Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.     

Effect of Sodium Bicarbonate Administration on Mortality in Patients with Lactic Acidosis: A Retrospective Analysis

Background

Lactic acidosis is a common cause of high anion gap metabolic acidosis. Sodium bicarbonate may be considered for an arterial pH <7.15 but paradoxically depresses cardiac performance and exacerbates acidosis by enhancing lactate production. This study aimed to evaluate the cause and mortality rate of lactic acidosis and to investigate the effect of factors, including sodium bicarbonate use, on death.

Methods

We conducted a single center analysis from May 2011 through April 2012. We retrospectively analyzed 103 patients with lactic acidosis among 207 patients with metabolic acidosis. We used SOFA and APACHE II as severity scores to estimate illness severity. Multivariate logistic regression analysis and Cox regression analysis models were used to identify factors that affect mortality.

Results

Of the 103 patients with a mean age of 66.1±11.4 years, eighty-three patients (80.6%) died from sepsis (61.4%), hepatic failure, cardiogenic shock and other causes. The percentage of sodium bicarbonate administration (p = 0.006), catecholamine use, ventilator care and male gender were higher in the non-survival group than the survival group. The non-survival group had significantly higher initial and follow-up lactic acid levels, lower initial albumin, higher SOFA scores and APACHE II scores than the survival group. The mortality rate was significantly higher in patients who received sodium bicarbonate. Sodium bicarbonate administration (p = 0.016) was associated with higher mortality. Independent factors that affected mortality were SOFA score (Exp (B) = 1.72, 95% CI = 1.12–2.63, p = 0.013) and sodium bicarbonate administration (Exp (B) = 6.27, 95% CI = 1.10–35.78, p = 0.039).

Conclusions

Lactic acidosis, which has a high mortality rate, should be evaluated in patients with metabolic acidosis. In addition, sodium bicarbonate should be prescribed with caution in the case of lactic acidosis because sodium bicarbonate administration may affect mortality.     

Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.     

Baicalein inhibits mitochondrial apoptosis induced by oxidative stress in cardiomyocytes by stabilizing MARCH5 expression

Abnormal mitochondrial fission and mitophagy participate in the pathogenesis of many cardiovascular diseases. Baicalein is a key active component in the roots of traditional Chinese medicinal herb Scutellaria baicalensis Georgi. It has been reported that baicalein can resist cardiotoxicity induced by several stress, but the mechanisms of baicalein operate in the protection of cardiomyocytes need to be researched further. Here we report that baicalein can promote cell survival under oxidative stress by up‐regulating the expression level of MARCH5 in cardiomyocytes. Pre‐treatment cells or mice with baicalein can stabilize the expression of MARCH5, which plays a crucial role in the regulation of mitochondrial network and mitophagy. Overexpressed MARCH5 is able to against H₂O₂ and ischaemia/reperfusion (I/R) stress by suppressing mitochondrial fission and enhancing mitophagy, and then attenuate cells apoptosis. Altogether, our present study investigated that baicalein exerts a protective effect through regulating KLF4‐MARCH5‐Drp1 pathway, our research also provided a novel theoretical basis for the clinical application of baicalein.     

Ultraviolet B(UVB)-induced cox-2 expression in murine skin: an immunohistochemical study

Cyclooxygenase (COX) is the rate-limiting enzyme in the production of prostaglandins from arachidonic acid. This enzyme exists in at least two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and plays various physiological roles. However, COX-2 expression is induced by a variety of agents, which include pro-inflammatory agents and mitogens. Evidence exists to indicate that increased expression of COX-2 occurs in several types of epithelial neoplasms. In this study, we show the effect of chronic exposure of murine skin to carcinogenic UVB on cutaneous COX-2 expression. SKH-1 mice were irradiated with 180 mJ/cm(2) UVB daily for five days a week for periods ranging from 1 to 20 weeks. Nontumor bearing skin areas of irradiated mice, skin of age-matched controls and benign papillomas and malignant tumors were assessed immunohistochemically for COX-2 expression in these mice. No epidermal staining occurred in any of the non-UVB-treated controls throughout the experiment. Epidermal COX-2 expression only occurred in UVB-irradiated mice. After 1 and 5 weeks of irradiation, patchy epidermal staining mostly confined to the granular layer and stratum corneum was observed. At week 9, staining intensity had increased, particularly in the granular layer. At week 13, staining was uniformly seen in all epidermal layers with particular prominence in the basal cell layer underlying areas of visible epidermal hyperplasia. It is of interest that the most intense staining was seen in the perinuclear region of keratinocytes and at the plasma membrane. At week 20, COX-2 staining was predominant in the granular layer, although in some tissue sections, the entire epidermis was positive. In benign papillomas, staining was confined to the superficial layers of the epidermis and in squamous cell carcinomas (SCCs), patchy staining in the granular and spinous layers predominated. In general, COX-2 expression was more intense in well-differentiated SCCs than in papillomas. In summary, our results indicate that COX-2 serves as an early marker of epidermal UVB exposure and its expression increases in benign papillomas and in SCCs. These results suggest that pharmacological intervention using specific COX-2 inhibitors could have anticarcinogenic effects in UVB-induced human skin cancer.     

Dynamics of Maize Carbon Contribution to Soil Organic Carbon in Association with Soil Type and Fertility Level

Soil type and fertility level influence straw carbon dynamics in the agroecosystems. However, there is a limited understanding of the dynamic processes of straw-derived and soil-derived carbon and the influence of the addition of straw carbon on soil-derived organic carbon in different soils associated with different fertility levels. In this study, we applied the in-situ carborundum tube method and ¹³C-labeled maize straw (with and without maize straw) at two cropland (Phaeozem and Luvisol soils) experimental sites in northeast China to quantify the dynamics of maize-derived and soil-derived carbon in soils associated with high and low fertility, and to examine how the addition of maize carbon influences soil-derived organic carbon and the interactions of soil type and fertility level with maize-derived and soil-derived carbon. We found that, on average, the contributions of maize-derived carbon to total organic carbon in maize-soil systems during the experimental period were differentiated among low fertility Luvisol (from 62.82% to 42.90), high fertility Luvisol (from 53.15% to 30.00%), low fertility Phaeozem (from 58.69% to 36.29%) and high fertility Phaeozem (from 41.06% to 16.60%). Furthermore, the addition of maize carbon significantly decreased the remaining soil-derived organic carbon in low and high fertility Luvisols and low fertility Phaeozem before two months. However, the increasing differences in soil-derived organic carbon between both soils with and without maize straw after two months suggested that maize-derived carbon was incorporated into soil-derived organic carbon, thereby potentially offsetting the loss of soil-derived organic carbon. These results suggested that Phaeozem and high fertility level soils would fix more maize carbon over time and thus were more beneficial for protecting soil-derived organic carbon from maize carbon decomposition.     

Serum lipoprotein (a) concentrations are inversely associated with T2D,prediabetes, and insulin resistance in a middle-aged and elderly Chinese population

Lipoprotein (a) [Lp(a)], an LDL-like particle, has been proposed as a causal risk factor for CVD among general populations. Meanwhile, both serum Lp(a) and diabetes increase the risk of CVD. However, the relationship between serum Lp(a) and T2D is poorly characterized, especially in the Asian population. Therefore, we conducted a cross-sectional study in 10,122 participants aged 40 years or older in Jiading District, Shanghai, China. Our study found that the prevalence of T2D was decreased from 20.9% to 15.0% from the lowest quartile to the highest quartile of serum Lp(a) concentrations (P for trend <0.0001). Logistic regression analyses showed that the odds ratios and 95% confidence intervals of prevalent T2D for quartiles 2–4 versus quartile 1 were 0.86 (0.73–1.01), 0.88 (0.75–1.04), and 0.76 (0.64–0.90) (P for trend = 0.0002), after adjustment for traditional confounding factors. Moreover, the risks for prevalent prediabetes, insulin resistance, and hyperinsulinemia were also decreased from the lowest to the top quartile. This inverse association between serum Lp(a) and T2D was not appreciably changed after we adjusted hypoglycemic medications or excluded the subjects with hypoglycemic and/or lipid-lowering agents and/or a history of self-reported CVD.     

Extreme Levels of HbA1c Increase Incident ESRD Risk in Chinese Patients with Type 2 Diabetes: Competing Risk Analysis in National Cohort of Taiwan Diabetes Study

Background

Whether HbA1c is a predictor of end-stage renal disease (ESRD) in type 2 diabetes patients remains unclear. This study evaluated relationship between HbA1c and ESRD in Chinese patients with type 2 diabetes.

Methods

Patients aged ≥ 30 years who were free of ESRD (n = 51 681) were included from National Diabetes Care Management Program from 2002–2003. Extended Cox proportional hazard model with competing risk of death served to evaluate association between HbA1c level and ESRD.

Results

A total of 2613 (5.06%) people developed ESRD during a follow-up period of 8.1 years. Overall incidence rate of ESRD was 6.26 per 1000 person-years. Patients with high levels of HbA1c had a high incidence rate of ESRD, from 4.29 for HbA1c of  6.0%–6.9% to 10.33 for HbA1c ≥ 10.0% per 1000 person-years. Patients with HbA1c < 6.0% particularly had a slightly higher ESRD incidence (4.34 per 1000 person-years) than those with HbA1c  of 6.0%–6.9%. A J-shaped relationship between HbA1c level and ESRD risk was observed. After adjustment, patients with HbA1c < 6.0% and ≥ 10.0% exhibited an increased risk of ESRD (HR: 1.99, 95% CI: 1.62–2.44; HR: 4.42, 95% CI: 3.80–5.14, respectively) compared with those with HbA1c of 6.0%–6.9%.

Conclusions

Diabetes care has focused on preventing hyperglycemia, but not hypoglycemia. Our study revealed that HbA1c level ≥ 7.0% was linked with increased ESRD risk in type 2 diabetes patients, and that HbA1c < 6.0% also had the potential to increase ESRD risk. Our study provides epidemiological evidence that appropriate glycemic control is essential for diabetes care to meet HbA1c targets and improve outcomes without increasing the risk to this population. Clinicians need to pay attention to HbA1c results on diabetic nephropathy.     

Discovery of a Novel Immune Gene Signature with Profound Prognostic Value in Colorectal Cancer: A Model of Cooperativity Disorientation Created in the Process from Development to Cancer

Immune response-related genes play a major role in colorectal carcinogenesis by mediating inflammation or immune-surveillance evasion. Although remarkable progress has been made to investigate the underlying mechanism, the understanding of the complicated carcinogenesis process was enormously hindered by large-scale tumor heterogeneity. Development and carcinogenesis share striking similarities in their cellular behavior and underlying molecular mechanisms. The association between embryonic development and carcinogenesis makes embryonic development a viable reference model for studying cancer thereby circumventing the potentially misleading complexity of tumor heterogeneity. Here we proposed that the immune genes, responsible for intra-immune cooperativity disorientation (defined in this study as disruption of developmental expression correlation patterns during carcinogenesis), probably contain untapped prognostic resource of colorectal cancer. In this study, we determined the mRNA expression profile of 137 human biopsy samples, including samples from different stages of human colonic development, colorectal precancerous progression and colorectal cancer samples, among which 60 were also used to generate miRNA expression profile. We originally established Spearman correlation transition model to quantify the cooperativity disorientation associated with the transition from normal to precancerous to cancer tissue, in conjunction with miRNA-mRNA regulatory network and machine learning algorithm to identify genes with prognostic value. Finally, a 12-gene signature was extracted, whose prognostic value was evaluated using Kaplan–Meier survival analysis in five independent datasets. Using the log-rank test, the 12-gene signature was closely related to overall survival in four datasets (GSE17536, n = 177, p = 0.0054; GSE17537, n = 55, p = 0.0039; GSE39582, n = 562, p = 0.13; GSE39084, n = 70, p = 0.11), and significantly associated with disease-free survival in four datasets (GSE17536, n = 177, p = 0.0018; GSE17537, n = 55, p = 0.016; GSE39582, n = 557, p = 4.4e-05; GSE14333, n = 226, p = 0.032). Cox regression analysis confirmed that the 12-gene signature was an independent factor in predicting colorectal cancer patient’s overall survival (hazard ratio: 1.759; 95% confidence interval: 1.126–2.746; p = 0.013], as well as disease-free survival (hazard ratio: 2.116; 95% confidence interval: 1.324–3.380; p = 0.002).