newly-differentiated Tregs suppress proliferation and IFN-γ production of CD4 + T cells and contribute to immunoregulatory milieu after MP pulse therapy., (Copyright © 2022. Published by Elsevier Inc.)

Background and Aims: The increased frequency of urinary tract infections in patients with primary biliary cholangitis (PBC) and the cross-reactivity between the lipoyl domains (LD) of human pyruvate dehydrogenase complex (hPDC-E2) and Escherichia coli PDC-E2 (ePDC-E2) have long suggested a role of E. coli in causality of PBC. This issue, however, has remained speculative. We hypothesized that by generating specific constructs of human and E. coli PDC-E2, we would be able to assess the specificity of autoantibody responses and define whether exposure to E. coli in susceptible hosts is the basis for the antimitochondrial antibody (AMA) response., Approach and Results: Importantly, the reactivity of hPDC-E2 LD (hPDC-E2LD) affinity-purified antibodies against hPDC-E2LD could only be removed by prior absorption with hPDC-E2LD and not ePDC-E2, suggesting the presence of unique human PDC-E2 epitopes distinct from E. coli PDC-E2. To identify the autoepitope(s) present in hPDC-E2LD, a more detailed study using a variety of PDC-E2 constructs was tested, including the effect of lipoic acid (LA) on ePDC-E2 conformation and AMA recognition. Individual recombinant ePDCE2 LD domains LD1, LD2 and LD3 did not react with either AMA or antibodies to LA (anti-LA), but in contrast, anti-LA was readily reactive against purified recombinant LD1, LD2, and LD3 expressed in tandem (LP); such reactivity increased when LP was precultured with LA. Moreover, when the three LD (LD1, LD2, LD3) domains were expressed in tandem in pET28a or when LD1 was expressed in another plasmid pGEX, they were lipoylated and reactive to PBC sera., Conclusions: In conclusion, our data are consistent with an exposure to E. coli that elicits specific antibody to ePDC-E2 resulting in determinant spreading and the classic autoantibody to hPDC-E2LD. We argue this is the first step to development of human PBC., (© 2021 by the American Association for the Study of Liver Diseases.)

Background: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterised by aberrant B cell hyperactivation, whose mechanism is partially understood., Methods: We performed whole transcriptome sequencing of B cells from three pSS patients and three matched healthy controls (HC). Differentially expression genes (DEGs) were confirmed with B cells from 40 pSS patients and 40 HC by quantitative PCR and western blot. We measured the proliferation potential and immunoglobulins production of siRNA-transfected or plasmid-transfected B cells stimulated with cytosine-phosphate-guanine (CpG) or anti-IgM. We also explored Toll-like receptor 9 (TLR9) signalling to reveal the potential mechanism of B cell hyperactivation in pSS., Results: We identified 77 upregulated and 32 downregulated DEGs in pSS B cells. We confirmed that epithelial stromal interaction (EPST1) expression in pSS B cells was significantly higher than that from HCs. EPSTI1-silencing B cells stimulated with CpG were less proliferated and produced lower level of IgG and IgM comparing with control B cells. EPSTI1-silencing B cells expressed lower level of p-p65 and higher level of IκBα, and B cells with overexpressed EPSTI1 showed higher level of p-p65 and lower level of IκBα. Finally, IκBα degradation inhibitor Dehydrocostus Lactone treatment attenuated p65 phosphorylation promoted by EPSTI1., Conclusion: Elevated EPSTI1 expression in pSS B cells promoted TLR9 signalling activation and contributed to the abnormal B cell activation, which was promoted by facilitating p65 phosphorylation and activation of NF-κB signalling via promoting IκBα degradation. EPSTI1 might be implicated in pSS pathogenesis and was a potential therapeutic target of pSS., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: To observe the effects of keratinocyte growth factor-2 (KGF-2) on the expressions of chemokine FKN and tight junction protein claudin-5 in lung tissue of rats with acute lung injury (ALI)., Methods: Healthy male Sprague-Dawley (SD) rats were randomly divided into normal saline (NS) control group, ALI model group and KGF-2 pretreatment group, with 10 rats in each group. The rat ALI model was reproduced by injection of 0.01 mL/kg oleic acid into the tail vein, and the rats in NS control group were injected with the same amount of NS. The rats in KGF-2 pretreatment group were instilled with 5 mg/kg KGF-2 in the airway at 72 hours before the model reproduction, and the rats in the NS control group and the ALI model group were instilled with the same amount of NS. The abdominal aortic blood of rats was collected at 8 hours after model reproduction, and then the rats were sacrificed, bronchoalveolar in left lung was lavage, and the bronchoalveolar lavage fluid (BALF) was collected for determination of protein levels in plasma and BALF, and the lung permeability index (LPI) was calculated. The lung tissues were harvested, after hematoxylin-eosin (HE) staining, the histopathological changes were observed under light microscope, and the ALI pathology score (LIS) was calculated. The lung wet/dry weight (W/D) ratio was determined. Immunohistochemistry and Western Blot were used to qualitatively and quantitatively analyze the expressions of FKN and claudin-5 in the lung tissue. The correlation between two variables was analyzed by linear or curve fitting correlation analysis., Results: In the ALI model group, the lung tissue was severely damaged, and obvious pathological changes were observed, including thickened alveolar space and inflammatory cell infiltration, and LIS score, lung W/D ratio and LPI were significantly higher than those of the NS control group (LIS: 3.56±0.28 vs. 0.62±0.36, lung W/D ratio: 6.37±0.29 vs. 4.39±0.33, LPI: 3.46±0.69 vs. 0.98±0.17, all P < 0.01). Compared with the NS control group, the positive expression of FKN in the lung tissue of the ALI model group was significantly increased, and the expression level was significantly increased (FKN/GAPDH: 0.97±0.18 vs. 0.62±0.04, P < 0.01); the positive expression of claudin-5 was significantly decreased, and the expression level was significantly decreased (claudin-5/GAPDH: 0.56±0.11 vs. 1.06±0.13, P < 0.01). There was a significant negative correlation between FKN and claudin-5 protein expression (r = -0.817, P = 0.025). After pretreatment with KGF-2, the degree of lung tissue damage was significantly reduced, and the pathological changes were significantly improved, and the LIS score, lung W/D ratio and LPI were significantly lower than those of the ALI model group (LIS: 2.41±0.17 vs. 3.56±0.28, lung W/D ratio: 5.45±0.55 vs. 6.37±0.29, LPI: 2.42±0.19 vs. 3.46±0.69, all P < 0.01). Compared with the ALI model group, the positive expression of FKN in the lung tissue of KGF-2 pretreatment group was significantly decreased, and the expression level was significantly decreased (FKN/GAPDH: 0.79±0.03 vs. 0.97±0.18, P < 0.01); the positive expression of claudin-5 was significantly increased, and the expression level was significantly increased (claudin-5/GAPDH: 0.80±0.05 vs. 0.56±0.11, P < 0.01). There was still a significant negative correlation between FKN and claudin-5 protein expression (r = -0.847, P = 0.012)., Conclusions: KGF-2 may restore the expression of tight junction protein claudin-5 by down-regulating the expression of chemokine FKN, thereby reducing the damage of blood barrier in ALI.