Synthesis and In Vivo Antiarrhythmic Activity Evaluation of Novel Scutellarein Analogues as Voltage-Gated Nav1.5 and Cav1.2 Channels Blockers.

Malignant cardiac arrhythmias with high morbidity and mortality have posed a significant threat to our human health. Scutellarein, a metabolite of Scutellarin which is isolated from Scutellaria altissima L., presents excellent therapeutic effects on cardiovascular diseases and could further be metabolized into methylated forms. A series of 22 new scutellarein derivatives with hydroxyl-substitution based on the scutellarin metabolite in vivo was designed, synthesized via the conjugation of the scutellarein scaffold with pharmacophores of FDA-approved antiarrhythmic medications and evaluated for their antiarrhythmic activity through the analyzation of the rat number of arrhythmia recovery, corresponding to the recovery time and maintenance time in the rat model of barium chloride-induced arrhythmia, as well as the cumulative dosage of aconitine required to induce VP, VT, VF and CA in the rat model of aconitine-induced arrhythmia. All designed compounds could shorten the time of the arrhythmia continuum induced by barium chloride, indicating that 4'-hydroxy substituents of scutellarein had rapid-onset antiarrhythmic effects. In addition, nearly all of the compounds could normalize the HR, RR, QRS, QT and QTc interval, as well as the P/T waves' amplitude. The most promising compound 10e showed the best antiarrhythmic activity with long-term efficacy and extremely low cytotoxicity, better than the positive control scutellarein. This result was also approved by the computational docking simulation. Most importantly, patch clamp measurements on Nav1.5 and Cav1.2 channels indicated that compound 10e was able to reduce the INa and ICa in a concentration-dependent manner and left-shifted the inactivation curve of Nav1.5. Taken together, all compounds were considered to be antiarrhythmic. Compound 10e even showed no proarrhythmic effect and could be classified as Ib Vaughan Williams antiarrhythmic agents. What is more, compound 10e did not block the hERG potassium channel which highly associated with cardiotoxicity.

Serum Uric Acid and High-Sensitivity C-Reactive Protein as Predictors of Cognitive Impairment in Patients with Cerebral Infarction.

BACKGROUND: Cognitive impairment induced by cerebral infarction has become a devastating health problem. More efficient predictors are required to evaluate the potential cognitive decline after cerebral infarction in clinic. Serum uric acid (UA) and high-sensitivity C-reactive protein (hs-CRP) are two factors reported to correlate with cognitive impairment. However, the understanding on serum UA and hs-CRP with cognitive dysfunction remains unclear. METHODS: Serum UA and hs-CRP were evaluated in patients with cerebral infarction (n = 197) using single factor analysis and multivariate logistic regression analysis. Clinical and pathological characteristics were analyzed by logistic regression, respectively, and the results demonstrated the correlation between the pathological characteristics and the cognitive impairment post cerebral infarction. Montreal Cognitive Assessment (MoCA) was used to evaluate the patients' cognitive function, and patients with a MoCA score <26 were recognized as with cognitive impairment. RESULTS: Clinical characteristics related to cognitive impairment, including age, gender, blood pressure, serum UA, and hs-CRP were collected and analyzed. Serum UA and hs-CRP were identified to be potential predictors for post-stroke cognitive dysfunction, with higher serum UA levels correlated with better cognitive function and higher hs-CRP levels correlated with worse cognitive impairment. CONCLUSION: Serum UA and hs-CRP are two predictors for cognitive impairment post cerebral infarction.

Rapid and non-destructive microbial quality prediction of fresh pork stored under modified atmospheres by using selected-ion flow-tube mass spectrometry and machine learning.

Volatile organic compounds (VOCs) indicative of pork microbial spoilage can be quantified rapidly at trace levels using selected-ion flow-tube mass spectrometry (SIFT-MS). Packaging atmosphere is one of the factors influencing VOC production patterns during storage. On this basis, machine learning would help to process complex volatolomic data and predict pork microbial quality efficiently. This study focused on (1) investigating model generalizability based on different nested cross-validation settings, and (2) comparing the predictive power and feature importance of nine algorithms, including Artificial Neural Network (ANN), k-Nearest Neighbors, Support Vector Regression, Decision Tree, Partial Least Squares Regression, and four ensemble learning models. The datasets used contain 37 VOCs' concentrations (input) and total plate counts (TPC, output) of 350 pork samples with different storage times, including 225 pork loin samples stored under three high-O2 and three low-O2 conditions, and 125 commercially packaged products. An appropriate choice of cross-validation strategies resulted in trustworthy and relevant predictions. When trained on all possible selections of two high-O2 and two low-O2 conditions, ANNs produced satisfactory TPC predictions of unseen test scenarios (one high-O2 condition, one low-O2 condition, and the commercial products). ANN-based bagging outperformed other employed models, when TPC exceeded ca. 6 log CFU/g. VOCs including benzaldehyde, 3-methyl-1-butanol, ethanol and methyl mercaptan were identified with high feature importance. This elaborated case study illustrates great prospects of real-time detection techniques and machine learning in meat quality prediction. Further investigations on handling low VOC levels would enhance the model performance and decision making in commercial meat quality control.

Targeting Grancalcin Accelerates Wound Healing by Improving Angiogenesis in Diabetes.

Chronic diabetic wounds are a serious complication of diabetes and often result in limb amputations and confer high mortality rates. The proinflammatory secretome in the wound perpetuates defective neovascularization and contributes to dysregulated tissue repair. This study aims to design a gelatin methacrylamide (GelMA) hydrogel to sustained the release of grancalcin-neutralizing antibody (GCA-NAb) and evaluate it as a potential scaffold to promote diabetic wound healing. Results show that the expression of grancalcin(GCA), a protein secreted by bone marrow-derived immune cells, is elevated in the wound sites of individuals and animals with diabetic ulcers. Genetic inhibition of grancalcin expression accelerates vascularization and healing in an animal model. Mechanistic studies show that grancalcin binds to transient receptor potential melastatin 8(TRPM8) and partially inactivates its downstream signaling pathways, thereby impairing angiogenesis in vitro and ex vivo. Systemic or topical administration of a GCA-NAb accelerate wound repair in mice with diabetes. The data suggest that GCA is a potential therapeutic target for the treatment of diabetic ulcers.

Myeloid-derived grancalcin instigates obesity-induced insulin resistance and metabolic inflammation in male mice.

The crosstalk between the bone and adipose tissue is known to orchestrate metabolic homeostasis, but the underlying mechanisms are largely unknown. Herein, we find that GCA + (grancalcin) immune cells accumulate in the bone marrow and release a considerable amount of GCA into circulation during obesity. Genetic deletion of Gca in myeloid cells attenuates metabolic dysfunction in obese male mice, whereas injection of recombinant GCA into male mice causes adipose tissue inflammation and insulin resistance. Mechanistically, we found that GCA binds to the Prohibitin-2 (PHB2) receptor on adipocytes and activates the innate and adaptive immune response of adipocytes via the PAK1-NF-κB signaling pathway, thus provoking the infiltration of inflammatory immune cells. Moreover, we show that GCA-neutralizing antibodies improve adipose tissue inflammation and insulin sensitivity in obese male mice. Together, these observations define a mechanism whereby bone marrow factor GCA initiates adipose tissue inflammation and insulin resistance, showing that GCA could be a potential target to treat metainflammation.

Identification of chalcone analogues as anti-inflammatory agents through the regulation of NF-κB and JNK activation.

To develop new anti-inflammatory agents with improved pharmaceutical profiles, a series of chalcone analogues were designed and synthesized. In vitro anti-inflammatory activity of these compounds was evaluated by screening their inhibitory effects on NO production in RAW264.7 cell lines. The most promising compounds 3h and 3l were selected for further investigation by assessment of their dose-dependent inhibitory activity against cytokines such as TNF-α, IL-1ß, and IL-6 and PGE2 release. The further study also indicated that 3h and 3l could significantly suppress the expression of iNOS and COX-2 through the NF-κB/JNK signaling pathway. Furthermore, compounds 3h and 3l could also remarkably inhibit the mRNA expression of inflammation-related genes. Meanwhile, 3h could also down-regulate ROS production. Docking simulation was conducted to position compounds 3h and 3l into the iNOS binding site to predict the probable binding mode. In conclusion, this series of chalcone analogues with reasonable drug-likeness obtained via in silico rapid prediction can be used as promising lead candidates.

Impact of cerebral microbleeds on cognitive functions and its risk factors in acute cerebral infarction patients.

BACKGROUND: Cerebral microbleeds (CMBs) are subclinical lesions of the brain parenchyma and an important marker for the clinical diagnosis of central nervous system vascular disease. However, the relationship between CMBs and cerebral infarction, cerebral hemorrhage, and cognitive impairment remains unclear. METHODS: In order to explore the cognitive function and risk factors of patients with acute cerebral infarction (ACI) complicated with cerebral microbleeds, 190 patients with ACI were collected. The patients were divided into groups with CMBs (n = 108) and groups without CMBs (n = 82) according to the presence or absence of CMBs. The general data, various examination indicators, Montreal Cognitive Assessment Scale (MoCA) scores of the two groups of patients were analyzed. Sixty healthy controls who underwent physical examination in our hospital during the same period were included as the healthy control group. RESULTS: ACI patients with CMBs had significantly higher rates of leukoaraiosis, hyperhomocysteinemia, hypercholesterolemia, and hypertension. Cognitive function was significantly lower in ACI patients with CMBs. Serum D-dimer, serum high-sensitivity C-reactive protein, serum neuron-specific enolase, and serum S100ß of ACI patients with CMBs were all negatively correlated with their MoCA scores. CONCLUSION: ACI patients with CMBs tended to have lower cognitive abilities than ACI patients without CMBs.

Selected-ion flow-tube mass spectrometry for the identification of volatile spoilage markers for fresh pork packaged under modified atmospheres.

Microbial behavior during meat storage leads to the generation of volatile organic compounds (VOCs) and unpleasant off-odors. This study focused on a novel real-time analytical method, selected-ion flow-tube mass spectrometry (SIFT-MS), to monitor VOC quality and identify spoilage indicators for fresh pork stored under different packaging atmospheres (air, 70/0/30, 70/30/0, 5/30/65, 0/30/70 - v/v% O2/CO2/N2) at 4 °C. A comprehensive selection methodology was used to identify compounds with good instrumental data quality as well as a strong relationship with microbial growth and olfactory rejection. Based on the volatolome quantified by SIFT-MS, storage periods and conditions can be discriminated using multivariate statistics. Acetoin (or ethyl acetate) represented a significant pork quality marker for high-O2 conditions, whereas ethanol, 3-methylbutanal and sulfur compounds can indicate the anaerobic storage progress. Considering the applicability in monitoring different VOC profiles, SIFT-MS is expected to be promising in many storage scenarios to improve analytical efficiency and ensure reliability.

Senescent immune cells accumulation promotes brown adipose tissue dysfunction during aging.

Brown adipose tissue (BAT)-mediated thermogenesis declines with age. However, the underlying mechanism remains unclear. Here we reveal that bone marrow-derived pro-inflammatory and senescent S100A8+ immune cells, mainly T cells and neutrophils, invade the BAT of male rats and mice during aging. These S100A8+ immune cells, coupled with adipocytes and sympathetic nerves, compromise axonal networks. Mechanistically, these senescent immune cells secrete abundant S100A8 to inhibit adipose RNA-binding motif protein 3 expression. This downregulation results in the dysregulation of axon guidance-related genes, leading to impaired sympathetic innervation and thermogenic function. Xenotransplantation experiments show that human S100A8+ immune cells infiltrate mice BAT and are sufficient to induce aging-like BAT dysfunction. Notably, treatment with S100A8 inhibitor paquinimod rejuvenates BAT axon networks and thermogenic function in aged male mice. Our study suggests that targeting the bone marrow-derived senescent immune cells presents an avenue to improve BAT aging and related metabolic disorders.

Bone Marrow-Derived GCA+ Immune Cells Drive Alzheimer's Disease Progression.

Alzheimer's disease (AD) is an age-related degenerative disease of the central nervous system (CNS), whereas the role of bone marrow immune cells in the pathogenesis of AD remains unclear. Here, the study reveals that compared to matched healthy individuals, AD patients have higher circulating grancalcin (GCA) levels, which negatively correlate with cognitive function. Bone marrow-derived GCA+ immune cells, which secret abundant GCA and increase during aging, preferentially invaded the hippocampus and cortex of AD mouse model in a C-C Motif Chemokine Receptor 10 (CCR10)-dependent manner. Transplanting GCA+ immune cells or direct stereotaxic injection of recombinant GCA protein intensified amyloid plaque load and aggravated cognitive and memory impairments. In contrast, genetic ablation of GCA in the hematopoietic compartment improves cognitive and memory function. Mechanistically, GCA competitively binds to the low-density lipoprotein receptor-related protein 1 (LRP1) in microglia, thus inhibiting phagocytosis and clearance of Aß and potentiating neuropathological changes. Importantly, GCA-neutralizing antibody treatment rejuvenated cognitive and memory function and constrained AD progression. Together, the study demonstrates a pathological role of GCA+ immune cells instigating cognitive and memory decline, suggesting that GCA+ immune cells can be a potential target for innovative therapeutic strategies in AD.

Identification of isothiazolones analogues as potent bactericidal agents against antibiotic resistant CRE and MRSA strains.

Carbapenem-resistant Enterobacterales (CRE) has emerged as a worldwide spread nosocomial superbug exhibiting antimicrobial resistance (AMR) to all current antibiotics, leaving limited options for treating its infection. To discovery novel antibiotics against CRE, we designed and synthesized a series of 14 isothiazol-3(2H)-one analogues subjected to antibacterial activity evaluation against Escherichia coli (E. coli) BL21 (NDM-1) and clinical strain E. coli HN88 for investigating their structure-activity relationships (SAR). The results suggested that 5-chloroisothiazolone core with an N-(4-chlorophenyl) substitution 5a was the most potent antibacterial activity against the E. coli BL21 (NDM-1) with MIC value of less than 0.032 µg/mL, which was at least 8000-fold higher than the positive control Meropenem (MRM). It also displayed 2048-fold potent than the positive control MRM against E. coli HN88. Additionally, SAR analysis supported the conclusion that compounds with a chloro-group substituted on the 5-position of the heterocyclic ring was much more potent than other positions. The board spectrum analysis suggested that compound 5a showed a promising antimicrobial activity on MRSA and CRE pathogens. Meanwhile, cytotoxicity study of compound 5a suggested that it had a therapeutic index value of 875, suggesting future therapeutic potential. In vivo efficacy study declared that compound 5a could also protect the BALB/c mice against American type culture collection (ATCC) 43,300. Further screening of our compounds against a collection of CRE strains isolated from patients indicated that compound 5 g displayed much stronger antibacterial activity compared with MRM. In conclusion, our studies indicated that isothiazolones analogues could be potent bactericidal agents against CRE and MRSA pathogens.

Astragaloside IV Exerts Anti-tumor Effect on Murine Colorectal Cancer by Re-educating Tumor-Associated Macrophage.

Astragaloside IV (AS-IV) has shown anti-tumorigenic properties in certain cancers for its effect of boosting the body's immune system, but its role in colorectal cancer (CRC) remains unclear. In this study, we investigated the therapeutic effect of AS-IV in CRC and explored its underlying mechanism. CT26 colon cancer cells and mouse model by injection of CT26 cells subcutaneously were used as in vitro and in vivo model. M1 and M2 macrophage-associated markers, mRNA and protein expression levels were analyzed after AS-IV treatment. Inflammatory factors and cytokines in the tumors from mouse model were detected. Repolarization effect of AS-IV in vitro on bone-marrow-derived macrophages was also detected. In vitro, AS-IV inhibited the proliferation of CT26 cells and induced cell apoptosis dose-dependently, and significantly reduced M2 macrophages and increased M1 macrophages. In mouse model, it suppressed tumor growth and decreased the production of anti-inflammatory factors such as TGF-ß, IL-10 and VEGF-A, while increased the production of pro-inflammatory factors like IFN-γ, IL-12 and TNF-α in tumor. Combination of AS-IV and checkpoint inhibitor aPD-1 exhibited synergistic antitumor effect by inhibiting tumor growth and increasing T cell infiltration. AS-IV could induce M2 macrophages polarization to the M1 phenotype. Its combination with immune checkpoint inhibitors could be expected to become a potential new strategy for the treatment of CRC.

One-step solid-state thermolysis of a metal-organic framework: a simple and facile route to large-scale of multiwalled carbon nanotubes.

We report a simple and facile solid-state approach to large-scale synthesis of multiwalled carbon nanotubes (MCNTs), for the first time, by one-step direct thermolysis of a metal-organic framework [Ni(3)(btc)(2).12H(2)O] (btc = benzene-1,3,5-tricarboxylato) in a one-end closed conventional horizontal tube furnace under relatively low temperature without using any additional carrier gas or catalyst.

Nrf1 is critical for redox balance and survival of liver cells during development.

The Nrf1 transcription factor belongs to the CNC subfamily of basic leucine zipper proteins. Knockout of Nrf1 is lethal in mouse embryos, but nothing is known about the cell types that absolutely require its function during development. We show by chimera analysis that Nrf1 is essential for the hepatocyte lineage. Mouse embryonic stem cells lacking Nrf1 developed normally and contributed to most tissues in adult chimeras where Nrf1 is normally expressed. Nrf1-deficient cells contributed to fetal, but not adult, liver cells. Loss of Nrf1 function resulted in liver cell apoptosis in late-gestation chimeric fetuses. Fetal livers from mutant embryos exhibited increased oxidative stress and impaired expression of antioxidant genes, and primary cultures of nrf1(-/-) fetal hepatocytes were sensitive to tert-butyl hydroperoxide-induced cell death, suggesting that impaired antioxidant defense may be responsible for the apoptosis observed in the livers of chimeric mice. In addition, cells deficient in Nrf1 were sensitized to the cytotoxic effects of tumor necrosis factor (TNF). Our results provide in vivo evidence demonstrating an essential role of Nrf1 in the survival of hepatocytes during development. Our results also suggest that Nrf1 may promote cell survival by maintaining redox balance and protecting embryonic hepatocytes from TNF-mediated apoptosis during development.

Graft-versus-leukemia target antigens in chronic myelogenous leukemia are expressed on myeloid progenitor cells.

PURPOSE: Donor lymphocyte infusion (DLI) reliably induces durable remission in 75% to 80% of patients with relapsed chronic myelogenous leukemia (CML) following allogeneic bone marrow transplantation. We previously reported the identification of a high titer-specific immunoglobulin G response against two novel leukemia-associated antigens, CML28 and CML66, which correlated with immune-induced remission. The present studies characterize expression of CML28 and CML66 in primary hematopoietic tissues. EXPERIMENTAL DESIGN: Specific monoclonal antibodies to CML28 and CML66 were developed and used to detect antigen expression in leukemia cell lines and primary leukemia tissue on Western blot and immunohistochemistry. Expression patterns were confirmed by antigen-specific real-time PCR. RESULTS: Both CML28 and CML66 were highly expressed in leukemic blasts from patients with acute myelogenous leukemia and CML blast crisis but barely detectable in normal bone marrow, normal peripheral blood, or leukemic cells from patients with stable-phase CML. In contrast, purified CD34+ progenitors from normal individuals and patients with stable-phase CML expressed high levels of CML28 and CML66 transcript and protein. Immunohistochemical staining for CML66 confirmed rare staining of myeloid precursors in normal marrow and diffuse staining of myeloblastic cells in acute myelogenous leukemia and blast crisis CML marrows. CONCLUSIONS: The expression patterns of CML28 and CML66 are strikingly similar and suggest that antigen expression may play a role in shaping the post-DLI antibody repertoire. The CD34+ restricted pattern of expression of CML28 and CML66 is particularly relevant in light of the notion that DLI likely exerts its curative effect by targeting antigens present in self-renewing malignant progenitor populations in CML.

CML28 is a broadly immunogenic antigen, which is overexpressed in tumor cells.

Donor lymphocyte infusion (DLI) reliably induces durable remission in 75-80% of patients with relapsed chronic myelogenous leukemia (CML) after allogeneic hematopoietic stem cell transplantation. To identify immunological targets of the graft-versus-leukemia response (GVL) after DLI, we used CML post-DLI responder sera to screen a CML cDNA expression library. One of the antigens identified in this screen is a M(r) 28,000 protein, termed CML28. CML28 is identical to hRrp46p, a component of the human exosome, a multiprotein complex involved in the 3' processing of RNA. Components of the human exosome include known autoantigens, such as PMScl-100, an autoantibody target in patients with polymyositis, scleroderma, or polymyositis-scleroderma overlap syndrome. Recombinant CML28-GST fusion protein was purified, and used in Western blot and ELISA to demonstrate the development of a high-titer CML28-specific IgG antibody response in a patient with relapsed CML who responded to DLI. Northern blotting demonstrated that CML28 is highly expressed in a variety of hematopoietic and epithelial tumor cell lines, but not in normal hematopoietic tissues or other normal tissue, with the exception of testis. Purified recombinant CML28 was used to generate a CML28-specific murine monoclonal antibody. Western blotting with CML28 monoclonal antibody against whole-cell lysates derived from blood and marrow of normal donors and patients with leukemia revealed high expression of this antigen in tumor but not in normal samples. Because CML28 was highly expressed in epithelial tumor cell lines, anti-CML28 responses were also examined in patients with solid tumors. By ELISA, we found specific serological responses in 10-33% of patients with lung cancer, melanoma, and prostate cancer. Our studies suggest that immunogenicity of CML28 is likely because of overexpression of this antigen in tumor cells. Moreover, given its expression and immunogenicity in a wide variety of malignancies, CML28 merits additional evaluation as a target for antigen-specific immunotherapy.

Liver-specific inactivation of the Nrf1 gene in adult mouse leads to nonalcoholic steatohepatitis and hepatic neoplasia.

Knockout studies have shown that the transcription factor Nrf1 is essential for embryonic development. Nrf1 has been implicated to play a role in mediating activation of oxidative stress response genes through the antioxidant response element (ARE). Because of embryonic lethality in knockout mice, analysis of this function in the adult knockout mouse was not possible. We report here that mice with somatic inactivation of nrf1 in the liver developed hepatic cancer. Before cancer development, mutant livers exhibited steatosis, apoptosis, necrosis, inflammation, and fibrosis. In addition, hepatocytes lacking Nrf1 showed oxidative stress, and gene expression analysis showed decreased expression of various ARE-containing genes, and up-regulation of CYP4A genes. These results suggest that reactive oxygen species generated from CYP4A-mediated fatty acid oxidation work synergistically with diminished expression of ARE-responsive genes to cause oxidative stress in mutant hepatocytes. Thus, Nrf1 has a protective function against oxidative stress and, potentially, a function in lipid homeostasis in the liver. Because the phenotype is similar to nonalcoholic steatohepatitis, these animals may prove useful as a model for investigating molecular mechanisms of nonalcoholic steatohepatitis and liver cancer.