In situ synthesis of luminescent dsDNA-Cu NCs stained with a dsDNA-lighted fluorophore for rapid and stable detection of histamine in food.

Poisonous histamine is accumulated in stale meat and fermented foods. The rapid and stable detection of histamine is essential for food safety. Herein, a ratiometric fluorometric method for histamine detection was designed through in situ preparing double-stranded DNA­copper nanoclusters (dsDNA-Cu NCs) stained with 4',6-diamidino-2-phenylindole (DAPI). dsDNA-Cu NCs with red emission were rapidly synthesized via mixing Cu2+, ascorbate and dsDNA at room temperature for 5 min. When DAPI was added during preparation, DAPI coordinated with the Cu element accompanied by the quenched red emission of dsDNA-Cu NCs, and DAPI bound to dsDNA together with the enhanced blue emission of DAPI. Upon adding DAPI and histamine simultaneously, the coordination of histamine with the Cu element further decreased the red emission of dsDNA-Cu NCs, and drove the movement of DAPI from the Cu element to dsDNA along with the enhanced blue emission of DAPI. Significantly, ratiometric fluorescence was insensitive to variations in instrument and environment, causing stable measurement. Meanwhile, in situ synthesis integrated probe preparation with analyte detection, reducing time consumption. Additionally, this method quantified histamine in the concentration range of 7-50 µM with a detection limit of 3.6 µM. It was applied to determining histamine in food with satisfactory accuracy and precision.

Analysis of the Immune Response by Standardized Whole-Blood Stimulation with Metabolism Modulation.

The immune system defends the body from infection and plays a vital role in a wide range of health conditions. Metabolism affects a series of physiological processes, including those linked to the function of human immune system. Cellular metabolism modulates immune cell activation and cytokine production. Understanding the relationship between metabolism and immune response has important implications for the development of immune-based therapeutics. However, the deployment of large-scale functional assays to investigate the metabolic regulation of immune response has been limited by the lack of standardized procedures. Here, we present a protocol for the analysis of immune response using standardized whole-blood stimulation with metabolism modulation. Diverse immune stimuli including pattern recognition receptor (PRR) ligands and microbial stimuli were incubated with fresh human whole blood. The metabolic inhibitors were used to modulate metabolic status in the immune cells. The variable immune responses after metabolic interventions were evaluated. We described in detail the main steps involved in the whole-blood stimulation and cytokines quantification, namely, collection and treatment of whole blood, preparation of samples and controls, cytokines detection, and stimulation with metabolic interventions. The metabolic inhibitors for anabolic pathways and catabolic pathways exert selective effects on the production of cytokines from immune cells. In addition to a robust and accurate assessment of immune response in cohort studies, the standardized whole-blood stimulation with metabolic regulation might provide new insights for modulating immunity. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00114-0.

Single cell multi-omics reveal intra-cell-line heterogeneity across human cancer cell lines.

Human cancer cell lines have long served as tools for cancer research and drug discovery, but the presence and the source of intra-cell-line heterogeneity remain elusive. Here, we perform single-cell RNA-sequencing and ATAC-sequencing on 42 and 39 human cell lines, respectively, to illustrate both transcriptomic and epigenetic heterogeneity within individual cell lines. Our data reveal that transcriptomic heterogeneity is frequently observed in cancer cell lines of different tissue origins, often driven by multiple common transcriptional programs. Copy number variation, as well as epigenetic variation and extrachromosomal DNA distribution all contribute to the detected intra-cell-line heterogeneity. Using hypoxia treatment as an example, we demonstrate that transcriptomic heterogeneity could be reshaped by environmental stress. Overall, our study performs single-cell multi-omics of commonly used human cancer cell lines and offers mechanistic insights into the intra-cell-line heterogeneity and its dynamics, which would serve as an important resource for future cancer cell line-based studies.

Deep Immunophenotyping of Human Whole Blood by Standardized Multi-parametric Flow Cytometry Analyses.

Immunophenotyping is proving crucial to understanding the role of the immune system in health and disease. High-throughput flow cytometry has been used extensively to reveal changes in immune cell composition and function at the single-cell level. Here, we describe six optimized 11-color flow cytometry panels for deep immunophenotyping of human whole blood. A total of 51 surface antibodies, which are readily available and validated, were selected to identify the key immune cell populations and evaluate their functional state in a single assay. The gating strategies for effective flow cytometry data analysis are included in the protocol. To ensure data reproducibility, we provide detailed procedures in three parts, including (1) instrument characterization and detector gain optimization, (2) antibody titration and sample staining, and (3) data acquisition and quality checks. This standardized approach has been applied to a variety of donors for a better understanding of the complexity of the human immune system. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-022-00092-9.

Salidroside facilitates therapeutic angiogenesis in diabetic hindlimb ischemia by inhibiting ferroptosis.

Hindlimb ischemia (HLI), in which blood perfusion to the hindlimb is obstructed, is one of the major complications of diabetes. Skeletal muscle cells are crucial for revascularization as they can secrete various angiogenic factors; however, hyperglycemia impairs their viability and subsequently their angiogenic potential. Salidroside can promote skeletal muscle cell viability under hyperglycemia; however, the molecular mechanism is still poorly understood. Here we revealed that salidroside could suppress hyperglycemia-induced ferroptosis in skeletal muscle cells by promoting GPX4 expression, thereby restoring their viability and paracrine functions. These in turn promoted the proliferation and migration potentials of blood vessel-forming cells. Furthermore, we showed that salidroside/GPX4-mediated ferroptosis inhibition is crucial for promoting angiogenesis and blood perfusion recovery in diabetic HLI mice. Together, we reveal a novel molecular mechanism of salidroside in enhancing skeletal muscle cells-mediated revascularization and blood perfusion recovery in diabetic HLI mice, further highlighting it as a potential compound for treating diabetic HLI.

SGLT2 inhibitor empagliflozin promotes revascularization in diabetic mouse hindlimb ischemia by inhibiting ferroptosis.

Gliflozins are known as SGLT2 inhibitors, which are used to treat diabetic patients by inhibiting glucose reabsorption in kidney proximal tubules. Recent studies show that gliflozins may exert other effects independent of SGLT2 pathways. In this study we investigated their effects on skeletal muscle cell viability and paracrine function, which were crucial for promoting revascularization in diabetic hindlimb ischemia (HLI). We showed that treatment with empagliflozin (0.1-40 µM) dose-dependently increased high glucose (25 mM)-impaired viability of skeletal muscle C2C12 cells. Canagliflozin, dapagliflozin, ertugliflozin, ipragliflozin and tofogliflozin exerted similar protective effects on skeletal muscle cells cultured under the hyperglycemic condition. Transcriptomic analysis revealed an enrichment of pathways related to ferroptosis in empagliflozin-treated C2C12 cells. We further demonstrated that empagliflozin and other gliflozins (10 µM) restored GPX4 expression in high glucose-treated C2C12 cells, thereby suppressing ferroptosis and promoting cell viability. Empagliflozin (10 µM) also markedly enhanced the proliferation and migration of blood vessel-forming cells by promoting paracrine function of skeletal muscle C2C12 cells. In diabetic HLI mice, injection of empagliflozin into the gastrocnemius muscle of the left hindlimb (10 mg/kg, every 3 days for 21 days) significantly enhanced revascularization and blood perfusion recovery. Collectively, these results reveal a novel effect of empagliflozin, a clinical hypoglycemic gliflozin drug, in inhibiting ferroptosis and enhancing skeletal muscle cell survival and paracrine function under hyperglycemic condition via restoring the expression of GPX4. This study highlights the potential of intramuscular injection of empagliflozin for treating diabetic HLI.

Therapeutic potential and molecular mechanisms of salidroside in ischemic diseases.

Rhodiola is an ancient wild plant that grows in rock areas in high-altitude mountains with a widespread habitat in Asia, Europe, and America. From empirical belief to research studies, Rhodiola has undergone a long history of discovery, and has been used as traditional medicine in many countries and regions for treating high-altitude sickness, anoxia, resisting stress or fatigue, and for promoting longevity. Salidroside, a phenylpropanoid glycoside, is the main active component found in all species of Rhodiola. Salidroside could enhance cell survival and angiogenesis while suppressing oxidative stress and inflammation, and thereby has been considered a potential compound for treating ischemia and ischemic injury. In this article, we highlight the recent advances in salidroside in treating ischemic diseases, such as cerebral ischemia, ischemic heart disease, liver ischemia, ischemic acute kidney injury and lower limb ischemia. Furthermore, we also discuss the pharmacological functions and underlying molecular mechanisms. To our knowledge, this review is the first one that covers the protective effects of salidroside on different ischemia-related disease.

Therapeutic angiogenesis-based strategy for peripheral artery disease.

Peripheral artery disease (PAD) poses a great challenge to society, with a growing prevalence in the upcoming years. Patients in the severe stages of PAD are prone to amputation and death, leading to poor quality of life and a great socioeconomic burden. Furthermore, PAD is one of the major complications of diabetic patients, who have higher risk to develop critical limb ischemia, the most severe manifestation of PAD, and thus have a poor prognosis. Hence, there is an urgent need to develop an effective therapeutic strategy to treat this disease. Therapeutic angiogenesis has raised concerns for more than two decades as a potential strategy for treating PAD, especially in patients without option for surgery-based therapies. Since the discovery of gene-based therapy for therapeutic angiogenesis, several approaches have been developed, including cell-, protein-, and small molecule drug-based therapeutic strategies, some of which have progressed into the clinical trial phase. Despite its promising potential, efforts are still needed to improve the efficacy of this strategy, reduce its cost, and promote its worldwide application. In this review, we highlight the current progress of therapeutic angiogenesis and the issues that need to be overcome prior to its clinical application.

Anti-biofouling Ti3C2TX MXene-holey graphene modified electrode for dopamine sensing in complex biological fluids.

With the development of new methods for the medical diagnosis, electrochemical sensors have attracted increasing attention. However, biofouling on the surface of the sensor significantly decreases sensor performance, thereby limiting the application of electrochemical sensors in complex biological fluids. Given the urgent need for anti-biofouling electrodes, a sensor based on a glass carbon electrode (GCE) modified with Ti3C2TX MXene and electrochemically reduced holey graphene (ERHG) was fabricated and demonstrated to have excellent electrochemical performance and anti-biofouling properties. ERHG provides abundant surface-active sites and imparts stability by hindering the agglomeration and oxidation of MXene. Furthermore, the excellent conductivity and hydrophilicity of MXene result in a high electron transfer rate and strong hydrophilicity. The MXene-ERHG/GCE sensor can detect dopamine with a wide linear range (0.2-125 µM) and a low detection limit of 0.044 µM in phosphate-buffer saline solution. Importantly, the hydrophilicity of MXene-ERHG reduces non-specific protein adsorption on the electrode surface, providing resistance to biofouling. After immersion in bovine serum albumin for 30 min, MXene-ERHG/GCE retained 85.90% of its initial peak current value, much higher than that of ERHG/GCE (17.75%). The MXene-ERHG/GCE sensor also showed good sensitivity for dopamine detection in serum and artificial cerebrospinal fluid (aCSF) containing bovine serum albumin. Moreover, MXene-ERHG/GCE exhibited excellent reproducibility and long-term stability in aCSF. The results demonstrate that MXene-ERHG/GCE has excellent anti-biofouling performance, and shows potential as an electrode material for application in biosensing.

Intramuscular injection of sotagliflozin promotes neovascularization in diabetic mice through enhancing skeletal muscle cells paracrine function.

Diabetes mellitus is associated with series of macrovascular and microvascular pathological changes that cause a wide range of complications. Diabetic patients are highly susceptible to hindlimb ischemia (HLI), which remains incurable. Evidence shows that skeletal muscle cells secrete a number of angiogenic factors to promote neovascularization and restore blood perfusion, this paracrine function is crucial for therapeutic angiogenesis in diabetic HLI. In this study we investigated whether sotagliflozin, an anti-hyperglycemia SGLT2 inhibitor, exerted therapeutic angiogenesis effects in diabetic HLI in vitro and in vivo. In C2C12 skeletal muscle cells, we showed that high glucose (HG, 25 mM) under hypoxia markedly inhibited cell viability, proliferation and migration potentials, which were dose-dependently reversed by pretreatment with sotagliflozin (5-20 µM). Sotagliflozin pretreatment enhanced expression levels of angiogenic factors HIF-1α, VEGF-A and PDGF-BB in HG-treated C2C12 cells under hypoxia as well as secreted amounts of VEGF-A and PDGF-BB in the medium; pretreatment with the HIF-1α inhibitor 2-methoxyestradiol (2-ME2, 10 µM) or HIF-1α knockdown abrogated sotagliflozin-induced increases in VEGF-A and PDGF-BB expression, as well as sotagliflozin-stimulated cell proliferation and migration potentials. Furthermore, the conditioned media from sotagliflozin-treated C2C12 cells in HG medium enhanced the migration and proliferation capabilities of vascular endothelial and smooth muscle cells, two types of cells necessary for forming functional blood vessels. In vivo study was conducted in diabetic mice subjected to excising the femoral artery of the left limb. After the surgery, sotagliflozin (10 mg/kg) was directly injected into gastrocnemius muscle of the left hindlimb once every 3 days for 3 weeks. We showed that intramuscular injection of sotagliflozin effectively promoted the formation of functional blood vessels, leading to significant recovery of blood perfusion in diabetic HLI mice. Together, our results highlight a new indication of SGLT2 inhibitor sotagliflozin as a potential therapeutic angiogenesis agent for diabetic HLI.

Discovery of Salidroside-Derivated Glycoside Analogues as Novel Angiogenesis Agents to Treat Diabetic Hind Limb Ischemia.

Therapeutic angiogenesis is a potential therapeutic strategy for hind limb ischemia (HLI); however, currently, there are no small-molecule drugs capable of inducing it at the clinical level. Activating the hypoxia-inducible factor-1 (HIF-1) pathway in skeletal muscle induces the secretion of angiogenic factors and thus is an attractive therapeutic angiogenesis strategy. Using salidroside, a natural glycosidic compound as a lead, we performed a structure-activity relationship (SAR) study for developing a more effective and druggable angiogenesis agent. We found a novel glycoside scaffold compound (C-30) with better efficacy than salidroside in enhancing the accumulation of the HIF-1α protein and stimulating the paracrine functions of skeletal muscle cells. This in turn significantly increased the angiogenic potential of vascular endothelial and smooth muscle cells and, subsequently, induced the formation of mature, functional blood vessels in diabetic and nondiabetic HLI mice. Together, this study offers a novel, promising small-molecule-based therapeutic strategy for treating HLI.

Endogenous Retrovirus-Derived Long Noncoding RNA Enhances Innate Immune Responses via Derepressing RELA Expression.

Endogenous retroviruses (ERVs) are transposable elements that cause host genome instability and usually play deleterious roles in disease such as tumorigenesis. Recent advances also suggest that this "enemy within" may encode a viral mimic to induce antiviral immune responses through viral sensors. Here, through whole-genome transcriptome analysis with RNA sequencing (RNA-Seq), we discovered that a full-length ERV-derived long noncoding RNA (lncRNA), designated lnc-EPAV (ERV-derived lncRNA positively regulates antiviral responses), was a positive regulator of NF-κB signaling. lnc-EPAV expression was rapidly upregulated by viral RNA mimics or RNA viruses to facilitate the expression of RELA, an NF-κB subunit that plays a crucial role in antiviral responses. Transcriptome analysis of lnc-EPAV-silenced macrophages showed that lnc-EPAV was critical for RELA target gene expression and innate immune responses. Consistently, lnc-EPAV-deficient mice exhibited reduced expression of type I interferons (IFNs) and, consequently, increased viral loads and mortality following lethal RNA virus infection. Mechanistically, lnc-EPAV promoted expression of RELA by competitively binding to and displacing SFPQ, a transcriptional repressor of Rela Altogether, our work demonstrates an alternative mechanism by which ERVs regulate antiviral immune responses.IMPORTANCE Endogenous retroviruses are transposable genetic elements comprising 8% to 10% of the human and mouse genomes. Although most ERVs have been inactivated due to deleterious mutations, some are still transcribed. However, the biological functions of transcribed ERVs are largely unknown. Here, we identified a full-length ERV-derived lncRNA, designated lnc-EPAV, as a positive regulator of host innate immune responses. We found that silencing lnc-EPAV impaired virus-induced cytokine production, resulting in increased viral replication in cells. The lnc-EPAV-deficient mice exhibited enhanced susceptibility to viral challenge. We also found that lnc-EPAV regulated expression of RELA, an NF-κB subunit that plays a critical role in antiviral responses. ERV-derived lncRNA coordinated with a transcription repressor, SFPQ, to control Rela transcription. Our report provides new insights into the previously unrecognized immune gene regulatory mechanism of ERV-derived lncRNAs.

Exploitation of nuclear protein SFPQ by the encephalomyocarditis virus to facilitate its replication.

The encephalomyocarditis virus (EMCV) is a single-stranded RNA virus that induces sudden death, diabetes, myocarditis and nervous disorders in non-human primates. The rapid development of xenografts such as heart transplantation from pig to human raises the issue of EMCV safety in human cells. SFPQ, a proline and glutamine rich splicing factor that participates in diverse molecular functions including paraspeckle formation, microRNA synthesis and transcription regulation, is known to regulate host innate immune response to viruses. However, the role of SFPQ in EMCV infection remains unclear. Here we reported that the SFPQ was essential for EMCV replication. Depletion of SFPQ impaired EMCV production, while forced expression of SFPQ promoted viral replication. Mechanistically, loss of SFPQ affected the transcription profile of host mitochondria pathway related genes. In addition, cellular SFPQ was exploited by EMCV and accumulated in cytoplasm and it interacted with eukaryotic initiation factors and ribosomal proteins to facilitate internal ribosome entry site (IRES)-dependent translation of EMCV protein. Altogether, our work discovered host SFPQ as a new target to inhibit EMCV replication and infection.

Engineering Clostridium absonum 7α-hydroxysteroid Dehydrogenase for Enhancing Thermostability Based on Flexible Site and ΔΔG Prediction.

BACKGROUND: Enhancing thermostability of the 7α-Hydroxysteroid dehydrogenases (7α-HSDHs) is beneficial to its industrial application broadly. For protein engineering to enhance thermostability the nonrational strategy, directed evolution, has been applied in obtaining more stable proteins through error-prone PCR or DNA rearrangement generating random mutations. However, the successful application of directed evolution needs to build a large mutant library. Site-directed mutations of CA 7α-HSDH had been performed to probe the relationship between the compactness increasing and thermostability enhancing. Although most of the mutations in ß-sheet core predicted by MAESTRO became more stable than wild type, unfortunately, all the mutations suffered dramatic activity loss. OBJECTIVE: The main objective of this study was to verify effects of the mutations in helices selected from the predicting results through MAESTRO on thermostability improving of CA 7α-HSDH. METHODS: Seven mutants, S22L, P124L, A125L, N171L, A195Q, L197E and Y259E were synthesized and verified through DNA sequencing in Sangon Biotech (Sangon, Shanghai, China). The two mutants, A104P and G105P were prepared by over-lapping PCR. The GST-fusion expression vector, pGEX-6p-1 (GE Healthcare), was used for protein expression with restriction sites BamH I and Not I. Thermostability was measured by circular dichroism (CD) spectrometer (MOS-450, BioLogic Inc). All the enzymes were diluted in PBS (pH 7.3, 10 mM) to OD222 value between 0.8 and 1, and temperature varied from 20°C to 95°C. Activity of enzyme was assayed by measuring the production of NADPH by UV-visible spectrophotometer at 340 nm. The activity assay was performed in 2 mL reaction mixture which contained PBS (pH 7.3, 10 mM), NADP+ (0.5 mM) and taurocholic acid (TCA) at 25°C. RESULTS: Based on unfolding free energy changes (ΔΔG) prediction seven mutations of Clostridium absonum (CA) 7α-HSDH were selected and experimentally verified, and these mutants fitted three-state denaturation model well, among which S22L located in the αA possessed the greatest Tm N→I increase (> 8°C). Mutants P124L, L197E, N171L and Y259E also became more stable than wild type CA 7α-HSDH with different ranges. Meanwhile, thermostability of the two mutants, A104P and G105P (in the coil between ßD and αD) resulting from the proline substitution method decreased significantly. Enzyme activity assays indicated that mutant L197E located in αF maximally maintained 28.7% of catalytic efficiency, and activity of the five mutants, P124L, A125L, N171L, A104P and G105P cannot be detected. CONCLUSION: Although all the mutants' activities decreased, the mutant L197E with the maximum activity retain suggested that the loop structure (residues 194 to 211) may be the favored candidate sites to enhance thermostability. In addition, CA 7α-HSDH may suffer structural destruction resulting from the proline substitution in A104 and G105.