Role of carnitine in adaptation of Chromohalobacter salexigens DSM 3043 and its mutants to osmotic and temperature stress in defined medium.

L-Carnitine is widespread in nature, but little information is available on its metabolism and physiological functions in moderate halophiles. In this study, we found that Chromohalobacter salexigens DSM 3043 could utilize carnitine not only as a nutrient, but also as an osmolyte. When grown at 37 °C under salt-stress conditions, the strain utilized carnitine as an osmoprotectant by enzymatically converting it into GB. When grown at low and high temperature, both carnitine and its metabolic intermediate GB were simultaneously accumulated intracellularly, serving as cryoprotectants and thermoprotectants. The genes (csal_3172, csal_3173, and csal_3174) which were predicted to participate in L-carnitine degradation to GB were deleted to construct the corresponding mutants. The effects of salinity and temperature on the growth rates and cytoplasmic solute pools of the C. salexigens wild-type and mutant strains were investigated. 13C-NMR analysis revealed that GB was still detected in the Δcsal_3172Δcsal_3173Δcsal_3174 mutant grown in a defined medium with added DL-carnitine, but not with L-carnitine, indicating that an unidentified D-carnitine degradation pathway exists in C. salexigens. Taken together, the data presented in this study expand our knowledge on carnitine metabolism and its physiological functions in C. salexigens exposed to single or multiple environmental abiotic stress.

COVID-19 patient with an incubation period of 27 d: A case report.

BACKGROUND: As a highly contagious disease, coronavirus disease 2019 (COVID-19) is wreaking havoc around the world due to continuous spread among close contacts mainly via droplets, aerosols, contaminated hands or surfaces. Therefore, centralized isolation of close contacts and suspected patients is an important measure to prevent the transmission of COVID-19. At present, the quarantine duration in most countries is 14 d due to the fact that the incubation period of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is usually identified as 1-14 d with median estimate of 4-7.5 d. Since COVID-19 patients in the incubation period are also contagious, cases with an incubation period of more than 14 d need to be evaluated. CASE SUMMARY: A 70-year-old male patient was admitted to the Department of Respiratory Medicine of The First Affiliated Hospital of Harbin Medical University on April 5 due to a cough with sputum and shortness of breath. On April 10, the patient was transferred to the Fever Clinic for further treatment due to close contact to one confirmed COVID-19 patient in the same room. During the period from April 10 to May 6, nucleic acid and antibodies to SARS-CoV-2 were tested 7 and 4 times, respectively, all of which were negative. On May 7, the patient developed fever with a maximum temperature of 39℃, and his respiratory difficulties had deteriorated. The results of nucleic acid and antibody detection of SARS-CoV-2 were positive. On May 8, the nucleic acid and antibody detection of SARS-CoV-2 by Heilongjiang Provincial Center for Disease Control were also positive, and the patient was diagnosed with COVID-19 and reported to the Chinese Center for Disease Control and Prevention. CONCLUSION: This case highlights the importance of the SARS-CoV-2 incubation period. Further epidemiological investigations and clinical observations are urgently needed to identify the optimal incubation period of SARS-CoV-2 and formulate rational and evidence-based quarantine policies for COVID-19 accordingly.

Standardization of critical care management of non-critically ill patients with COVID-19.

The large global outbreak of coronavirus disease 2019 (COVID-19) has seriously endangered the health care system in China and globally. The sudden surge of patients with severe acute respiratory syndrome coronavirus 2 infection has revealed the shortage of critical care medicine resources and intensivists. Currently, the management of non-critically ill patients with COVID-19 is performed mostly by non-intensive care unit (ICU) physicians, who lack the required professional knowledge, training, and practice in critical care medicine, especially in terms of continuous monitoring of the respiratory function, intervention, and feedback on treatment effects. This clinical problem needs an urgent solution. Therefore, here, we propose a series of clinical strategies for non-ICU physicians aimed at the standardization of the management of non-critically ill patients with COVID-19 from the perspective of critical care medicine. Isolation management is performed to facilitate the implementation of hierarchical monitoring and intervention to ensure the reasonable distribution of scarce critical care medical resources and intensivists, highlight the key patients, timely detection of disease progression, and early and appropriate intervention and organ function support, and thus improve the prognosis. Different management objectives are also set based on the high-risk factors and the severity of patients with COVID-19. The approaches suggested herein will facilitate the timely detection of disease progression, and thus ensure the provision of early and appropriate intervention and organ function support, which will eventually improve the prognosis.

Functional Metabolomics Analysis Elucidating the Metabolic Biomarker and Key Pathway Change Associated With the Chronic Glomerulonephritis and Revealing Action Mechanism of Rhein.

Chronic glomerulonephritis (CGN) as the culprit of kidney failure can increase the mortality of critically ill patients and seriously threatens people's health all over the world. This study using metabolomics strategy is to reveal the potential therapeutic mechanism-related targets to evaluate the effects of rhein (RH) on CGN rats. Changes of serum metabolites and pathways were analyzed by non-targeted metabolomic method based on liquid chromatography-mass spectrometry (LC-MS) combined with ingenuity pathway analysis. In addition, the levels of biochemical indicators were also detected. A total of 25 potential biomarkers were identified to express serum metabolic turbulence in CGN animal model, and then 16 biomarkers were regulated by RH trending to the normal states. From metabolite enrichment and pathway analysis, pharmacological activity of RH on CGN were mainly involved in six vital metabolic pathways including phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arachidonic acid metabolism, tricarboxylic acid cycle (TCA cycle), alanine, aspartate, and glutamate metabolism, arginine and proline metabolism. It suggested CGN treatment with RH, which may be mediated via interference with metabolic pathway such as amino acid metabolism, arachidonic acid metabolism, and TCA cycle to regulating inflammation, oxidation response and immune regulation against CGN. It showed that metabolomics method offer deeply insight into the therapeutic mechanisms of natural product.

Role of N,N-Dimethylglycine and Its Catabolism to Sarcosine in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow on N,N-dimethylglycine (DMG) as the sole C, N, and energy source and utilize sarcosine as the sole N source under aerobic conditions. However, little is known about the genes and enzymes involved in the conversion of DMG to sarcosine in this strain. In the present study, gene disruption and complementation assays indicated that the csal_0990, csal_0991, csal_0992, and csal_0993 genes are responsible for DMG degradation to sarcosine. The csal_0990 gene heterologously expressed in Escherichia coli was proven to encode an unusual DMG dehydrogenase (DMGDH). The enzyme, existing as a monomer of 79 kDa with a noncovalently bound flavin adenine dinucleotide, utilized both DMG and sarcosine as substrates and exhibited dual coenzyme specificity, preferring NAD+ to NADP+ The optimum pH and temperature of enzyme activity were determined to be 7.0 and 60°C, respectively. Kinetic parameters of the enzyme toward its substrates were determined accordingly. Under high-salinity conditions, the presence of DMG inhibited growth of the wild type and induced the production and accumulation of trehalose and glucosylglycerate intracellularly. Moreover, exogenous addition of DMG significantly improved the growth rates of the four DMG- mutants (Δcsal_0990, Δcsal_0991, Δcsal_0992, and Δcsal_0993) incubated at 37°C in S-M63 synthetic medium with sarcosine as the sole N source. 13C nuclear magnetic resonance (13C-NMR) experiments revealed that not only ectoine, glutamate, and N-acetyl-2,4-diaminobutyrate but also glycine betaine (GB), DMG, sarcosine, trehalose, and glucosylglycerate are accumulated intracellularly in the four mutants.IMPORTANCE Although N,N-dimethylglycine (DMG) dehydrogenase (DMGDH) activity was detected in cell extracts of microorganisms, the genes encoding microbial DMGDHs have not been determined until now. In addition, to our knowledge, the physiological role of DMG in moderate halophiles has never been investigated. In this study, we identified the genes involved in DMG degradation to sarcosine, characterized an unusual DMGDH, and investigated the role of DMG in Chromohalobacter salexigens DSM 3043 and its mutants. Our results suggested that the conversion of DMG to sarcosine is accompanied by intramolecular delivery of electrons in DMGDH and intermolecular electron transfer between DMGDH and other electron acceptors. Moreover, an unidentified methyltransferase catalyzing the production of glycine betaine (GB) from DMG but sharing no homology with the reported sarcosine DMG methyltransferases was predicted to be present in the cells. The results of this study expand our understanding of the physiological role of DMG and its catabolism to sarcosine in C. salexigens.

Hemin inhibits NLRP3 inflammasome activation in sepsis-induced acute lung injury, involving heme oxygenase-1.

NLRP3 inflammasome activation contributes to acute lung injury (ALI), accelerating caspase-1 maturation, and resulting in IL-1ß and IL-18 over-production. Heme oxygenase-1 (HO-1) plays a protective role in ALI. This study investigated the effect of hemin (a potent HO-1 inducer) on NLRP3 inflammasome in sepsis-induced ALI. The sepsis model of cecal ligation and puncture (CLP) was used in C57BL6 mice. In vivo induction and suppression of HO-1 were performed by pretreatment with hemin and zinc protoporphyrin IX (ZnPP, a HO-1 competitive inhibitor) respectively. CLP triggered significant pulmonary damage, neutrophil infiltration, increased levels of IL-1ß and IL-18, and edema formation in the lung. Hemin pretreatment exerted inhibitory effect on lung injury and attenuated IL-1ß and IL-18 secretion in serum and lung tissue. In lung tissues, hemin down-regulated mRNA and protein levels of NLRP3, ASC and caspase-1. Moreover, hemin reduced malondialdehyde and reactive oxygen species production, and inhibited NF-κB and NLRP3 inflammasome activity. Meanwhile, hemin significantly increased HO-1 mRNA and protein expression and HO-1 enzymatic activity. In contrast, no significant differences were observed between the CLP and ZnPP groups. Our study suggests that hemin-inhibited NLRP3 inflammasome activation involved HO-1, reducing IL-1ß and IL-18 secretion and limiting the inflammatory response.

Glycopolymer brushes for the affinity adsorption of RCA120: effects of thickness, grafting density, and epitope density.

The interactions between glycopolymer brushes and lectin are very important for the development of affinity membrane chromatography in protein separation. Here, we report the combination of surface-initiated atom transfer radical polymerization (SI-ATRP) and surface plasmon resonance (SPR) to investigate the relationship between the structure of glycopolymer brushes and the affinity adsorption of lectin. The glycopolymer brushes were fabricated from self-assembly of 11-mercapto-1-undecanol (MUD)/1-undecanethiol (UDT) mixture, immobilization of ATRP initiators, and then SI-ATRP of 2-lactobionamidoethyl methacrylate (LAMA). Brush thickness and grafting density were adjusted by controlling polymerization time and thiol ratio in MUD/UDT mixture, respectively. Sugar epitope density was also controlled through copolymerization of 2-hydroxylethyl methacrylate (HEMA) with LAMA. Ricinus communis agglutinin (RCA(120)), one kind of lectin that can bind galactose specifically, was chosen to study the effects of brush architectures on lectin adsorption. SPR results indicate not only the thickness but also the grafting density and the epitope density of glycopolymer brushes can achieve the best performance of sugar cluster effect in affinity adsorption of lectin. In addition, the mass transport effect is crucial in the adsorption process. We propose that it is important to keep the balance between the sugar cluster effect and the mass transport effect in the preparation of high-performance affinity membrane chromatography.

Poly(2-hydroxyethyl methacrylate) brush surface for specific and oriented adsorption of glycosidases.

We present a detailed picture to screen general ligands from simple chemicals for fabricating affinity surface to glycosidase enzymes. The surface was constructed by grafting poly(2-hydroxyethyl methacrylate) (PHEMA) brush on SPR gold chip via surface-initiated atom-transfer radical polymerization, after which poly(methoxyethyl methacrylate) (PMEMA) and poly(oligo(ethylene glycol) methacrylate) (POEGMA) brushes were also prepared for comparison. SPR measurements were adopted to monitor the early-stage adsorption of two glycosidases and three other typical proteins. PHEMA resists the adsorption of lysozyme, bovine serum albumin, and fibrinogen, while it is capable of specifically adsorbing ß-glucosidase (GLU) and ß-galactosidase (GAL). These are quite different from the nonspecific adsorption of PMEMA and the anti-nonspecific adsorption of POEGMA to the studied proteins, because PHEMA is the acceptor substrate of the glycosidases. About 69.6 and 93.7 ng/cm(2) of GAL and GLU are adsorbed on the PHEMA brush surface, of which more than 49.6 ng/cm(2) is remained after washing with PBS. The specific adsorption process is appropriately described by Freundlich isothermal model rather than Langmuir one, and is also indicated to be spontaneous, endothermic, and entropy driven through thermodynamic studies. Taking into account all stated results above, we propose that molecular recognition takes place between the hydroxyl groups of PHEMA and the active sites of glycosidases, which subsequently enables the oriented adsorption of glycosidases on the brush surface. The adsorbed enzyme can be effectively eluted with 1.0 M aqueous solution of ethanol. Our findings open the door to the further development in the design of novel acceptor substrate-ligand affinity chromatography for enzyme purification.

Imbalance between tissue inhibitor of metalloproteinase 1 and matrix metalloproteinase 9 after cardiopulmonary resuscitation.

AIMS: This study aimed to determine whether (a) there was an imbalance between matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) after cardiopulmonary resuscitation (CPR) in a canine model of prolonged ventricular fibrillation (VF); (b) with the duration of VF, the degree of the imbalance would be greater; and (c) there was a relationship between the level of MMP-9 or TIMP-1 and the cardiac function. METHODS AND RESULTS: Ventricular fibrillation was electrically induced in 24 dogs. The animals were randomly divided into 3 groups (sham control, n = 8; 8-minute VF, n = 8; 12-minute VF, n = 8). Echocardiographic measurement and hemodynamic variables were recorded before VF and after return of spontaneous circulation. Tissue inhibitor of metalloproteinase 1 (TIMP-1) and MMP-9 were analyzed by Western blot and immunohistochemistry. Compared with sham controls, dogs under VF and CPR showed significantly decreased level of TIMP-1 (P < .001), and with the duration of VF, the level of TIMP-1 declined (P < .01). The level of MMP-9 did not achieve statistical significance in the 3 groups (P > .05); however, they were higher in VF and longer duration VF groups. The ratios of TIMP-1/MMP-9 were lower in VF groups (P < .05). There was a negative correlation between TIMP-1 and left atrium dimension and left ventricular diastolic dimensions (r = -0.83 and r = -0.96, respectively; P < .01) and a positive correlation between TIMP-1 and left ventricular ejection fraction (r = 0.85; P < .01). CONCLUSIONS: There was an imbalance between TIMP-1 and MMP-9 after CPR. It may partly contribute to the postresuscitation cardiac dysfunction.

Initial defibrillation versus initial chest compression in a 4-minute ventricular fibrillation canine model of cardiac arrest.

OBJECTIVE: Previous laboratory and clinical studies have demonstrated that chest compression preceding defibrillation in prolonged ventricular fibrillation (VF) increases the likelihood of successful cardiac resuscitation. The lower limit of VF duration when preshock chest compression provides no benefit has not been specifically studied. We aimed to study the effect of order of defibrillation and chest compression on defibrillation and cardiac resuscitation in a 4-minute VF canine model of cardiac arrest. DESIGN: Prospective, randomized animal study. SETTING: Key Laboratory of Cardiovascular Remodeling and Function Research and Department of Cardiology, QiLu Hospital. SUBJECTS: Twenty-four domestic dogs. INTERVENTIONS: VF was induced in anesthetized and ventilated canines. After 4 minutes of untreated VF, animals were randomly assigned to receive shock first or chest compression first. Animals in the shock-first group received an immediate single countershock of 360 J for <10 seconds, then 200 immediate compressions before pulse check or rhythm reanalysis. The ratio of compression to ventilation was 30:2. Interruptions to deliver rescue breaths were eliminated in this study. Animals in the chest compression-first group received 200 chest compressions before a single countershock; the other interventions were the same as for the shock-first group. End points were restoration of spontaneous circulation (ROSC), defined as spontaneous systolic arterial pressure >50 mm Hg, when epinephrine (0.02 mg/kg intravenously) was given, and resuscitation, defined as maintaining systolic arterial pressure >50 mm Hg at the 24-hour study end point. MEASUREMENTS AND MAIN RESULTS: In the shock-first group, all animals achieved ROSC, and ten of 12 survived at the 24-hour study end point. In the chest compression-first group, 11 of 12 animals achieved ROSC, and nine of 12 survived at the 24-hour study end point. CONCLUSIONS: In this 4-minute VF canine model of cardiac arrest, the order of initial defibrillation or initial chest compression does not affect cardiac resuscitation.