Detecting fa leptin receptor mutation in Zucker rats with tetra-primer amplification-refractory mutation system (ARMS)-PCR.

Due to the genetic mutation (fa) in the gene encoding for leptin receptor, homozygous Zucker rats (fa-/-) develop excessive adiposity and become an experimental animal model in obesity and metabolic-related diseases research. Based on tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), we developed a method to quickly genotype Zucker rats with a mutated fa allele from their wildtype littermates. The three genotypes are clearly discriminated on 2.0% agarose gel. Our method can be used as a reliable tool to set up and maintain the breeding colony in animal facilities as well as assign animals to control and treatment groups based on their genotypes for animal studies.

Global Landscape of Alternative Splicing in Maize Response to Low Temperature.

Maize (Zea mays L.) is an important food crop planted across the world, and low-temperature stress can affect maize germination. Alternative splicing (AS) is widely present in plants under abiotic stress; however, the response of AS to low-temperature stress in maize remains unclear. In this study, a genome-wide analysis of AS during maize response to low temperatures was performed. AS events were distributed on each chromosome, approximately 2.05-2.09 AS events per gene. Seven genes only had AS in low-temperature-resistant inbred lines. A total of 278 KEGGs and 46 GOs were enriched based on overlapping AS genes, which were associated with hormone and oxidoreductase activity. The mutant was used to verify the function of AS gene ZmWRKY48, and the RGR, RSL, RRL, and RRSA of the mutant decreased by 15.16%-19.87% compared with the normal line. These results contribute to subsequent analysis of the regulatory mechanism of maize in response to low-temperature stress.

Association of Thoracic Aortic Aneurysm Size With Long-term Patient Outcomes: The KP-TAA Study.

Importance: The risk of adverse events from ascending thoracic aorta aneurysm (TAA) is poorly understood but drives clinical decision-making. Objective: To evaluate the association of TAA size with outcomes in nonsyndromic patients in a large non-referral-based health care delivery system. Design, Setting, and Participants: The Kaiser Permanente Thoracic Aortic Aneurysm (KP-TAA) cohort study was a retrospective cohort study at Kaiser Permanente Northern California, a fully integrated health care delivery system insuring and providing care for more than 4.5 million persons. Nonsyndromic patients from a regional TAA safety net tracking system were included. Imaging data including maximum TAA size were merged with electronic health record (EHR) and comprehensive death data to obtain demographic characteristics, comorbidities, medications, laboratory values, vital signs, and subsequent outcomes. Unadjusted rates were calculated and the association of TAA size with outcomes was evaluated in multivariable competing risk models that categorized TAA size as a baseline and time-updated variable and accounted for potential confounders. Data were analyzed from January 2018 to August 2021. Exposures: TAA size. Main Outcomes and Measures: Aortic dissection (AD), all-cause death, and elective aortic surgery. Results: Of 6372 patients with TAA identified between 2000 and 2016 (mean [SD] age, 68.6 [13.0] years; 2050 female individuals [32.2%] and 4322 male individuals [67.8%]), mean (SD) initial TAA size was 4.4 (0.5) cm (828 individuals [13.0% of cohort] had initial TAA size 5.0 cm or larger and 280 [4.4%] 5.5 cm or larger). Rates of AD were low across a mean (SD) 3.7 (2.5) years of follow-up (44 individuals [0.7% of cohort]; incidence 0.22 events per 100 person-years). Larger initial aortic size was associated with higher risk of AD and all-cause death in multivariable models, with an inflection point in risk at 6.0 cm. Estimated adjusted risks of AD within 5 years were 0.3% (95% CI, 0.3-0.7), 0.6% (95% CI, 0.4-1.3), 1.5% (95% CI, 1.2-3.9), 3.6% (95% CI, 1.8-12.8), and 10.5% (95% CI, 2.7-44.3) in patients with TAA size of 4.0 to 4.4 cm, 4.5 to 4.9 cm, 5.0 to 5.4 cm, 5.5 to 5.9 cm, and 6.0 cm or larger, respectively, in time-updated models. Rates of the composite outcome of AD and all-cause death were higher than for AD alone, but a similar inflection point for increased risk was observed at 6.0 cm. Conclusions and Relevance: In a large sociodemographically diverse cohort of patients with TAA, absolute risk of aortic dissection was low but increased with larger aortic sizes after adjustment for potential confounders and competing risks. Our data support current consensus guidelines recommending prophylactic surgery in nonsyndromic individuals with TAA at a 5.5-cm threshold.

Using a high density bin map to analyze quantitative trait locis of germination ability of maize at low temperatures.

Low temperatures in the spring often lead to a decline in the emergence rate and uniformity of maize, which can affect yield in northern regions. This study used 365 recombinant inbred lines (RILs), which arose from crossing Qi319 and Ye478, to identify low-temperature resistance during the germination stage by measuring eight low-temperature-related traits. The quantitative trait locis (QTLs) were mapped using R/qtl software by combining phenotypic data, and the genotyping by sequencing (GBS) method to produce a high-density genetic linkage map. Twenty QTLs were detected during QTL mapping, of which seven QTLs simultaneously detected a consistent 197.10-202.30 Mb segment on chromosome 1. The primary segment was named cQTL1-2, with a phenotypic variation of 5.18-25.96% and a physical distance of 5.2 Mb. This combines the phenotype and genotype with the identification of seven chromosome segment substitution lines (CSSLs), which were derived from Ye478*Qi319 and related to cQTL1-2. The physical distance of cQTL1-2 was reduced to approximately 1.9 Mb. The consistent meta-QTL mQTL1 was located at 619.06 cM on chromosome 1, had a genetic distance of 7.27 cM, and overlapped with cQTL1-2. This was identified by combining the results of previous QTL studies assessing maize tolerance to low temperatures at the germination stage. An assessment of the results of the RIL population, CSSLs, and mQTL1 found the consistent QTL to be LtQTL1-1. It was identified in bin1.06-1.07 at a confidence interval of between 200,400,148 and 201,775,619 bp. In this interval, qRT-PCR found that relative expression of the candidate genes GRMZM2G082630 and GRMZM2G115730 were both up-regulated in low-temperature tolerant lines and down-regulated in sensitive lines (P < 0.01).

Inflammation Induced by Lipopolysaccharide and Palmitic Acid Increases Cholesterol Accumulation via Enhancing Myeloid Differentiation Factor 88 Expression in HepG2 Cells.

Recently, multiple studies have shown that chronic inflammation disturbs cholesterol homeostasis and promotes its accumulation in the liver. The underlying molecular mechanism remains to be revealed. The relationship between the toll-like receptor 4 (TLR4) inflammatory signaling pathway and cholesterol accumulation was investigated in HepG2 cells treated with lipopolysaccharide (LPS) or palmitic acid (PA) for different lengths of time. In addition, the effects of pretreatment with 20µmol/L ST2825 (MyD88 inhibitor) were also studied in LPS- or PA-treated HepG2 cells and myeloid differentiation factor 88 (MyD88)-overexpressing HEK293T cells. The intracellular total and free cholesterol levels were measured using a commercial kit and filipin staining, respectively. The expression levels of sterol regulatory element-binding protein-2 (SREBP-2) and components in the TLR4 signaling pathway were determined using Western blotting. The treatments with LPS for 12 h and with PA for 24 h significantly increased the contents of intracellular total and free cholesterol, as well as the expression levels of SREBP-2 and components in the TLR4 signaling pathway. The inhibition of MyD88 by ST2825 significantly decreased the cholesterol content and the expression levels of SREBP-2 and components of the TLR4/MyD88/NF-κB pathway in HepG2 cells, as well as MyD88-overexpressing HEK293T cells. These results indicated that LPS and PA treatments increase SREBP-2-mediated cholesterol accumulation via the activation of the TLR4/MyD88/NF-κB signaling pathway in HepG2 cells.

Reduction in the Dietary VA Status Prevents Type 2 Diabetes and Obesity in Zucker Diabetic Fatty Rats.

We hypothesized that the vitamin A (VA) status regulates type 2 diabetes (T2D) development in Zucker diabetic fatty (ZDF) rats. Zucker Lean and ZDF rats at weaning were fed a VA deficient with basal fat (VAD-BF, no VA and 22.1% fat energy), VA marginal with BF (VAM-BF, 0.35 mg retinyl palmitate (RP)/kg), VA sufficient with BF (VAS-BF, 4.0 mg RP/kg), VAD with high fat (VAD-HF, 60% fat energy), VAM-HF or VAS-HF diet for 8 weeks, including an oral glucose tolerance test (OGTT) at week 7.5. The hepatic mRNA and proteins levels were determined using real-time PCR and Western blot, respectively. The VAD-BF/HF and VAM-BF/HF diets prevented peripheral hyperglycemia and attenuated obesity in ZDF rats, which occurred in the presence of the VAS-BF/HF diets. This lowered VA status reduced venous blood hyperglycemia, hyperinsulinemia and hyperlipidemia, and improved OGTT and homeostasis model assessment for insulin resistance results in ZDF rats. The expression levels of key hepatic genes for glucose and fat metabolism were regulated by VA status and dietary fat contents. An interaction between VA and HF condition was also observed. We conclude that the reduction in the dietary VA status in both BF and HF conditions prevents T2D and obesity in ZDF rats.

The Extraction of ß-Carotene from Microalgae for Testing Their Health Benefits.

ß-carotene, a member of the carotenoid family, is a provitamin A, and can be converted into vitamin A (retinol), which plays essential roles in the regulation of physiological functions in animal bodies. Microalgae synthesize a variety of carotenoids including ß-carotene and are a rich source of natural ß-carotene. This has attracted the attention of researchers in academia and the biotech industry. Methods to enrich or purify ß-carotene from microalgae have been investigated, and experiments to understand the biological functions of microalgae products containing ß-carotene have been conducted. To better understand the use of microalgae to produce ß-carotene and other carotenoids, we have searched PubMed in August 2021 for the recent studies that are focused on microalgae carotenoid content, the extraction methods to produce ß-carotene from microalgae, and the bioactivities of ß-carotene from microalgae. Articles published in peer-reviewed scientific journals were identified, screened, and summarized here. So far, various types and amounts of carotenoids have been identified and extracted in different types of microalgae. Diverse methods have been developed overtime to extract ß-carotene efficiently and practically from microalgae for mass production. It appears that methods have been developed to simplify the steps and extract ß-carotene directly and efficiently. Multiple studies have shown that extracts or whole organism of microalgae containing ß-carotene have activities to promote lifespan in lab animals and reduce oxidative stress in culture cells, etc. Nevertheless, more studies are warranted to study the health benefits and functional mechanisms of ß-carotene in these microalgae extracts, which may benefit human and animal health in the future.

The Potential Use of Vitamin C to Prevent Kidney Injury in Patients with COVID-19.

The newly found SARS-CoV-2 has led to the pandemic of COVID-19, which has caused respiratory distress syndrome and even death worldwide. This has become a global public health crisis. Unfortunately, elders and subjects with comorbidities have high mortality rates. One main feature of COVID-19 is the cytokine storm, which can cause damage in cells and tissues including the kidneys. Here, we reviewed the current literature on renal impairments in patients with COVID-19 and analyzed the possible etiology and mechanisms. In addition, we investigated the potential use of vitamin C for the prevention of renal injury in those patients. It appears that vitamin C could be helpful to improve the outcomes of patients with COVID-19. Lastly, we discussed the possible protective effects of vitamin C on renal functions in COVID-19 patients with existing kidney conditions.

Vitamin A and Diabetes.

Recently, research data have shown that vitamin A (VA, retinol) as a micronutrient participates in the regulation of glucose and lipid metabolism. Since diabetes is a metabolic disease, it is imperative to reveal the relationship of VA and diabetes. This review was aimed to summarize the current understanding of VA and its metabolites in diabetes. Since April of 2020, the authors have searched the PubMed using key words and retrieved articles that focused on diabetes and VA or its metabolites. Based on the published data, it appears that the development of type 1 diabetes leads to reduction of blood VA level in human and animals, and increase of hepatic VA store in experimental animals. On the other hand, the mutual impacts of type 2 diabetes and VA intake and blood VA level on each other appear to be uncertain. Retinoic acid, the active metabolite of VA, has been studied extensively for the treatment of diabetic complications. The current data appear to indicate that the development of diabetes is associated with changes of VA metabolism. More carefully designed clinical and laboratory experiments are needed to reveal the impacts of diabetes on VA metabolism and the role of VA in the development and treatment of diabetes.

Current understanding of glucose transporter 4 expression and functional mechanisms.

Glucose is used aerobically and anaerobically to generate energy for cells. Glucose transporters (GLUTs) are transmembrane proteins that transport glucose across the cell membrane. Insulin promotes glucose utilization in part through promoting glucose entry into the skeletal and adipose tissues. This has been thought to be achieved through insulin-induced GLUT4 translocation from intracellular compartments to the cell membrane, which increases the overall rate of glucose flux into a cell. The insulin-induced GLUT4 translocation has been investigated extensively. Recently, significant progress has been made in our understanding of GLUT4 expression and translocation. Here, we summarized the methods and reagents used to determine the expression levels of Slc2a4 mRNA and GLUT4 protein, and GLUT4 translocation in the skeletal muscle, adipose tissues, heart and brain. Overall, a variety of methods such real-time polymerase chain reaction, immunohistochemistry, fluorescence microscopy, fusion proteins, stable cell line and transgenic animals have been used to answer particular questions related to GLUT4 system and insulin action. It seems that insulin-induced GLUT4 translocation can be observed in the heart and brain in addition to the skeletal muscle and adipocytes. Hormones other than insulin can induce GLUT4 translocation. Clearly, more studies of GLUT4 are warranted in the future to advance of our understanding of glucose homeostasis.

Conductance of connexin hemichannels segregates with the first transmembrane segment.

Gap junction channels are intercellular channels that mediate the gated transfer of molecules between adjacent cells. To identify the domain determining channel conductance, the first transmembrane segment (M1) was reciprocally exchanged between Cx46 and Cx32E(1)43. The resulting chimeras exhibited conductances similar to that of the respective M1 donor. Furthermore, a chimera with the carboxy-terminal half of M1 in Cx46 replaced by that of Cx32 exhibited a conductance similar to that of Cx32E(1)43, whereas the chimera with only the amino-terminal half of M1 replaced retained the unitary conductance of wild-type Cx46. Extending the M1 domain swapping to other connexins by replacing the carboxy-terminal half of M1 in Cx46 with that of Cx37 yielded a chimera channel with increased unitary conductance close to that of Cx37. Furthermore, a point mutant of Cx46, with leucine substituted by glycine in position 35, displayed a conductance much larger than that of the wild type. Thus, the M1 segment, especially the second half, contains important determinants of conductance of the connexin channel.