Irisin alters D-galactose-induced apoptosis by increasing caveolin-1 expression in C2C12 myoblasts and skeletal muscle fibroblasts.

Muscle atrophy and skeletal muscle fibrosis are significant pathological manifestations of primary sarcopenia. The regulation of C2C12 myoblast and skeletal muscle fibroblast apoptosis is associated with these pathological changes. Previous studies have indicated that irisin, the cleaved form of fibronectin type III domain-containing protein 5 (FNDC5), can alleviate primary sarcopenia. However, the mechanisms of the effect of irisin in age-related apoptosis remain unknown. Our present research aimed to explore the effect of irisin and the underlying mechanism of D-galactose (D-gal)-induced apoptosis in skeletal muscle fibroblasts and C2C12 myoblasts. We found the opposite effects of D-gal on C2C12 myoblasts and fibroblasts. We also found that irisin suppressed C2C12 cell apoptosis and promoted fibroblast apoptosis. Mechanistically, irisin altered D-gal-induced apoptosis by increasing caveolin-1 expression. Taken together, these findings further demonstrated that irisin is a potential agent that can treat aged-relative muscle atrophy and fibrosis.

Bidirectional effect of uric acid on C2C12 myotubes and its partial mechanism.

AIM: To explore the effects and mechanisms of different concentrations of uric acid on skeletal muscle cells. METHODS: C2C12 myoblasts were differentiated into myotubes and then exposed to a medium containing uric acid (0 µM, 200 µM, 400 µM, 600 µM, 800 µM, 1000 µM, 1200 µM, 1400 µM). The myotube diameters were observed under light microscopy; the expressions of myosin heavy chain (MyHC), autophagy-related proteins (LC3BII/LC3BI, P62), cGAS, and p-Sting/Sting proteins were analyzed using Western blotting or immunoprecipitation; and oxidative stress and mitochondrial damage were evaluated using ROS, mtDNA and JC-1 assays. Cell viability was measured via CCK8 assay, and 1000-µM uric acid was selected for follow-up experiments. Furthermore, C2C12 myotubes were divided into a blank control group (Ctrl), a high-uric-acid group (HUA), and an HUA plus cGASn inhibitor group (HUA + RU.521). Then, the myotube diameter was observed, oxidative stress and mitochondrial damage were evaluated, and MyHC and autophagy-related protein expressions were analysed. RESULTS: C2C12 myotubes cultured in 400-µM uric acid medium had the greatest myotube diameter and the highest MyHC protein expression. At 1000-µM uric acid, the diameter and MyHC protein expression were significantly decreased, LCB3II/LCB3I expression was notably increased, and the level of p62 protein expression was considerably decreased. RU.521 partially alleviated the HUA-induced C2C12 myotubes changes. CONCLUSIONS: Uric acid bidirectionally affected C2C12 myotubes: 400-µΜ uric acid promoted myotube growth, while 1000-µΜ uric acid triggered myotube atrophy with increased autophagy. Inhibiting cGAS-Sting signaling attenuated HUA-induced C2C12 myotube autophagy and atrophy. Geriatr Gerontol Int 2024; 24: 430-439.

Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants.

Cas12a, also known as Cpf1, is a highly versatile CRISPR-Cas enzyme that has been widely used in genome editing. Unlike its well-known counterpart, Cas9, Cas12a has unique features that make it a highly efficient genome editing tool at AT-rich genomic regions. To enrich the CRISPR-Cas12a plant genome editing toolbox, we explored 17 novel Cas12a orthologs for their genome editing capabilities in plants. Out of them, Ev1Cas12a and Hs1Cas12a showed efficient multiplexed genome editing in rice and tomato protoplasts. Notably, Hs1Cas12a exhibited greater tolerance to lower temperatures. Moreover, Hs1Cas12a generated up to 87.5% biallelic editing in rice T0 plants. Both Ev1Cas12a and Hs1Cas12a achieved effective editing in poplar T0 plants, with up to 100% of plants edited, albeit with high chimerism. Taken together, the efficient genome editing demonstrated by Ev1Cas12a and Hs1Cas12a in both monocot and dicot plants highlights their potential as promising genome editing tools in plant species and beyond.

Sarcopenia is an independent risk factor for all-cause mortality rate in patients with diabetic foot ulcers.

Objective: This study aimed to determine whether sarcopenia affects the all-cause mortality rate of patients with diabetic foot ulcers (DFUs). Research design and methods: The clinic-based observational study included 217 patients treated at the Department of Endocrinology, the First Affiliated Hospital of Chongqing Medical University during a 4-year period. All subjects underwent dual-energy X-ray absorptiometry to determine their body composition during hospitalization. Diagnosis of sarcopenia was based on the Baumgartner diagnostic criteria. Patients were followed up regularly by phone calls until April 1, 2019, and their survival status was recorded.Univariate and multivariate Cox risk ratio regression models were used to analyze factors influencing the all-cause mortality rate of patients with DFUs. Results: Of the 217 patients, 158 people survived (82.7%), 33 died (17.3%), and 26 were lost to follow-up. The median follow-up time was 23 (Range 11-34) months. The majority of patients were male (68.6%), with a mean age of 67.29 ± 11.14 years. The 5-year survival rate was 68.3% and 45.9% for all study patients (n = 217) and sarcopenia patients (n = 81), respectively. Multivariate Cox risk regression model showed that age (HR 1.042[95%CI:1.006, 1.078], P = 0.021), sarcopenia (HR 5.051[95%CI:1.968, 12.961], P = 0.001), and serum creatinine (HR 1.007[95%CI: 1.003, 1.010], P < 0.001) were independent risk factors for all-cause mortality rate of patients with DFUs. Kaplan-Meier survival curve indicated that the survival rate of patients with sarcopenia was significantly lower than non-sarcopenia patients (P < 0.001). Conclusions: Sarcopenia is an independent risk factor for all-cause mortality of patients with DFUs and hence an important prognostic factor for patients with DFUs. Active prevention and improvement of sarcopenia can potentially improve the survival outcomes of this patient population.

Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants.

BACKGROUND: Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. RESULTS: To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. CONCLUSIONS: Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

Automated, High-Throughput Protoplast Transfection for Gene Editing and Transgene Expression Studies.

In an era of cost-efficient gene synthesis and high-throughput construct assembly, the onus of scientific experimentation is on the rate of in vivo testing for the identification of top performing candidates or designs. Assay platforms that are relevant to the species of interest and in the tissue of choice are highly desirable. A protoplast isolation and transfection method that is compatible with a large repertoire of species and tissues would be the platform of choice. A necessary aspect of this high-throughput screening approach is the need to handle many delicate protoplast samples at the same time, which is a bottleneck for manual operation. Such bottlenecks can be mitigated with the use of automated liquid handlers for the execution of protoplast transfection steps. The method described within this chapter utilizes a 96-well head for simultaneous, high-throughput initiation of transfection. While initially developed and optimized for use with etiolated maize leaf protoplasts, the automated protocol has also been demonstrated to be compatible with other established protoplast systems, such as soybean immature embryo derived protoplast, similarly described within. This chapter also includes instructions for a sample randomization design to reduce the impact of edge effects, which might be present when microplates are used for fluorescence readout following transfection. We also describe a streamlined, expedient, and cost-effective protocol for determining gene editing efficiencies using the T7E1 endonuclease cleavage assay with a publicly available image analysis tool.

Irisin ameliorates D-galactose-induced skeletal muscle fibrosis via the PI3K/Akt pathway.

Primary sarcopenia is a multicausal skeletal muscle disease associated with muscle strength and mass loss. Skeletal muscle fibrosis is one of the significant pathological manifestations associated with the development of age-related sarcopenia. Irisin, which is cleaved by the extracellular domain of fibronectin type Ⅲ domain-containing protein 5 (FNDC5), has previously been reported to exert antifibrotic effects on the heart, liver, and pancreas, but whether it can rescue skeletal muscle fibrosis remains unknown. In this study, we examined the effects of irisin on D-galactose (D-gal)-induced skeletal muscle fibroblasts. We found that D-gal-induced senescence, fibrosis, and redox imbalance were inhibited by irisin treatment. Mechanistically, irisin or FNDC5 overexpression attenuated D-gal-induced senescence, redox imbalance, and fibrosis by regulating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Overall, irisin might be a promising therapeutic candidate for age-related skeletal muscle fibrosis.

Genome-wide investigation of multiplexed CRISPR-Cas12a-mediated editing in rice.

Clustered regularly interspaced short palindromic repeats (CRISPR) nucleases like Cas9 and Cas12a are revolutionizing plant basic research and crop breeding. A major advantage of CRISPR over earlier nucleases systems is its capability of multiplexed genome editing. However, it remains unknown about the potential off-target effects when multiple concurrent DNA double-strand breaks (DSBs) are induced in a crop genome. Here, we investigated this important question in rice (Oryza sativa) using a highly multiplexed CRISPR-Cas12a system. With whole-genome sequencing, we first revealed high genome editing specificity of Mb2Cas12a and protospacer adjacent motif promiscuity of LbCas12a. We discovered large chromosomal rearrangement events in edited rice plants that endured many (e.g., >50) simultaneous DSBs, but not in plants that endured lower order DSBs (e.g., <10). Our results shed important light on the analysis and regulation of engineered crops derived from CRISPR-Cas mediated multiplexed genome editing.

Preparation, Mechanical Properties and Strengthening Mechanism of W-Re Alloys: A Review.

W-Re alloys are one of the most important refractory materials with excellent high-temperature performance that were developed to improve the brittleness of tungsten. In the present work, we firstly summarized the research progress on the preparation and strengthening methods of a W-Re alloy. Then, the strengthening mechanisms of the W-Re alloy were discussed, including the influence of Re, solid solution strengthening, second-phase reinforcement and fine-grain strengthening. The results showed that the softening effect of Re was mainly related to the transformation of the preferred slip plane and the introduction of additional d-valence electrons. Some transition elements and refractory metal elements effectively strengthened the W-Re alloy. Carbides can significantly enhance the high-temperature mechanical properties of W-Re alloys, and the reasons are twofold: one is the interaction between carbides and dislocations, and the other is the synergistic strengthening effect between carbides and Re. The objective of this work was to enhance the comprehension on W-Re alloys and provide future research directions for W-Re alloys.

Comparative Muscle Tolerability of Different Types and Intensities of Statins: A Network Meta-Analysis of Double-Blind Randomized Controlled Trials.

PURPOSE: The benefits of statins for ischemic cardio-cerebrovascular diseases are well known. However, concerns around muscle adverse events still exist. We therefore aimed to compare the muscle safety of individual statins in adults. METHODS: PubMed, Embase, Cochrane Central Register of Controlled Trials and Web of Science were searched to include double-blind randomized controlled trials (RCTs) comparing one statin with another or with control treatment. Pairwise meta-analyses and network meta-analyses were undertaken with Stata 14.0 software. Relative risk (RR) with 95% confidence intervals (CIs) was adopted for each outcome. RESULTS: A total of 83 RCTs were included. In the pairwise meta-analysis, statins were significantly associated with only a slight increase in muscle symptoms compared with control (RR=1.05; 95% CI=1.01-1.09). In the drug-level network meta-analyses, no statistically significant difference was found between individual statins in the incidence of muscle symptoms, myalgia, myopathy, rhabdomyolysis, creatine kinase (CK) >10 times the upper limit of normal (ULN) or discontinuation due to muscle adverse events. In the dose-level network meta-analyses, there were no statistically significant dose-dependent effects on any outcomes except that moderate-intensity statins had a higher incidence of muscle symptoms than control (RR=1.13; 95% CI=1.01-1.27). Moderate simvastatin (RR=6.57; 95% CI=1.26-34.41) and moderate pravastatin (RR=5.96; 95% CI=1.00-35.44) had a statistically significantly higher incidence of CK >10×ULN compared with moderate atorvastatin. Lipophilic statins and statins metabolized by liver cytochrome P450 3A4 were not associated with an increased risk of muscle adverse events. CONCLUSION: Statins may be generally safe on muscle. Moderate atorvastatin may be superior to equivalent simvastatin and pravastatin in muscle tolerability.

Highly efficient CRISPR systems for loss-of-function and gain-of-function research in pear calli.

CRISPR/Cas systems have been widely used for genome engineering in many plant species. However, their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only a few reports on the application of the CRISPR/Cas9 system in pear have been documented, and have shown very low editing efficiency. Here we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand the targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taking these results together, we have built a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear.

Expanding the targeting scope of FokI-dCas nuclease systems with SpRY and Mb2Cas12a.

CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system was developed that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system was developed that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.

Association among calf circumference, physical performance, and depression in the elderly Chinese population: a cross-sectional study.

BACKGROUND: Depression and sarcopenia are common diseases in the elderly population. However, the association between them is controversial. Based on the Chinese Longitudinal Healthy Longevity Survey (CLHLS) database, a cross-sectional study was conducted to explore the relationship of calf circumference and physical performance with depression. METHODS: From the 8th wave of CLHLS conducted in 2018, data on calf circumference, physical performance, depressive symptoms, and demographic, socioeconomic, and health-related characteristics were collected. Multiple logistic regression was conducted to explore the impact of calf circumference, physical performance and their combination on depressive symptoms. RESULTS: We enrolled a total of 12,227 participants aged 83.4 ± 11.0 years, including 5689 (46.5%) men and 6538 (53.5%) women. Patients with depression were more likely to have low calf circumference (2274 [68.2%] vs. 5406 [60.8%], p<0.001) and poor physical performance (3[0, 6] vs. 1[0, 4], p<0.001). A significant multiplicative interaction was found between calf circumference and physical performance in their effect on depression. After adjusting for confounding factors, multiple logistic regression showed that a significant inverse correlation persisted between physical performance and depressive symptoms in normal (odds ratio [OR] = 1.20, 95% confidence interval [CI]: 1.15-1.26, p<0.001) and low (OR = 1.14, 95% CI: 1.11-1.18, p<0.001) calf circumference group, while the association between calf circumference and depression disappeared. Participants with low calf circumference and poor physical performance were 2.21 times more likely to have depression than those with normal calf circumference and physical performance. All results were found to be robust in sensitivity analyses. CONCLUSIONS: Physical performance was significantly associated with depression in the elderly Chinese population. Attention should be paid to assess depressive symptoms in patients with poor physical performance.

Projected climate-driven changes in pollen emission season length and magnitude over the continental United States.

Atmospheric conditions affect the release of anemophilous pollen, and the timing and magnitude will be altered by climate change. As simulated with a pollen emission model and future climate data, warmer end-of-century temperatures (4-6 K) shift the start of spring emissions 10-40 days earlier and summer/fall weeds and grasses 5-15 days later and lengthen the season duration. Phenological shifts depend on the temperature response of individual taxa, with convergence in some regions and divergence in others. Temperature and precipitation alter daily pollen emission maxima by -35 to 40% and increase the annual total pollen emission by 16-40% due to changes in phenology and temperature-driven pollen production. Increasing atmospheric CO2 may increase pollen production, and doubling production in conjunction with climate increases end-of-century emissions up to 200%. Land cover change modifies the distribution of pollen emitters, yet the effects are relatively small (<10%) compared to climate or CO2. These simulations indicate that increasing pollen and longer seasons will increase the likelihood of seasonal allergies.

Highly Efficient Genome Editing in Plant Protoplasts by Ribonucleoprotein Delivery of CRISPR-Cas12a Nucleases.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing is a powerful approach for crop improvement. Traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects. CRISPR delivered as ribonucleoproteins (RNPs) can prevent exogenous DNA integration, minimize off-target effects, and reduce cellular toxicity. Although RNP delivered CRISPR genome editing has been demonstrated in many plant species, optimization strategies that yield high editing efficiencies have not been thoroughly investigated. Using rice and citrus protoplast systems we demonstrated highly efficient genome editing using Cas12a delivered as RNPs. Four Cas12a variants, including LbCas12a, LbCas12a-E795L, AsCas12a, and AsCas12a Ultra, were investigated. Nearly 100% editing efficiency was observed for three out of four target sites by LbCas12a, LbCas12a-E795L, and AsCas12a Ultra, as measured by restriction fragment length polymorphism (RFLP) and verified by next generation sequencing of PCR amplicons. RNP delivery resulted in higher editing efficiencies than plasmid delivery at 32°C and 25°C. LbCas12a and LbCas12a-E795L demonstrated increased editing efficiencies in comparison to AsCas12a and AsCas12a Ultra, especially when used at lower RNP concentrations. In addition, we discovered that a 1:1 Cas12a:crRNA molar ratio is sufficient to achieve efficient genome editing. Nuclear localization signals (NLSs) are essential for efficient RNP-based genome editing. However, the different crRNA modifications tested did not significantly improve genome editing efficiency. Finally, we applied the Cas12a RNP system in citrus protoplasts and obtained similarly high editing efficiencies at the target site. Our study provides a comprehensive guideline for Cas12a-mediated genome editing using RNP delivery in plant cells, setting the foundation for the generation of transgene-free genome edited plants.

Effect of pH on the Corrosion and Repassivation Behavior of TA2 in Simulated Seawater.

The effect of pH on the corrosion and repassivation behavior of TA2 in simulated seawater was studied using electrochemical tests, immersion experiments, and surface morphology topology analysis. The results show that Ecorr and Rf increased while ipass and weight loss rate decreased as the pH of simulated seawater increased. The TA2 passive film was determined to be mainly composed of a large amount of TiO2 and a small amount of TiO. The repassivation function of TA2 can be expressed as E = -0.1375 + 0.0532ln(t - 1.241) for a simulated seawater pH of 8.2. The parameter b, which represents the slope of the potential-time curve during the friction electrode test, was used to evaluate the repassivation behavior of TA2. The increase in pH value was observed to promote the repassivation speed of the passive film, which is beneficial to the corrosion resistance of TA2.

Construct design for CRISPR/Cas-based genome editing in plants.

CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing.