Sestrin 2 attenuates sepsis‐associated encephalopathy through the promotion of autophagy in hippocampal neurons

Sepsis‐associated encephalopathy (SAE) has typically been associated with a poor prognosis. Although sestrin 2 (SESN2) plays a crucial role in metabolic regulation and the stress response, its expression and functional roles in SAE are still unclear. In the present study, SAE was established in mice through caecal ligation and puncture (CLP). The adeno‐associated virus 2 (AAV2)‐mediated SESN2 expression (ie overexpression and knockdown) system was injected into the hippocampi of mice with SAE, and subsequently followed by electron microscopic analysis, the Morris water maze task and pathological examination. Our results demonstrated an increase of SESN2 in the hippocampal neurons of mice with SAE, 2‐16 hours following CLP. AAV2‐mediated ectopic expression of SESN2 attenuated brain damage and loss of learning and memory functions in mice with SAE, and these effects were associated with lower pro‐inflammatory cytokines in the hippocampus. Mechanistically, SESN2 promoted unc‐51‐like kinase 1 (ULK1)‐dependent autophagy in hippocampal neurons through the activation of the AMPK/mTOR signalling pathway. Finally, AMPK inhibition by SBI‐0206965 blocked SESN2‐mediated attenuation of SAE in mice. In conclusion, our findings demonstrated that SESN2 might be a novel pharmacological intervention strategy for SAE treatment through promotion of ULK1‐dependent autophagy in hippocampal neurons.     

Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury

The mechanism of how SARS-CoV-2 causes severe multi-organ failure is largely unknown. Acute kidney injury(AKI) is one of the frequent organ damage in severe COVID-19 patients. Previous studies have shown that human renal tubule cells could be the potential host cells targeted by SARS-CoV-2. Traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. Animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. There is an unmet need for normal human epithelial cells for disease modeling. In this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells(KPTECs) in 2 D and 3 D culture systems using conditional reprogramming(CR) and organoids techniques.These cells had the ability to differentiate and repair DNA damage, and showed no transforming property. Importantly, the CR KPTECs maintained lineage function with expression of specific transporters(SLC34 A3 and cubilin). They also expressed angiotensin-converting enzyme 2(ACE2), a receptor for SARS-CoV and SARS-CoV-2. In contrast, cancer cell line did not express endogenous SLC34 A3, cubilin and ACE2. Very interestingly, ACE2 expression was around twofold higher in 3 D organoids culture compared to that in 2 D CR culture condition. Pseudovirion assays demonstrated that SARS-CoV spike(S) protein was able to enter CR cells with luciferase reporter. This integrated 2 D CR and 3 D organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation.     

Highly efficient soluble expression and purification of recombinant human basic fibroblast growth factor (hbFGF) by fusion with a new collagen-like protein (Scl2) in Escherichia coli

Abstract

Human basic fibroblast growth factor (hbFGF) is involved in a wide range of biological activities that affect the growth, differentiation, and migration. Due to its wound healing effects and therapy, hbFGF has the potential as therapeutic agent. Therefore, large-scale production of biologically active recombinant hbFGF with low cost is highly desirable. However, the complex structure of hbFGF hinders its high-level expression as the soluble and functional form. In the present study, an efficient, cost-effective, and scalable method for producing recombinant hbFGF was developed. The modified collagen-like protein (Scl2-M) from Streptococcus pyogenes was used as the fusion tag for producing recombinant hbFGF for the first time. After optimization, the expression level of Scl2-M-hbFGF reached approximately 0.85?g/L in the shake flask and 7.7?g/L in a high cell-density fermenter using glycerol as a carbon source. Then, the recombinant Scl2-M-hbFGF was readily purified using one-step acid precipitation and the purified Scl2-M-hbFGF was digested with enterokinase. The digested mixture was further subject to ion-exchange chromatography, and the final high-purity (96%) hbFGF product was prepared by freeze-drying. The recovery rate of the whole purification process attained 55.0%. In addition, the biological activity of recombinant hbFGF was confirmed by using L929 and BALB/c3T3 fibroblasts. Overall, this method has the potential for large scale production of recombinant hbFGF.     

Flavonoid 3′-hydroxylase of Camellia nitidissima Chi. promotes the synthesis of polyphenols better than flavonoids

Molecular Biology Reports - Camellia nitidissima Chi. is an ornamental plant of the genus Camellia L. Its flowers contain a lot of flavonoids and polyphenols. Flavonoid 3′-hydroxylase...     

Antifungal Effect of Long Noncoding RNA 9708-1 in the Vulvovaginal Candidiasis Murine Model

Mycopathologia - Vulvovaginal candidiasis (VVC) caused by Candida spp. affects 70–75% of women at least once during their lives. We aim to elucidate the potential mechanism of VVC and...     

Increased kynurenine-to-tryptophan ratio in the serum of patients infected with SARS-CoV2: An observational cohort study.

Immune dysregulation is a hallmark of patients infected by SARS-CoV2 and the balance between immune reactivity and tolerance is a key determinant of all stages of infection, including the excessive inflammatory state causing the acute respiratory distress syndrome. The kynurenine pathway (KP) of tryptophan (Trp) metabolism is activated by pro-inflammatory cytokines and drives mechanisms of immune tolerance. We examined the state of activation of the KP by measuring the Kyn:Trp ratio in the serum of healthy subjects (n = 239), and SARS-CoV2-negative (n = 305) and -positive patients (n = 89). Patients were recruited at the Emergency Room of St. Andrea Hospital (Rome, Italy). Kyn and Trp serum levels were assessed by HPLC/MS-MS. Compared to healthy controls, both SARS-CoV2-negative and -positive patients showed an increase in the Kyn:Trp ratio. The increase was larger in SARS-CoV2-positive patients, with a significant difference between SARS-CoV2-positive and -negative patients. In addition, the increase was more prominent in males, and positively correlated with age and severity of SARS-CoV2 infection, categorized as follows: 1 = no need for intensive care unit (ICU); 2 ≤ 3 weeks spent in ICU; 3 ≥ 3 weeks spent in ICU; and 4 = death. The highest Kyn:Trp values were found in SARS-CoV2-positive patients with severe lymphopenia. These findings suggest that the Kyn:Trp ratio reflects the level of inflammation associated with SARS-CoV2 infection, and, therefore, might represent a valuable biomarker for therapeutic intervention.     

The optimum dietary methionine requirement of juvenile humpback grouper (Cromileptes altivelis): effects on growth,micromorphology, protein and lipid metabolism

An 8-week feeding trial was conducted to evaluate optimum dietary methionine (Met) requirement of juvenile humpback grouper (Cromileptes altivelis) and the influence of dietary methionine (Met) supplementations on growth, gut micromorphology, protein and lipid metabolism. Seven isoproteic (48.91%) and isolipidic diets (10%) were made to contain 0.70, 0.88, 1.04, 1.27 1.46, 1.61 and 1.76% of dry matter Met levels. Results showed that lower survival, weight gain (WG%), protein efficiency ratio (PER), protein productive value (PPV) but higher daily feed intake (DFI) and feed conversion ratio (FCR) were observed in the Met deficient groups (0.70 and 0.88%). Optimum dietary Met requirement for humpback grouper was found to be 1.07% through the straight-broken line analysis of WG% against Met. Fish fed Met deficient diets (0.70, 0.88%) exhibited lower mRNA levels of growth hormone (GH), growth hormone receptor (GHR), insulin-like growth factor-I (IGF-1), target of rapamycin (TOR) as well as S6 kinase 1 (S6K1) than other dietary groups. Whereas, expression of genes related to general control nonderepressible (GCN2) kinase i.e., GCN2 and C/EBPβ enhancer-binding protein β was upregulated in fish fed low Met diets (P < 0.05). The mRNA expression of hepatic fatty acid synthase (FAS) and sterol regulatory element-binding protein-1 (SREBP-1) were higher in fish fed 0.70 and 0.88% dietary Met group and the lipolytic genes, hepatic peroxisome proliferator-activated receptor α (PPARα) and carnitine palmitoyl transferase-1 (CPT-1) showed an opposite variation tendency as FAS or SREBP1. Generally, the optimum Met requirement for humpback grouper was predicted to be 1.07% of dry matter.

     

Microcella flavibacter sp. nov., isolated from marine sediment,and reclassification of Chryseoglobus frigidaquae,Chryseoglobus indicus,and Yonghaparkia alkaliphila as Microcella frigidaquae comb. nov., Microcella indica nom. nov., and Microcella alkalica nom. nov.

A novel Gram-staining positive, aerobic, rod-shaped, non-motile and yellow-pigmented actinobacterium, designated strain WY83^T, was isolated from a marine sediment of Indian Ocean. Strain WY83^T grew optimally at 30–35 °C, pH 7–8 and with 0–3% (w/v) NaCl. The predominant menaquinones were MK-10, MK-11 and MK-12, and the major fatty acids were C_19:1 ω9c/C19:1 ω11c, anteiso-C_15:0, C_17:0 3OH, and iso-C_16:0. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol and one unidentified glycolipid. The cell-wall peptidoglycan contained lysine as a diamino acid. The DNA G?+?C content was 72.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences and ninety-two bacterial core genes indicated that strain WY83^T formed an evolutionary lineage with Chryseoglobus frigidaquae JCM 14730^T, Chryseoglobus indicus CTD02-10-2^T, Yonghaparkia alkaliphila JCM 15138^T, Microcella alkaliphila DSM 18851^T and Microcella putealis DSM 19627^T within the radiation enclosing members of the family Microbacteriaceae. All pairwise percentage of conserved proteins between strain WY83^T and the closely related phylogenetic neighbors were greater than 65%. The average nucleotide identity and in silico DNA–DNA hybridization values were both below the thresholds used for the delineation of a new species. On the basis of the evidence presented, strains WY83^T, Y. alkaliphila JCM 15138^T, C. frigidaquae JCM 14730^T, M. alkaliphila DSM 18851^T and M. putealis DSM 19627^T should belong to different species of the same genus. Strain WY83^T represents a novel species of the genus Microcella, for which the name Microcella flavibacter sp. nov. is proposed. The type strain is WY83^T (=?KCTC 39637^T?=?MCCC 1A07099^T). Furthermore, Chryseoglobus frigidaquae, Chryseoglobus indicus, and Yonghaparkia alkaliphila were reclassified as Microcella frigidaquae comb. nov., Microcella indica nom. nov., and Microcella alkalica nom. nov., respectively.