Kielin/chordin-like protein deficiency aggravates pressure overload-induced cardiac dysfunction and remodeling via P53/P21/CCNB1 signaling in mice.

Targeting cardiac remodeling is regarded as a key therapeutic strategy for heart failure. Kielin/chordin-like protein (KCP) is a secretory protein with 18 cysteine-rich domains and associated with kidney and liver fibrosis. However, the relationship between KCP and cardiac remodeling remains unclear. Here, we aimed to investigate the role of KCP in cardiac remodeling induced by pressure overload and explore its potential mechanisms. Left ventricular (LV) KCP expression was measured with real-time quantitative PCR, western blotting, and immunofluorescence staining in pressure overload-induced cardiac remodeling in mice. Cardiac function and remodeling were evaluated in wide-type (WT) mice and KCP knockout (KO) mice by echocardiography, which were further confirmed by histological analysis with hematoxylin and eosin and Masson staining. RNA sequence was performed with LV tissue from WT and KO mice to identify differentially expressed genes and related signaling pathways. Primary cardiac fibroblasts (CFs) were used to validate the regulatory role and potential mechanisms of KCP during fibrosis. KCP was down-regulated in the progression of cardiac remodeling induced by pressure overload, and was mainly expressed in fibroblasts. KCP deficiency significantly aggravated pressure overload-induced cardiac dysfunction and remodeling. RNA sequence revealed that the role of KCP deficiency in cardiac remodeling was associated with cell division, cell cycle, and P53 signaling pathway, while cyclin B1 (CCNB1) was the most significantly up-regulated gene. Further investigation in vivo and in vitro suggested that KCP deficiency promoted the proliferation of CFs via P53/P21/CCNB1 pathway. Taken together, these results suggested that KCP deficiency aggravates cardiac dysfunction and remodeling induced by pressure overload via P53/P21/CCNB1 signaling in mice.

A Novel 165 Kb Duplication Involving the α-Globin Gene Cluster Is Identified by Low-Pass Whole Genome Sequencing in a Chinese Thalassemia Intermedia Patient.

Copy number variations (CNVs) involving the α-globin gene cluster can lead to an imbalance in the proportion of α- and ß-globin chains and consequently cause clinical symptoms of ß-thalassemia. In our case, a 6-year-old boy, clinically diagnosed with ß thalassemia intermedia, was admitted for further genetic diagnosis with his family. Targeted sequencing and third generation sequencing (TGS) were used to detect the possible variants of the thalassemia genes. Low-pass whole genome sequencing (lpWGS) was conducted to specify the exact location of relevant CNVs across the genome, which was then validated by multiplex ligation-dependent probe amplification.The results revealed that the patient had a heterozygous ß0 mutation of Codon17 (A > T) and a full duplication of the α-globin gene cluster, inherited from his mother and father, respectively. Besides, a novel point mutation within the 5' untranslated region of ß-Globin (HBB: c. -175 (G > A) was only detected in the patient. This study suggests that lpWGS seems a powerful alternative to detect large CNVs related to thalassemia with second intention for more information of the breakpoints and a simultaneous genome-scale detection of other pathogenic CNVs.

Arachis hypogaea L. from Acid Soils of Nanyang (China) Is Frequently Associated with Bradyrhizobium guangdongense and Occasionally with Bradyrhizobium ottawaense or Three Bradyrhizobium Genospecies.

Henan Province is a major area of peanut production in China but the rhizobia nodulating the crop in this region have not been described. A collection of 217 strains of peanut rhizobia was obtained from six field sites across four soil types in Henan Province, North China, by using peanut as a trap host under glasshouse conditions. The 217 strains separated into 8 distinct types on PCR-RFLP analysis of their IGS sequences. Phylogenetic analysis of the 16S rRNA, recA, atpD, and glnII genes of 11 representative strains of the 8 IGS types identified Bradyrhizobium guangdongense, B. ottawaense and three novel Bradyrhizobium genospecies. Bradyrhizobium guangdongense was dominant, accounting for 75.0% of the total isolates across the field sites while B. ottawaense covered 5.1% and the three novel Bradyrhizobium genospecies 4.1 to 8.8% of the total. The symbiosis-related nodA and nifH gene sequences were not congruent with the core genes on phylogenetic analysis and separated into three groups, two of which were similar to sequences of Bradyrhizobium spp. isolated from peanut in south-east China and the third identical to that of B. yuanmingense isolated from Lespedeza cuneata in northern China. A canonical correlation analysis between the distribution of IGS genotypes and soil physicochemical characteristics and climatic factors indicated that the occurrence of IGS types/species was mainly associated with soil pH and available phosphorus.

Expression and functional identification of recombinant SARS-CoV-2 receptor binding domain (RBD) from E. coli system.

The receptor binding domain (RBD) of SARS-CoV-2 is located in the C-terminal of S1 subunit of the spike (S) protein which is responsible for recognizing and binding to the angiotensin-converting enzyme 2 (ACE2) receptor. The DNA encoding the SARS-CoV-2 RBD was inserted into pET-28a (+) to construct expression plasmid pET-28a (+)/RBD. The desired RBD protein was produced in E. coli Rosetta (DE) and purified by a Ni-NTA column. The recombinant RBD was analyzed by SDS-PAGE and Western blot. The flow cytometry analysis indicated that the recombinant RBD is capable of binding to human ACE2 (hACE2) in the ACE2-overexpressed HEK293A-hACE2 cells. Our results demonstrated that recombinant RBD expressed in E. coli Rosetta (DE) strain has bioactivities and can be used as an antigen for diagnosis and as a tool for the development of novel anti-viral drugs against SASR-CoV-2.

An efficient system to generate truncated human angiotensin converting enzyme 2 (hACE2) capable of binding RBD and spike protein of SARS-CoV2.

Human angiotensin converting enzyme 2 (hACE2) mediates the cell entry of both SARS-CoV and SARS-CoV2 and can be used as a drug target. The DNA encoding the truncated hACE2 (30-356aa) was cloned into pET-28a (+) and expressed in Escherichia coli Rosetta (DE3). The recombinant hACE2 (rhACE2) was purified by affinity chromatography on a Ni-NTA column and characterized with SDS-PAGE and Western blot. The binding activity of rhACE2 to Spike protein of SARS-CoV2 was evaluated in S protein-overexpressed HEK293A cells (HEK293A-SP cells) through flow cytometry. The prokaryotic expression system is able to produce approximately 75 mg protein per liter, which would be useful for infection mechanism study, and drug screening and development of SARS-CoV2.

The characteristics and trend of adverse events following immunization reported by information system in Jiangsu province, China, 2015-2018.

OBJECTIVE: Adverse events following immunization is an important factor influencing public trust in vaccination. Publicizing its incidence timely can increase public trust. The aim of this study is to describe the incidence and characteristics of adverse events following immunization in Jiangsu province of China from 2015 to 2018. METHODS: All information of adverse events following immunization (AEFIs) was gained from Jiangsu Province Vaccination Integrated Service Management Information System. The reported AEFI trend was analyzed using Chi-square test. RESULTS: A total of 77,980 AEFI cases were reported through the AEFI system; Among which, 77,731 were classified as non-serious AEFI cases and 249 were serious AEFI cases. The male to female ratio was 1.31:1, cases less than 7 years old accounted for 97.7%. The total estimated AEFI rate was 62.70/100,000 doses. By severity, 60.75/100,000, 4.46/100,000 and 0.11/100,000 AEFI cases were common vaccine reaction, rare vaccine reaction, and serious rare vaccine reaction, respectively. The top two serious AEFI were thrombocytopenic purpura and febrile. The incidence rates showed the increasing trend and the linear trend of the increasing incidence rates passed the significant test at 0.05 levels. CONCLUSION: The sensitivity of AEFI monitoring in Jiangsu Province is increasing and higher than the national average and most countries. The majority of AEFI cases were common adverse reactions, while the serious vaccine reactions caused by vaccines were extremely low. To elevate the sensitivity of AEFI surveillance may reduce the incidence of developing serious AEFI cases.

miRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy.

Dendritic cell (DC) based immunotherapy is a promising approach to clinical cancer treatment. miRNAs are a class of small non-coding RNA molecules that bind to RNAs to mediate multiple events which are important in diverse biological processes. miRNA mimics and antagomirs may be potent agents to enhance DC-based immunotherapy against cancers. miRNA array analysis was used to identify a representative miR-5119 potentially regulating PD-L1 in DCs. We evaluated levels of ligands of immune cell inhibitory receptors (IRs) and miR-5119 in DCs from immunocompetent mouse breast tumor-bearing mice, and examined the molecular targets of miR-5119. We report that miRNA-5119 was downregulated in spleen DCs from mouse breast cancer-bearing mice. In silico analysis and qPCR data showed that miRNA-5119 targeted mRNAs encoding multiple negative immune regulatory molecules, including ligands of IRs such as PD-L1 and IDO2. DCs engineered to express a miR-5119 mimic downregulated PD-L1 and prevented T cell exhaustion in mice with breast cancer homografts. Moreover, miR-5119 mimic-engineered DCs effectively restored function to exhausted CD8+ T cells in vitro and in vivo, resulting in robust anti-tumor cell immune response, upregulated cytokine production, reduced T cell apoptosis, and exhaustion. Treatment of 4T1 breast tumor-bearing mice with miR-5119 mimic-engineered DC vaccine reduced T cell exhaustion and suppressed mouse breast tumor homograft growth. This study provides evidence supporting a novel therapeutic approach using miRNA-5119 mimic-engineered DC vaccines to regulate inhibitory receptors and enhance anti-tumor immune response in a mouse model of breast cancer. miRNA/DC-based immunotherapy has potential for advancement to the clinic as a new strategy for DC-based anti-breast cancer immunotherapy.

High expression of NEK2 promotes lung cancer progression and drug resistance and is regulated by mutant EGFR.

Activating mutations within the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR) gene are observed in 10 ~ 30% of the patients diagnosed with non-small cell lung cancer (NSCLC), and are causally related to NSCLC initiation and progression. Treatments with tyrosine kinase inhibitors (TKIs) targeting EGFR significantly improve the outcome of NSCLC patients with EGFR mutation, but are often associated with drug resistance, which is the main cause of treatment failure and cancer relapse. In the present study, by screening the transcriptome of NSCLC patients, we found that EGFR activation is highly correlated with the up-regulation of mitotic regulator, never in mitosis gene A-related kinase 2 (NEK2). NEK2 overexpression is associated with the poor survival of EGFR-mutant patients but not the wild-type patients. Further functional validation revealed that EGFR mutation induces NEK2 expression by activating ERK signaling pathway. Elevated NEK2 level promotes the rapid cell cycle progression and favors the rapid proliferation of EGFR-mutant NSCLC cells. Of note, NEK2 overexpression also impairs the efficacy of TKI treatment via inhibiting apoptosis, while depleting NEK2 suppresses cell growth and restored the sensitivity of TKI in NSCLC cells. Taken together, our study revealed that NEK2 is an oncogene regulated by EGFR mutation and is involved in disease progression and treatment response in NSCLC with EGFR mutation. These findings will pave the road for optimizing personalized treatment strategies to overcome drug resistance and improve the prognosis of lung cancer patients with EGFR mutation.

Reliability and validity of the mandarin version of the supportive care needs survey short-form (SCNS-SF34) and the head and neck cancer-specific supportive care needs (SCNS-HNC) module.

BACKGROUND: This study aimed to translate the English version of the supportive care needs scale of head and neck cancer patients (SCNS-HNC) questionnaire into Mandarin and to test the reliability and validity of the SCNS-SF34 and SCNS-HNC module in head and neck cancer patients. METHODS: The Mandarin version of the Supportive Care Needs Survey Short-Form (SCNS-SF34) and SCNS-HNC scales were used to assess 206 patients with head and neck cancer in Chengdu, China. Among them, 51 patients were re-tested 2 or 3 days after the first survey. The internal consistency of the scale was evaluated by Cronbach's alpha coefficient, the retest reliability of the scale was evaluated by retest correlation coefficient r, the structural validity of the scale was evaluated by exploratory factor analysis, and the ceiling and floor effects of the scale were evaluated. RESULTS: The Mandarin version of the SCNS-HNC had Cronbach's alpha coefficients greater than 0.700 (0.737 ≤ 0.962) for all of the domains. Except for the psychological demand dimension (r = 0.674) of the SCNS-SF34 scale, the retest reliability of the other domains was greater than 0.8. Three common factors were extracted by exploratory factor analysis, and the cumulative variance contribution rate was 64.39%. CONCLUSIONS: The Mandarin version of the SCNS-SF34 and SCNS-HNC demonstrated satisfactory reliability and validity and is able to measure the supportive care needs of Chinese patients with head and neck cancer. TRIAL REGISTRATION: ChiCTR, ChiCTR1900026635 . Registered 16 October 2019- Retrospectively registered.

Rhizobium changzhiense sp. nov., isolated from effective nodules of Vicia sativa L. in North China.

Three fast-growing rhizobial strains isolated from effective nodules of common vetch (Vicia sativa L.) were characterized using a polyphasic approach. All three strains were assigned to the genus Rhizobium on the basis of the results of 16S rRNA gene sequence analysis. Phylogenetic analysis based on concatenated atpD-recA genes separated the strains into a distinct lineage represented by WYCCWR 11279T, which showed average nucleotide identity values of 95.40 and 93.61 % with the most similar phylogenetic type strains of Rhizobium sophorae CCBAU 03386T and Rhizobium laguerreae FB TT, respectively. The digital DNA-DNA hybridization relatedness values between WYCCWR 11279T and the closest related type strains were less than 70 %. Therefore, a novel rhizobial species is proposed, Rhizobium changzhiense sp. nov., and strain WYCCWR 11279T (=HAMBI 3709T=LMG 31534T) is designated as the type strain for the novel species.

A new cancer immunotherapy via simultaneous DC-mobilization and DC-targeted IDO gene silencing using an immune-stimulatory nanosystem.

The activity of negative immune regulatory molecules, such as indoleamine 2,3-oxygenase (IDO), significantly attenuates DC (Dendritic cells)-mediated immunotherapy. We have previously reported that knockdown of IDO using siRNA can reinstall anti-tumor immunity. However, a DC-targeted siRNA delivery system for in vivo mobilized DCs remains to be developed, while gene silencing in mobilized DCs for cancer immunotherapy has never been explored. In our study, we developed a novel DC-targeted siRNA delivery system, man-GNR-siIDO, using as a nanocarrier of siRNA specific for IDO (siIDO) and mannose (man) as a guide molecule for targeting DCs. We explored the immunostimulatory man-GNR-siIDO nano-construct in DCs mobilized by Flt3-L, a receptor-type tyrosine kinase ligand, for lung cancer immunotherapy. In vivo DC-targeted gene silencing of IDO resulted in robust anti-tumor immunity as evidenced by promoting DC maturation, up-regulating tumor antigen-specific T-cell proliferation and enhancing tumor-specific cytotoxicity. A combinatorial treatment for Lewis Lung Carcinoma (LLC)-bearing mice, with man-GNR-siIDO and Flt3-L, significantly attenuated tumor growth and delayed tumor formation, suggesting the treatment feasibility of the man-GNR-siIDO system in Flt3-L mobilized DCs in the immunotherapy of lung cancer. Therefore, our study highlights a clinical potential for a first-in-class anti-cancer immunotherapy through simultaneous DC-mobilization and DC-targeted gene silencing of IDO with man-GNR-siIDO and Flt3-L treatments.

(E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides as tubulin polymerization inhibitors: Structure-based bioisosterism design, synthesis, biological evaluation, molecular docking and in silico ADME prediction.

A series of (E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides have been synthesized and evaluated for their anticancer activity in human hepatocellular liver carcinoma HepG2 and breast adenocarcinoma MCF-7 cell lines. Among all the tested compounds, compound 3a, 3e and 3n displayed more activity than lead compound with IC50 value of 0.26-0.61 µM. Meanwhile, these compounds (3a, 3e and 3n) showed potent antiproliferative activity against a panel of cancer cells and the HCT-8/T multidrug resistant cell line with IC50 values in the range of 0.077- 7.44 µM. Flow cytometric analyses revealed that compound 3n induced cell cycle arrest in G2/M phases in a dose dependent manner. The compound 3n also displayed potent tubulin polymerization inhibition with an IC50 value of 0.9 µM, with ten folds more active than colchicine (IC50 = 9 µM). Molecular docking studies revealed that compound 3n efficiently interacted with the colchicine binding site of tubulin through hydrophobic, cation-π and hydrogen bond interaction. Furthermore, in silico pharmacokinetic prediction shown that these compounds have a good ADME-related physicochemical parameters. These results demonstrate that 3n exhibits potent cytotoxicity in cancer cells by targeting the colchicine binding site of tubulin and potentially acts as a therapeutic lead compound for the development of anticancer drugs.

Targeted-gene silencing of BRAF to interrupt BRAF/MEK/ERK pathway synergized photothermal therapeutics for melanoma using a novel FA-GNR-siBRAF nanosystem.

Melanoma is significantly associated with mutant BRAF gene, a suitable target for siRNA-based anti-melanoma therapy. However, a tumor-specific delivery system is a major hurdle for clinical applications. Here, we developed a novel nano-carrier, FA-GNR-siBRAF for safe topical application, which consists of folic acid (FA) as the tumor-targeting moiety, golden nanorods (GNR) providing photothermal capability to kill tumor cells under laser irradiation, and siRNA specifically silencing BRAF (siBRAF). The in vitro and in vivo results revealed that FA-GNR-siBRAF displayed high transfection rates, and subsequently induced remarkable gene knockdown of BRAF, resulting in suppression of melanoma growth due to the interruption of the MEK/ERK pathway. Combinatorial photothermal effects and BRAF knockdown by FA-GNR-siBRAF effectively killed tumor cells through apoptosis, with enhanced efficiency than individual treatments. Therefore, the FA-GNR-siBRAF simultaneously induced BRAF gene silencing and photothermal effects which achieved synergistic efficacy in the treatment of melanoma, paving a new path for developing clinical treatment methods for melanoma.

Increased production of omega-3 fatty acids protects retinal ganglion cells after optic nerve injury in mice.

Injury to the central nervous system causes progressive degeneration of injured axons, leading to loss of the neuronal bodies. Neuronal survival after injury is a prerequisite for successful regeneration of injured axons. In this study, we investigated the effects of increased production of omega-3 fatty acids and elevation of cAMP on retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) crush injury in adult mice. We found that increased production of omega-3 fatty acids in mice enhanced RGC survival, but not axonal regeneration, over a period of 3 weeks after ON injury. cAMP elevation promoted RGC survival in wild type mice, but no significant difference in cell survival was seen in mice over-producing omega-3 fatty acids and receiving intravitreal injections of CPT-cAMP, suggesting that cAMP elevation protects RGCs after injury but does not potentiate the actions of the omega-3 fatty acids. The observed omega-3 fatty acid-mediated neuroprotection is likely achieved partially through ERK1/2 signaling as inhibition of this pathway by PD98059 hindered, but did not completely block, RGC protection. Our study thus enhances our current understanding of neural repair after CNS injury, including the visual system.

Anaerobic Digestion of Algae Biomass to Produce Energy during Wastewater Treatment.

Water resource recovery facilities (WRRFs) are asked to improve both energy efficiency and nutrient removal efficacy. Integration of algaculture offers several potential synergies that could address these goals, including an opportunity to leverage anaerobic digestion at WRRFs. In this study, bench-scale experiments are used to measure methane yield during co-digestion of Scenedesmus dimorphus or mixed WRRF-grown algae with WRRF biosolids. The results indicate that normalized methane yield decreases with increasing algae content in a manner than can be reasonably well fit using linear regression (R(2) = 67%). It is thus possible to predict methane yield for any mixture of algae and biosolids based on the methane yield of the biosolids alone. Using revised methane yields, the energy return on investment of a typical WRRF increases from 0.53 (without algae) to 0.66 (with algae). Thus, algae-based wastewater treatment may hold promise for improving WRRF energy efficiency without compromising effluent quality.

A dual-functional oncolytic adenovirus ZD55-aPD-L1 scFv armed with PD-L1 inhibitor potentiates its antitumor activity.

BACKGROUND: Clinical data indicate that a substantial portion of cancer patients, though eligible for immune checkpoint inhibitor (ICI) therapy, cannot fully benefit from ICI monotherapy due to the poor immunogenicity of tumors and lack of tumor-infiltrating lymphocytes within the 'cold' tumor microenvironment (TME). In addition to poor antibody penetrance into the TME, systemic delivery of ICIs is associated with immune-related adverse events (irAEs) among recipients, some of which are life-threatening. Oncolytic virotherapy is a potentially viable approach to improving the efficacy of ICI therapy because of their ability to selectively replicate and lyse tumor cells, release tumor-associated antigens (TAAs), induce inflammatory response and promote lymphocyte infiltration in tumors. METHODS: A recombinant oncolytic adenoviruses (OAd), denoted ZD55-aPD-L1 scFv, which carried the expression cassette for anti-PD-L1 scFv was constructed by molecular cloning. Western blot and ELISA assay were performed to detect aPD-L1 scFv expression. Flow cytometry were used to analyse PD-L1 expression and count tumor cells. Co-culture assay of human peripheral blood mononuclear cells (PBMCs) with tumor cells in vitro and triple-negative breast cancer (TNBC) MDA-MB-231 tumor-bearing model in vivo were evaluated the antitumor effects of recombinant oncolytic adenoviruses ZD55-aPD-L1 scFv. RESULTS: We found that cells infected with recombinant oncolytic adenovirus ZD55-aPD-L1 scFv can effectively express aPD-L1 scFv, which function similarly to its full-length anti-PD-L1 antibody. PBMCs have inherently very limited killing effect on tumor cells even with administration of anti-PD-L1 antibody as observed from our in vitro co-cultures. Treatment consisting of ZD55 alone or ZD55 combined with anti-PD-L1 antibody yielded mediocre antitumor efficacy in subsequent in vitro and in vivo investigations, but were all substantially surpassed by the synergistic antitumor effects observed with ZD55-aPD-L1 scFv treatment. We show that the concomitant direct oncolysis by the recombinant OAd and localized autocrine/paracrine interception of PD-1:PD-L1 checkpoint interaction mediated by ZD55-aPD-L1 scFv-infected cells is exceedingly superior to co-administration of ZD55 and anti-PD-L1 antibody in the human TNBC mice model. CONCLUSIONS: Our results provided evidence for the development of novel strategies, in this case an anti-PD-L1 scFv-armed OAd, to bolster immune responses to 'cold' tumors and to improve therapeutic responsiveness to ICIs.

A PD-L1 tropism-expanded oncolytic adenovirus enhanced gene delivery efficiency and anti-tumor effects.

Recombinant adenovirus serotype 5 (Ad5)-mediated virotherapy is a maturing technique in cancer treatment. However, the utility of adenovirus (Ad) has been limited by low expression of coxsackievirus and adenovirus receptor (CAR) in cancer cells resulting in poor infectivity of Ads. To overcome the problem, we aimed to develop a novel tropism-modified oncolytic adenovirus, ZD55-F-HI-sPD-1-EGFP, which contains the epitope of PD-1 (70-77aa) at the HI-loop of Ad fiber. Trimerization of Fiber-sPD-1 was confirmed by immunoblot analysis. ZD55-F-HI-sPD-1-EGFP shows a remarkable improvement in viral infection rate and gene transduction efficiency in the PD-L1-positive cancer cells. Competition assays with a PD-L1 protein reveals that cell internalization of ZD55-F-HI-sPD-1-EGFP is mediated by both CAR and PD-L1 at a high dose. The progeny virus production capacity showed that sPD-1 incorporated fiber-modified oncolytic Ad replication was not affected. Furthermore, treating with ZD55-F-HI-sPD-1-EGFP significantly increased viral infection rate and enhanced anti-tumor effect in vivo. This study demonstrates that the strategy to expand tropism of oncolytic Ad may significantly improve therapeutic profile for cancer treatment.