Molecular identification and studies on genetic diversity and structure-related GC heterogeneity of Spatholobus Suberectus based on ITS2.

To determine the role of internal transcribed spacer 2 (ITS2) in the identification of Spatholobus suberectus and explore the genetic diversity of S. suberectus. A total of 292 ITS2s from S. suberectus and 17 other plant species were analysed. S. suberectus was clustered separately in the phylogenetic tree. The genetic distance between species was greater than that within S. suberectus. Synonymous substitution rate (Ks) analysis revealed that ITS2 diverged the most recently within S. suberectus (Ks = 0.0022). These findings suggested that ITS2 is suitable for the identification of S. suberectus. The ITS2s were divided into 8 haplotypes and 4 evolutionary branches on the basis of secondary structure, indicating that there was variation within S. suberectus. Evolutionary analysis revealed that the GC content of paired regions (pGC) was greater than that of unpaired regions (upGC), and the pGC showed a decreasing trend, whereas the upGC remained unchanged. Single-base mutation was the main cause of base pair substitution. In both the initial state and the equilibrium state, the substitution rate of GC was higher than that of AU. The increase in the GC content was partly attributed to GC-biased gene conversion (gBGC). High GC content reflected the high recombination and mutation rates of ITS2, which is the basis for species identification and genetic diversity. We characterized the sequence and structural characteristics of S. suberectus ITS2 in detail, providing a reference and basis for the identification of S. suberectus and its products, as well as the protection and utilization of wild resources.

Regulatory network of flavonoids, phenolic acids and terpenoids biosynthesis in Zizyphus jujuba Mill. cv. Goutou jujube fruits.

Flavonoids, phenolic acids and terpenoids are important active ingredients that are biomarkers for evaluating the quality of Zizyphus jujuba Mill. Cv. Goutou jujube fruit. Nevertheless, regulatory network of these active ingredients biosynthesis in jujube fruit is still unclear. Here, integrated metabolomics and transcriptomics analyses were conducted at four different stages during the ripening of jujube fruits. Cytochrome P450 enzymes (CYP450s) and uridine 5'-diphospho-glucuronosyltransferases (UGTs) are pivotal enzymes for flavonoids, phenolic acids and terpenoids biosynthesis in plants. Benzoylmalic acid, a phenolic acid, 4', 5, 7-trihydroxyflavanon and quercetin-3-O-(6″-p-coumaroyl), two flavonoid metabolites, and jujuboside B1, a triterpenoid metabolite were targeted as they were correlated with both CYP450s and UGTs. Furthermore, networks of TFs, CYP450s and UGTs involved in the target metabolites biosynthesis were elucidated. NAC_1 and bZIP2 up-regulated CYP71A7 expression, while G2-like2 and bHLH_1 positively regulated the CSE expression contributing to promoted benzoylmalic acid biosynthesis. G2-like2, bHLH_1 and bHLH_2 indicated a positive relationship with CYP93D1, CYP86C2/3 or UGT71A16 which were positively correlated with 4', 5, 7-trihydroxyflavanon biosynthesis. MYB1/2/3, C2H2_2 and WRKY positively regulated expression of CYP82A4 or UGT_1 resulted in increased quercetin-3-O-(6″-p-coumaroyl) galactoside biosynthesis. G2-like2 and bHLH_1 up-regulated 4 C L, CYP93D1 or UGT71A16 was the reason for an increase of jujuboside B1 biosynthesis. The findings provide new insight into molecular breeding of high-quality jujube fruits.

Au(I)/Au(III)-Catalyzed Sulfonylation of Aryl Iodides for the Synthesis of Various Functionalized Aryl Sulfones.

A gold-catalyzed sulfonylation reaction of aryl iodides with different sodium sulfinates facilitated by the ligand-enabled Au(I)/Au(III) redox catalysis was developed. In the reaction of gold-catalyzed C-S coupling, a variety of functionalized sodium sulfinates, such as CF3, CHF2, CH3, and alkyl groups, can react smoothly with aryl iodides to directly construct diversely functionalized aryl sulfones. This gold-catalyzed sulfonylation offers a complementary method for synthesizing functionalized aryl sulfones with electron-donating groups.

Voltage Gated Ion Channels and Sleep.

Ion channels are integral components of the nervous system, playing a pivotal role in shaping membrane potential, neuronal excitability, synaptic transmission and plasticity. Dysfunction in these channels, such as improper expression or localization, can lead to irregular neuronal excitability and synaptic communication, which may manifest as various behavioral abnormalities, including disrupted rest-activity cycles. Research has highlighted the significant impact of voltage gated ion channels on sleep parameters, influencing sleep latency, duration and waveforms. Furthermore, these ion channels have been implicated in the vulnerability to, and the pathogenesis of, several neurological and psychiatric disorders, including epilepsy, autism, schizophrenia, and Alzheimer's disease (AD). In this comprehensive review, we aim to provide a summary of the regulatory role of three predominant types of voltage-gated ion channels-calcium (Ca2+), sodium (Na+), and potassium (K+)-in sleep across species, from flies to mammals. We will also discuss the association of sleep disorders with various human diseases that may arise from the dysfunction of these ion channels, thereby underscoring the potential therapeutic benefits of targeting specific ion channel subtypes for sleep disturbance treatment.

Asymmetric Kosmotropism-Stabilized Double-Layer Hydrogel for Low-Cost Neutral Zinc-Air Battery.

Zinc air battery (ZAB) provides a low-cost and high-energy density power source, particularly in wearable and portable devices. Despite the extensive research on air cathode catalysts, their practical application is hindered by low zinc utilization rate and severe corrosion and passivation in liquid-based alkaline electrolytes. Herein, a double-layer gel (DLKgel) is developed by leveraging the distinct kosmotropic properties of ZnCl2 and ZnSO4. Through phase separation induced by the kosmotropic differentiation (instead of membrane in decoupled systems), this DLKgel electrolyte serves a dual purpose of shielding cathode from irreversible reaction products and protecting Zn anode from passivation. Neutral ZABs with DLKgel demonstrate high zinc utilization rate of 89.3% and stable cycling over 800 h under a current density of 0.1 mA cm-2. The integration of DLKgel-based ZABs into a flexible GPS tracking device is demonstrated, highlighting the potential for broad adoption of flexible ZABs in wearable and logistics applications.

Multi-dataset identification of innovative feature genes and molecular mechanisms in keratoconus.

This study aimed to identify feature genes and explore the molecular mechanisms of keratoconus (KC). We downloaded data files from NCBI GEO public database. The Limma package was used for differential expression analysis of gene profiles. Lasso regression was used to identify the feature genes. The CIBERSORT algorithm was used to infer the proportion of immune-infiltrating cells and analyse the correlation between gene expression levels and immune cells. Related transcription factors and miRNAs of key genes were predicted using the Cistrome DB and Mircode databases. Analysis of expression differences in disease genes was based on the GeneCards database. The CMap was used to analyse targeted therapeutic drugs. IHC was performed to verify the expression levels of ATOH7 and MYRF in corneas. Exactly 593 upregulated and 473 downregulated genes were identified. Lasso regression analysis identified ATOH7, DBNDD1, RNF217-AS1, ARL11, MYRF and SNORA74B as feature genes for KC. All key genes were correlated with immune infiltration and the levels of activated memory CD4+ T cells and plasma cells were significantly increased. miRNA, IRF and STAT families were correlated to feature genes. The expression levels of key genes were significantly correlated to KC-related genes. Entinostat, ochratoxin-a, diphencyprone and GSK-3-inhibitor-II were predicted as potential KC medications. The expression of MYRF was significantly higher in the KC samples, contrary to the expression of ATOH7. KC is related to both immune infiltration and genetic factors. MYRF and ATOH7 were newly identified and verified feature genes of KC.

Investigation and Confirmation of PYCARD as a Potential Biomarker for the Management of Psoriasis Disease.

Purpose: Psoriasis is not yet completely curable, and its etiology and pathogenesis are unclear. Necroptosis, also known as programmed necrosis, is a regulated mode of necrotic cell death. The interaction between inflammatory diseases and necrotic apoptosis has recently attracted significant attention. We explored the molecular mechanisms of necrotic apoptosis-related genes in psoriasis using bioinformatics methods to identify potential biomarkers for psoriasis. Patients and Methods: In this study, we screened psoriasis differentially expressed genes from the datasets GSE13355 and GSE14905 and took intersections with necrotic apoptosis-related genes for the next analysis. We used multiple machine learning algorithms to screen key genes and perform enrichment analysis. In addition, we performed an immune infiltration analysis. Transcription factors were predicted by the R package "RcisTarget". We also observed the cellular clustering of key genes in different cell types at the single-cell sequencing level. We used real-time fluorescence-based quantitative-polymerase chain reaction, Western blot, and immunohistochemistry to analyze gene expression in clinical samples. We constructed an imiquimod-induced psoriasis-like dermatitis model in mice for further validation. Results: Seven key genes were screened as follows: AIM2, CARD6, HPSE, MYD88, PYCARD, RAI14, and TNFSF10. Enrichment analysis showed that the key genes were mainly involved in inflammatory pathways. Immune infiltration analysis showed significantly higher levels of CD8 T cells, CD4 initial T cells, and CD4 memory-activated T cells in the disease group's samples than in the normal patients' samples. The key gene expression in single cells analyzed showed that PYCARD was significantly expressed in keratinocytes. PYCARD was selected for gene expression analysis; the results showed that its expression was significantly elevated in the skin lesion tissues of patients with psoriasis. We also verified that PYCARD might play a vital role in the development of psoriasis skin lesions using animal experiments. Conclusion: PYCARD plays a vital role in psoriasis development and is a potential biomarker for psoriasis.

Identification of feature genes and molecular mechanisms involved in cell communication in uveal melanoma through analysis of single­cell sequencing data.

Uveal melanoma (UM) is a highly metastatic cancer with resistance to immunotherapy. The present study aimed to identify novel feature genes and molecular mechanisms in UM through analysis of single-cell sequencing data. For this purpose, data were downloaded from The Cancer Genome Atlas and National Center for Biotechnology Information Gene Expression Omnibus public databases. The statistical analysis function of the CellPhoneDB software package was used to analyze the ligand-receptor relationships of the feature genes. The Metascape database was used to perform the functional annotation of notable gene sets. The randomForestSRC package and random survival forest algorithm were applied to screen feature genes. The CIBERSORT algorithm was used to analyze the RNA-sequencing data and infer the relative proportions of the 22 immune-infiltrating cell types. In vitro, small interfering RNAs were used to knockdown the expression of target genes in C918 cells. The migration capability and viability of these cells were then assessed by gap closure and Cell Counting Kit-8 assays. In total, 13 single-cell sample subtypes were clustered by t-distributed Stochastic Neighbor Embedding and annotated by the R package, SingleR, into 7 cell categories: Tissue stem cells, epithelial cells, fibroblasts, macrophages, natural killer cells, neurons and endothelial cells. The interactions in NK cells|Endothelial cells, Neurons|Endothelial cells, CD74_APP, and SPP1_PTGER4 were more significant than those in the other subsets. T-Box transcription factor 2, tropomyosin 4, plexin D1 (PLXND1), G protein subunit α I2 (GNAI2) and SEC14-like lipid binding 1 were identified as the feature genes in UM. These marker genes were found to be significantly enriched in pathways such as vasculature development, focal adhesion and cell adhesion molecule binding. Significant correlations were observed between key genes and immune cells as well as immune factors. Relationships were also observed between the expression levels of the key genes and multiple disease-related genes. Knockdown of PLXND1 and GNAI2 expression led to significantly lower viability and gap closure rates of C918 cells. Therefore, the results of the present study uncovered cell communication between endothelial cells and other cell types, identified innovative key genes and provided potential targets of gene therapy in UM.

Improving Triethylamine-Sensing Performance of WO3 Nanoplates through In Situ Heterojunction Construction.

Surface engineering techniques can be used to develop high-performance gas sensing materials and advance the development of sensors. In this study, we improved the gas sensing performance of two-dimensional (2D) WO3 nanoplates by combining surface Zn modification and the in situ formation of ZnWO4/WO3 heterojunctions. Introducing Zn atoms by surface modification can reconstruct the atomic surface of 2D WO3 nanoplates, creating additional active sites. This allowed for the preparation of various types of ZnWO4/WO3 heterojunctions on the surface of the WO3 nanoplates, which improved the selectivity and sensitivity to the target gas triethylamine. The sensor exhibited good gas sensing performance for triethylamine even at low operating temperatures and strongly resisted humidity changes. The ZnWO4/WO3 material we prepared demonstrated a nearly threefold improvement in the triethylamine (TEA) response, with a gas sensing responsivity of 40.75 for 10 ppm of TEA at 250 °C. The sensor based on ZnWO4/WO3 has a limit of detection (LOD) for TEA of 200 ppb in practical measurements (its theoretical LOD is even as low as 31 ppb). The method of growing ZnWO4 on the surface of WO3 nanoplates using surface modification techniques to form surface heterojunctions differs from ordinary composites. The results suggest that the in situ construction of surface heterojunctions using surface engineering strategies, such as in situ modifying, is a practical approach to enhance the gas sensing properties and resistance to the humidity changes of metal oxide materials.

Prognosis of early-stage HFpEF in the community-dwelling elderly: The Northern Shanghai Study.

OBJECTIVE: Heart failure with preserved ejection fraction (HFpEF) is a growing concern among the elderly population, significantly impacting morbidity and mortality rates. This study aimed to screen and investigate the characteristics and prognosis of early-stage HFpEF in the elderly. METHODS: A total of 1789 community-dwelling individuals aged over 65 from northern Shanghai were enrolled. According to American Heart Association (AHA) guidelines, participants were classified into four groups: HFpEF stage 0, HFpEF stage A, HFpEF stage B and HFpEF stage C. Major endpoints included major adverse cardiovascular events (MACEs), all-cause death and cardiovascular death. RESULTS: After a mean follow-up period of 7.10 ± 1.27 years, 1623 elderly subjects were included [HFpEF stage 0 (10.3%), HFpEF stage A (16.3%), HFpEF stage B (60.6%) and HFpEF stage C (12.8%)]. Patients with HFpEF stage A, HFpEF stage B and HFpEF stage C exhibited more MACEs than those in HFpEF stage 0 (P < 0.01). Patients with HFpEF stage C had a significantly higher cardiovascular (P < 0.001) and all-cause death ratio (P < 0.01). With HFpEF stage 0 as a reference, the increases in MACEs were significantly associated with HFpEF stage A [hazard ratio (HR): 2.97, 95% confidence interval (CI) (1.13, 7.82), P < 0.05], HFpEF stage B [HR: 2.69, 95% CI (1.09, 6.64), P < 0.05] and HFpEF stage C [HR: 4.86, 95% CI (1.88, 12.59), P < 0.01] in the Cox regression analysis. Our finding remains unaltered in the sensitivity analysis, with no interaction for effectiveness. CONCLUSIONS: Compared with those with HFpEF stage 0, patients with HFpEF, whether in stage B or C, exhibit significantly higher cardiovascular and all-cause mortality in the elderly. This study underscores the importance of early-stage HFpEF screening, particularly in older, asymptomatic stage B individuals.

Estimating the effect of inclisiran on hypercholesterolemia and primary prevention of cardiovascular disease: the NHANES 1999-2018 study.

BACKGROUND: Hypercholesterolemia has been identified as an independent predictor of cardiovascular disease (CVD). Inclisiran, an innovative small interfering RNA agent, is anticipated to result in a notable reduction of approximately 50% in low-density lipoprotein cholesterol (LDL-C) levels. Given its transformative impact, this study scrutinized the eligibility of the US population for inclisiran treatment and evaluated its potential effects on hypercholesterolemia and the primary prevention of CVD. METHODS: This study applied the eligibility criteria from the ORION 10 and 11 trials to the 1999-2018 National Health and Nutrition Examination Survey (NHANES) dataset to estimate the size of the eligible population for atherosclerotic cardiovascular disease (ASCVD) and ASCVD-risk equivalents. Utilizing the reduction in LDL-C levels from ORION 10, this study predicted the impact of inclisiran on LDL-C levels among ASCVD patients. Similarly, leveraging the changes in lipid levels from ORION 11, this study predicted inclisiran's effect on the 10-year change in CVD risk and preventable CVD events in the ASCVD-risk equivalents population, employing the Framingham CVD Risk Score. RESULTS: The study identified 579 ASCVD patients (5 million) and 382 ASCVD-risk equivalents (2.66 million) who met the eligibility criteria from ORION 10 and 11. Among the ASCVD population, 3.5 million (70.2%) would achieve a ≥ 50% reduction in LDL-C levels after treatment. Furthermore, 4.6 million (91.3%) would achieve LDL-C levels < 70 mg/dL, and 3.8 million (75%) would achieve LDL-C levels < 55 mg/dL after treatment. For the ASCVD-risk equivalents population, the estimated 10-year CVD risk would decrease from 25.3 to 17.7%, an absolute reduction of 7.6% and a relative reduction of 30% following inclisiran treatment, potentially preventing 202,353 CVD events over a decade, including 138,084 coronary heart disease cases, 37,351 strokes, and 23,894 congestive heart failure cases. CONCLUSIONS: Inclisiran has the potential to substantially reduce the prevalence of hypercholesterolemia and prevent nearly 200,000 CVD events in eligible US adults.

Role of SbNRT1.1B in cadmium accumulation is attributed to nitrate uptake and glutathione-dependent phytochelatins biosynthesis.

Phytoremediation of cadmium (Cd)-polluted soil by using sweet sorghum displays a tremendous potential as it is a fast-growing, high biomass and Cd tolerant energy plant. Previous study has demonstrated SbNRT1.1B expression change is in accordance with enhanced Cd accumulation by external nitrate supply in sweet sorghum. Nevertheless, underlying mechanism of SbNRT1.1B response to Cd stress is still elusive. SbNRT1.1B exhibited a positive response to Cd stress in sweet sorghum. Overexpressing SbNRT1.1B increased primary root length, shoot fresh weight, nitrate and chlorophyll concentrations compared with Col-0 under Cd stress, while complementary SbNRT1.1B rescued these decreased values in mutant chl1-5. Cd concentrations in overexpressing SbNRT1.1B, complementary SbNRT1.1B and Col-0 lines were 3.2-4.1, 2.5-3.1 and 1.2-2.1 folds of that in chl1-5. Consistent with Cd concentrations, non-protein thiol (NPT), reduced glutathione (GSH) and phytochelatins (PCs) concentrations as well as the related genes expression levels showed the same trends under Cd stress. GSH biosynthesis inhibitor failed to reverse the patterns of GSH-dependent PCs concentrations changes in different lines, suggesting that SbNRT1.1B plays an upstream role in GSH-dependent PCs biosynthesis under Cd treatment. Altogether, SbNRT1.1B enhances nitrate concentrations contributing to increased chlorophyll concentrations and GSH-dependent PCs metabolites biosynthesis, thereby improving growth and Cd concentrations in plants.

Transcriptome analysis of potassium-mediated cadmium accumulation in sweet sorghum.

Cadmium (Cd) pollution in the soil is a serious environmental issue worldwide. Phytoextraction of Cd-polluted soil is a cost-effective, sustainable and environmentally-friendly strategy. Agricultural fertilizer management is beneficial for promoting the Cd phytoremediation efficiency. Potassium (K) is the nutrient required in the largest amount cation by plants. Sweet sorghum exhibits a substantial phytoremediation potential of Cd-polluted soil. Clarifying the mechanism of K-mediated Cd accumulation in sweet sorghum is imperative. Sweet sorghum plants were grown hydroponically with an extra K supply in the presence or absence of Cd treatment. An extra K application significantly increased plant growth under non-Cd addition, while K lost the profitable effect under Cd stress. K supplementation remarkably enhanced Cd concentrations and Cd accumulation in shoots and roots of sweet sorghum. Transcriptome analysis demonstrated that zinc ion transport, cysteine and methionine metabolism, flavonoid biosynthesis and phenylpropanoid biosynthesis pathways might contribute to the increased Cd accumulation as affected by an extra K supply. Furthermore, SbZIP9, SbSTP8, SbYS1, SbMAG and SbFOMT-like were targeted as they closely correlated with both plant growth and Cd stress in sweet sorghum. SbFOMT-like showed an independent pathway, while SbZIP9, SbSTP8, SbYS1 and SbMAG displayed positive correlations mutually. Notably, SbZIP9 and SbFOMT-like were highly expressed when compared with other target genes. Taken together, SbZIP9 and SbFOMT-like were upregulated and downregulated by an extra K supply under Cd stress, suggesting that SbZIP9 and SbFOMT-like enhances and declines Cd accumulation as regulated by K addition in sweet sorghum respectively.

Photosensitized Reductive Elimination of Gold(III) to Enable Esterification of Aryl Iodides with Carboxylic Acids.

Compared to the well-established transition metal-catalyzed cross-coupling reactions, Au(I)/Au(III)-catalyzed cross-coupling reactions have lagged behind. Despite some advancements, achieving gold-catalyzed C-O coupling with carboxylic acids via an Au(III) carboxylate intermediate remains challenging due to the thermal unfavorability of the critical reductive elimination step. Here, we present the first photosensitized reductive elimination of gold(III) to enable esterification of aryl iodides with carboxylic acids. In the presence of a (P, N)-gold(I) catalyst and a photosensitizer benzophenone under blue LED irradiation, esterification derivatives were obtained from aryl iodides with both aryl and alkyl (1°, 2°, 3°) carboxylic acids. Mechanistic and modeling studies support that energy transfer (EnT) from a photosensitizer produces an excited-state gold(III) complex that couples aryl iodides with carboxylic acids. This photoinduced energy-transfer strategy has been applied in several other photosensitized gold catalysis reactions, indicating its potential for further applications.

Rational Design of Non-Covalent Imprinted Polymers Based on the Combination of Molecular Dynamics Simulation and Quantum Mechanics Calculations.

Molecular imprinting is a promising approach for developing polymeric materials as artificial receptors. However, only a few types of molecularly imprinted polymers (MIPs) are commercially available, and most research on MIPS is still in the experimental phase. The significant limitation has been a challenge for screening imprinting systems, particularly for weak functional target molecules. Herein, a combined method of quantum mechanics (QM) computations and molecular dynamics (MD) simulations was employed to screen an appropriate 2,4-dichlorophenoxyacetic acid (2,4-D) imprinting system. QM calculations were performed using the Gaussian 09 software. MD simulations were conducted using the Gromacs2018.8 software suite. The QM computation results were consistent with those of the MD simulations. In the MD simulations, a realistic model of the 'actual' pre-polymerisation mixture was obtained by introducing numerous components in the simulations to thoroughly investigate all non-covalent interactions during imprinting. This study systematically examined MIP systems using computer simulations and established a theoretical prediction model for the affinity and selectivity of MIPs. The combined method of QM computations and MD simulations provides a robust foundation for the rational design of MIPs.

From onset to blindness: a comprehensive analysis of RPGR-associated X-linked retinopathy in a large cohort in China.

BACKGROUND: Variants in the RPGR are the leading cause of X-linked retinopathies (XLRPs). Further in-depth investigation is needed to understand the natural history. METHODS: Review of all case records, molecular genetic testing results, best-corrected visual acuity (BCVA), retinal imaging data (including fundus autofluorescence imaging and optical coherence tomography (OCT)), static visual field (VF) assessments and full-field electroretinogram. RESULTS: Genetic testing was conducted on 104 male patients from 89 family pedigrees, identifying 22 novel variants and 1 de novo variant. The initial symptoms appeared in 78.2% of patients at a median age of 5 years. BCVA declined at a mean rate of 0.02 (IQR, 0-0.04) logarithm of the minimum angle of resolution per year, with a gradual, non-linear decrease over the first 40 years. Autofluorescence imaging revealed macular atrophy at a median age of 36.1 (IQR, 29.9-43.2) years. Patients experienced blindness at a median age of 42.5 (IQR, 32.9-45.2) years according to WHO visual impairment categories. OCT analysis showed a mean ellipsoid zone narrowing rate of 23.3 (IQR, -1.04-22.29) µm/month, with an accelerated reduction in the first 40 years (p<0.01). The median age at which ERG no longer detected a waveform was 26.5 (IQR, 20.5-32.8) years. Comparison by variant location indicated faster progression in patients with exon 1-14 variants during the initial two decades, while those with ORF15 variants showed accelerated progression from the third decade. CONCLUSIONS: We provide a foundation for determining the treatment window and an objective basis for evaluating the therapeutic efficacy of gene therapy for XLRP.

A convenient broad-host counterselectable system endowing rapid genetic manipulations of Kluyveromyces lactis and other yeast species.

Being generally regarded as safe, Kluyveromyces lactis has been widely taken for food, feed, and pharmaceutical applications, owing to its ability to achieve high levels of protein secretion and hence being suitable for industrial production of heterologous proteins. Production platform strains can be created through genetic engineering; while prototrophic cells without chromosomally accumulated antibiotics resistance genes have been generally preferred, arising the need for dominant counterselection. We report here the establishment of a convenient counterselection system based on a Frs2 variant, Frs2v, which is a mutant of the alpha-subunit of phenylalanyl-tRNA synthase capable of preferentially incorporating a toxic analog of phenylalanine, r-chloro-phenylalanine (4-CP), into proteins to bring about cell growth inhibition. We demonstrated that expression of Frs2v from an episomal plasmid in K. lactis could make the host cells sensitive to 2 mM 4-CP, and a Frs2v-expressing plasmid could be efficiently removed from the cells immediately after a single round of cell culturing in a 4-CP-contianing YPD medium. This Frs2v-based counterselection helped us attain scarless gene replacement in K. lactis without any prior engineering of the host cells. More importantly, counterselection with this system was proven to be functionally efficient also in Saccharomyces cerevisiae and Komagataella phaffii, suggestive of a broader application scope of the system in various yeast hosts. Collectively, this work has developed a strategy to enable rapid, convenient, and high-efficiency construction of prototrophic strains of K. lactis and possibly many other yeast species, and provided an important reference for establishing similar methods in other industrially important eukaryotic microbes.

Etiology, pathology, and treatment of osteonecrosis of the femoral head in adolescents: A comprehensive review.

Femoral head necrosis is a common refractory disease in orthopedics, and shows a trend of getting younger. The occurrence of femoral head necrosis in adolescents is related to the use of glucocorticoids, autoimmune diseases, trauma, and other factors. Because adolescent patients are in the period of physical development, high activity requirements, and have fertility needs in the future, treatment is relatively difficult. Early artificial joint replacement may have problems such as wear and loosening, so total hip replacement is not the preferred treatment for adolescent patients with femoral head necrosis. This article will elaborate the research progress of femoral head necrosis in adolescents from 3 aspects, and summarize the benefits and side effects of core decompression combined with autologous stem cell transplantation in the treatment of early femoral head necrosis, so as to provide clinical ideas for the treatment of femoral head necrosis in adolescents.

Exogenous iron caused osteocyte apoptosis, increased RANKL production, and stimulated bone resorption through oxidative stress in a murine model.

Iron overload is a risk factor for osteoporosis due to its oxidative toxicity. Previous studies have demonstrated that an excessive amount of iron increases osteocyte apoptosis and receptor activator of nuclear factor κ-B ligand (RANKL) production, which stimulates osteoclast differentiation in vitro. However, the effects of exogenous iron supplementation-induced iron overload on osteocytes in vivo and its role in iron overload-induced bone loss are unknown. This work aimed to develop an iron overloaded murine model of C57BL/6 mice by intraperitoneal administration of iron dextran for two months. The iron levels in various organs, bone, and serum, as well as the microstructure and strength of bone, apoptosis of osteocytes, oxidative stress in bone tissue, and bone formation and resorption, were assessed. The results showed that 2 months of exogenous iron supplementation significantly increased iron levels in the liver, spleen, kidney, bone tissue, and serum. Iron overload negatively affected bone microstructure and strength. Osteocyte apoptosis and empty lacunae rate were elevated by exogenous iron. Iron overload upregulated RANKL expression but had no significant impact on osteoprotegerin (OPG) and sclerostin levels. Static and dynamic histologic analyses and serum biochemical assay showed that iron overload increased bone resorption without significantly affecting bone formation. Exogenous iron promoted oxidative stress in osteocytes in vivo and in vitro. Additional supplementation of iron chelator (deferoxamine) or N-acetyl-l-cysteine (NAC) partially alleviated bone loss, osteocyte apoptosis, osteoclast formation, and oxidative stress due to iron overload. These findings, in line with prior in vitro studies, suggest that exogenous iron supplementation induces osteoclastogenesis and osteoporosis by promoting osteocyte apoptosis and RANKL production via oxidative stress.

CRISPR/Cas12a-triggered ordered concatemeric DNA probes signal-on/off multifunctional analytical sensing system for ultrasensitive detection of thalassemia.

Based on CRISPR/Cas12a triggered ordered concatemeric DNA probes, a "on/off" self-powered biosensor is developed to achieve highly sensitive detection of thalassemia gene CD142 through open-circuit potential-assisted visual signal output. The ingeniously constructed glucose oxidase (GOD)-functionalized ordered concatemeric DNA probe structure can significantly amplify signal output, while the coupled CRISPR/Cas12a system is served as a "signal switch" with excellent signal-transducing capabilities. When the ordered concatemeric DNA probe structure is anchored on electrode, the response signal of the sensing system is in the "signal on" mode. While, the presence of the target activates the non-specific cleavage activity of the CRISPR/Cas12a system, causing the sensing system to switch to the "signal off" mode. In the detection system, GOD catalyzes the oxidation of glucose to produce hydrogen peroxide, which further catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to form a color product, enabling visual signal of the target through naked-eye color contrast. By employing a multifunctional analytical mode combining electrochemical and visual signal outputs, accurate determination of the target is achieved, with linear ranges of 0.0001-100 pM, and detection limits of 48.1 aM (S/N = 3). This work provides a reference method for sensitive detection of thalassemia genes and holds great diagnostic potential in biomedical applications.