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d -2-Hydroxyglutarate ( d -2-HG) is a metabolite involved in many physiological metabolic processes. When d -2-HG is aberrantly accumulated due to mutations in isocitrate dehydrogenase or d -2-HG dehydrogenase, it functions in a pro-oncogenic manner and is thus considered a therapeutic target and biomarker in many cancers. In this study, DhdR from Achromobacter denitrificans NBRC 15125 is identified as an allosteric transcriptional factor that negatively regulates d -2-HG dehydrogenase expression and responds to the presence of d -2-HG. Based on the allosteric effect of DhdR, a d -2-HG biosensor is developed by combining DhdR with amplified luminescent proximity homogeneous assay (AlphaScreen) technology. The biosensor is able to detect d -2-HG in serum, urine, and cell culture medium with high specificity and sensitivity. Additionally, this biosensor is used to identify the role of d -2-HG metabolism in lipopolysaccharide biosynthesis of Pseudomonas aeruginosa , demonstrating its broad usages. d -2-hydroxyglutarate ( d -2-HG) is a metabolite that can be aberrantly accumulated and acts as a biomarker in many cancers. Here the authors report a d -2-HG biosensor based on the allosteric transcription factor DhdR which they use for detection in serum and urine.

l -2-Hydroxyglutarate ( l- 2-HG) plays important roles in diverse physiological processes, such as carbon starvation response, tumorigenesis, and hypoxic adaptation. Despite its importance and intensively studied metabolism, regulation of l- 2-HG metabolism remains poorly understood and none of regulator specifically responded to l- 2-HG has been identified. Based on bacterial genomic neighborhood analysis of the gene encoding l -2-HG oxidase (LhgO), LhgR, which represses the transcription of lhgO in Pseudomonas putida W619, is identified in this study. LhgR is demonstrated to recognize l -2-HG as its specific effector molecule, and this allosteric transcription factor is then used as a biorecognition element to construct an l -2-HG-sensing FRET sensor. The l -2-HG sensor is able to conveniently monitor the concentrations of l -2-HG in various biological samples. In addition to bacterial l- 2-HG generation during carbon starvation, biological function of the l -2-HG dehydrogenase and hypoxia induced l- 2-HG accumulation are also revealed by using the l -2-HG sensor in human cells. L-2-hydroxyglutarate (L-2-HG) is an important metabolite but its regulation is poorly understood. Here the authors report an L-2-HG FRET biosensor based on the allosteric transcription factor, LhgR, to monitor L-2-HG in cells and biological samples.

The tumor microenvironment (TME) contains cells that regulate medication response and cancer growth in a major way. Tumor immunology research has been rejuvenated and cancer treatment has been changed by immunotherapy, a rapidly developing therapeutic approach. The growth patterns of tumor cells in vivo and the heterogeneity, complexity, and individuality of tumors produced from patients are not reflected in traditional two-dimensional tumor cell profiles. On the other hand, an in vitro three-dimensional (3D) model called the organoid model is gaining popularity. It can replicate the physiological and pathological properties of the original tissues in vivo . Tumor cells are the source of immune organoids. The TME characteristics can be preserved while preserving the variety of tumors by cultivating epithelial tumor cells with various stromal and immunological components. In addition to having genetic and physical similarities to human diseases and the ability to partially reconstruct the complex structure of tumors, these models are now widely used in research fields including cancer, developmental biology, regenerative mechanisms, drug development, disease modeling, and organ transplantation. This study reviews the function of organoids in immunotherapy and the tumor immune milieu. We also discuss current developments and suggest translational uses of tumor organoids in immuno-oncology research, immunotherapy modeling, and precision medicine.

Middle East respiratory syndrome coronavirus (MERS-CoV) currently spreads in humans and causes ∼36% fatality in infected patients. Believed to have originated from bats, MERS-CoV is genetically related to bat coronaviruses HKU4 and HKU5. To understand how bat coronaviruses transmit to humans, we investigated the receptor usage and cell entry activity of the virus-surface spike proteins of HKU4 and HKU5. We found that dipeptidyl peptidase 4 (DPP4), the receptor for MERS-CoV, is also the receptor for HKU4, but not HKU5. Despite sharing a common receptor, MERS-CoV and HKU4 spikes demonstrated functional differences. First, whereas MERS-CoV prefers human DPP4 over bat DPP4 as its receptor, HKU4 shows the opposite trend. Second, in the absence of exogenous proteases, both MERS-CoV and HKU4 spikes mediate pseudovirus entry into bat cells, whereas only MERS-CoV spike, but not HKU4 spike, mediates pseudovirus entry into human cells. Thus, MERS-CoV, but not HKU4, has adapted to use human DPP4 and human cellular proteases for efficient human cell entry, contributing to the enhanced pathogenesis of MERS-CoV in humans. These results establish DPP4 as a functional receptor for HKU4 and host cellular proteases as a host range determinant for HKU4. They also suggest that DPP4-recognizing bat coronaviruses threaten human health because of their spikes’ capability to adapt to human cells for cross-species transmissions.

A novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), has caused outbreaks of a SARS-like illness with high case fatality rate. The reports of its person-to-person transmission through close contacts have raised a global concern about its pandemic potential. Here we characterize the six-helix bundle fusion core structure of MERS-CoV spike protein S2 subunit by X-ray crystallography and biophysical analysis. We find that two peptides, HR1P and HR2P, spanning residues 998–1039 in HR1 and 1251–1286 in HR2 domains, respectively, can form a stable six-helix bundle fusion core structure, suggesting that MERS-CoV enters into the host cell mainly through membrane fusion mechanism. HR2P can effectively inhibit MERS-CoV replication and its spike protein-mediated cell–cell fusion. Introduction of hydrophilic residues into HR2P results in significant improvement of its stability, solubility and antiviral activity. Therefore, the HR2P analogues have good potential to be further developed into effective viral fusion inhibitors for treating MERS-CoV infection. MERS-CoV is a novel human coronavirus that has recently caused outbreaks of respiratory illness with high case fatality rate. Here the authors characterize the membrane fusion apparatus of MERS-CoV and develop a peptide that can inhibit virus fusion and replication in vitro .

Background Although significant advances have been made in intensive care medicine and antibacterial treatment, sepsis is still a common disease with high mortality. The condition of sepsis patients changes rapidly, and each hour of delay in the administration of appropriate antibiotic treatment can lead to a 4–7% increase in fatality. Therefore, early diagnosis and intervention may help improve the prognosis of patients with sepsis. Methods We obtained single-cell sequencing data from 12 patients. This included 14,622 cells from four patients with bacterial infectious sepsis and eight patients with sepsis admitted to the ICU for other various reasons. Monocyte differentiation trajectories were analyzed using the “monocle” software, and differentiation-related genes were identified. Based on the expression of differentiation-related genes, 99 machine-learning combinations of prognostic signatures were obtained, and risk scores were calculated for all patients. The “scissor” software was used to associate high-risk and low-risk patients with individual cells. The “cellchat” software was used to demonstrate the regulatory relationships between high-risk and low-risk cells in a cellular communication network. The diagnostic value and prognostic predictive value of Enah/Vasp-like ( EVL ) were determined. Clinical validation of the results was performed with 40 samples. The “CBNplot” software based on Bayesian network inference was used to construct EVL regulatory networks. Results We systematically analyzed three cell states during monocyte differentiation. The differential analysis identified 166 monocyte differentiation-related genes. Among the 99 machine-learning combinations of prognostic signatures constructed, the Lasso + CoxBoost signature with 17 genes showed the best prognostic prediction performance. The highest percentage of high-risk cells was found in state one. Cell communication analysis demonstrated regulatory networks between high-risk and low-risk cell subpopulations and other immune cells. We then determined the diagnostic and prognostic value of EVL stabilization in multiple external datasets. Experiments with clinical samples demonstrated the accuracy of this analysis. Finally, Bayesian network inference revealed potential network mechanisms of EVL regulation. Conclusions Monocyte differentiation-related prognostic signatures based on the Lasso + CoxBoost combination were able to accurately predict the prognostic status of patients with sepsis. In addition, low EVL expression was associated with poor prognosis in sepsis.

Background As an attracted compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) showed great potentials in various field. However, lower productivity and high saline medium seriously hinder its wide applications. Results The entire ectoine metabolism, including pathways for ectoine synthesis and catabolism, was identified in the genome of an ectoine-excreting strain Halomonas hydrothermalis Y2. By in-frame deletion of genes encoding ectoine hydroxylase (EctD) and (or) ectoine hydrolase (DoeA) that responsible for ectoine catabolism, the pathways for ectoine utilization were disrupted and resulted in an obviously enhanced productivity. Using an optimized medium containing 100 g L −1 NaCl in a 500-mL flask, the double mutant of Y2/ ΔectD / ΔdoeA synthesized 3.13 g L −1 ectoine after 30 h cultivation. This is much higher than that of the wild type strain (1.91 g L −1 ), and also exceeds the production of Y2/ ΔectD (2.21 g L −1 ). The remarkably enhanced accumulation of ectoine by Y2/ ΔectD / ΔdoeA implied a critical function of Doe pathway in the ectoine catabolism. Furthermore, to reduce the salinity of fermentation medium and overcome the wastewater treatment difficulty, mutants that lacking key Na + /H + antiporter, Mrp and (or) NhaD2, were constructed based on strain Y2/ ΔectD / ΔdoeA . As a result, the Mrp-deficient strain could synthesize equal amount of ectoine (around 7 g L −1 or 500 mg (g DCW) −1 ) in the medium containing lower concentration of NaCl. During a fed-batch fermentation process with 60 g L −1 NaCl stress, a maximum 10.5 g L −1 ectoine was accumulated by the Mrp-deficient strain, with a specific production of 765 mg (g DCW) −1 and a yield of 0.21 g g −1 monosodium glutamate. Conclusion The remarkably enhanced production of ectoine by Y2/ ΔectD / ΔdoeA implied the critical function of Doe pathway in the ectoine catabolism. Moreover, the reduced salinity requirement of Mrp-deficient strain implied a feasible protocol for many compatible solute biosynthesis, i.e., by silencing some Na + /H + antiporters in their halophilic producers and thus lowering the medium salinity.

Background Integrin α5β1 plays a crucial role in the invasion of nonphagocytic cells by Staphylococcus aureus ( S. aureus ), thereby facilitating infection development. Lipid nanoparticles (LNPs) serve as an effective vehicle for delivering small interfering ribonucleic acids (siRNA) that represent a method to knockdown integrin α5β1 in the lungs through nebulization, thereby potentially mitigating the severity of S. aureus pneumonia. The aim of this study was to harness LNP-mediated targeting to precisely knockdown integrin α5β1, thus effectively addressing S. aureus -induced pneumonia. Methods C57 mice (8 week-old females) infected with S. aureus via an intratracheal nebulizing device were utilized for the experiments. The LNPs were synthesized via microfluidic mixing and characterized by their size, polydispersity index, and encapsulation efficiency. Continuous intratracheal nebulization was employed for consistent siRNA administration, with the pulmonary function metrics affirming biosafety. The therapeutic efficacy of LNP-encapsulated siRNAs against pneumonia was assessed through western blotting, bacterial count measurement, quantitative polymerase chain reaction, and histological analyses. Results LNPs, which have an onion-like structure, retained integrity post-nebulization, ensuring prolonged siRNA stability and in vivo safety. Intratracheal nebulization delivery markedly alleviated the severity of S. aureus -induced pneumonia, as indicated by reduced bacterial load and bolstered immune response, thereby localizing the infection to the lungs and averting systemic dissemination. Conclusions Intratracheal nebulization of LNP-encapsulated siRNAs targeting integrin α5β1 significantly diminished the S. aureus -mediated cellular invasion and disease progression in the lungs, presenting a viable therapeutic approach for respiratory infections.

Smoking and infection are the most common risk factors for acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Irisin is a hormone-like substance that helps reduce oxidative stress and inflammation in lung diseases. This study aims to explore the potential regulatory mechanism of irisin in AECOPD induced by CSE combined with LPS. Autophagy-deficient models were established in mice and MH-S cells. Pathological changes in mouse lung tissues were assessed using HE staining, while Irisin expression was detected via immunofluorescence. Levels of IL-1β, IL-6, and TNF-α in mouse BALF and MH-S cell supernatants were measured by ELISA. Autophagic flux in MH-S cells was monitored using the Ad-mCherry-GFP-LC3B dual-fluorescence system and transmission electron microscopy. Western blotting was performed to analyze autophagy-related proteins in mice and MH-S cells, elucidating the molecular mechanism of Irisin’s protective effects. Irisin can reduce the accumulation of the autophagy marker protein P62 and promote the conversion of LC3-I to LC3-II through the AMPK-Beclin1 pathway, increase autophagosome-lysosome formation, effectively restore damaged autophagic flux, and lower the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α elevated by CSE + LPS. Irisin activates protective autophagy through the AMPK-Beclin1 pathway, restores the autophagy imbalance induced by CSE + LPS in the lungs, reduces lung inflammation levels, and provides a new therapeutic target for the treatment of AECOPD.

Anaerobic fermentation using lignocellulosic hydrolysates as co-substrates is an economically attractive method to enhance 1,3-propanediol (1,3-PD) production by increasing the conversion yield from glycerol. Lignocellulosic hydrolysates contain the mixed sugars that are primarily glucose, xylose and arabinose. Therefore, these three individual sugars were used, separately, as co-substrates with glycerol, in 1,3-PD production by a Clostridium diolis strain DSM 15410, resulting in an 18%–28% increase in the 1,3-PD yield. Co-fermentation of the mixed sugars and glycerol obtained a higher intracellular NADH/NAD + ratio and increased the 1,3-PD yield by 22% relative to fermentation of glycerol alone. Thereafter, two kinds of lignocellulosic hydrolysates, corn stover hydrolysate and corncob molasses, were individually co-fermented with glycerol. The maximum 1,3-PD yield from glycerol reached 0.85 mol/mol. Fed-batch co-fermentation was also performed, improving the 1,3-PD yield (from 0.62 mol/mol to 0.82 mol/mol). These results demonstrate that the co-fermentation strategy is an efficient and economical way to produce 1,3-PD from glycerol.