Multiplex competitive annealing mediated isothermal amplification with high fidelity DNA polymerase (HiFi-CAMP).

Various isothermal amplification methods have been developed for point-of-care testing (POCT) of various infectious diseases. Here, we proposed a novel isothermal amplification method, named as 5'-half complementary primers mediated isothermal amplification (HCPA). Because of the similarity of our method to the previous method competitive annealing mediated isothermal amplification (CAMP) in primer design, we also use the name CAMP for our method. We demonstrated that CAMP is mediated by both a linear isothermal amplification pattern and a loop-mediated isothermal amplification pattern. To improve the specificity and enable multiplex detection, we further developed HiFi-CAMP method that uses a small amount of high-fidelity DNA polymerase to cut HFman probe to release fluorescent signal. The HiFi-CAMP method was demonstrated to have a good specificity and sensitivity, and fast amplification speed in detection of three human respiratory viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus A (RSV-A) and influenza A viruses (IAV). When compared with gold standard RT-qPCR assays, the HiFi-CAMP assays showed sensitivities of 90.0 %, 71.4 % and 78.1 %, specificities of 100 %, 100 % and 95.5 %, and consistencies of 93.0 %, 93.3 % and 88.2 % for SARS-CoV-2, RSV-A and IAV, respectively. Furthermore, a duplex HiFi-CAMP assay was also developed to simultaneously detect RSV-A and SARS-CoV-2. The HiFi-CAMP will provide a promising candidate for POCT diagnosis in resource-limited settings.

First report of Diaporthe tulliensis causing leaf spot on Millettia speciosa in China.

Millettia speciosa Champ, renowned for its diverse applications in traditional medicine, is extensively cultivated in the Guangxi region of China, spanning roughly 5,973 hectares. In July 2021, a plantation in Yulin, Guangxi, China (22°64'N; 110°29'E), exhibited severe leaf spot disease on M. speciosa. Notably, a 46,690 square meters area had over 40% leaf spot incidence. Initially, symptoms appeared as small, circular, pale-yellow lesions on the leaves, then turned into irregular, dark brown spots with yellow halos, leading to the wilt and defoliation of leaves. To identify the responsible pathogen, a total of five symptomatic leaves were collected and sterilized systematically. Small tissue segments (5×5 mm) from lesion peripheries were aseptically excised, then surface sterilized with 75% ethanol for 10 s, and 1% sodium hypochlorite (NaClO) for 3 min. Following this, the sterilized tissues were triple-rinsed with sterile water and cultured on potato dextrose agar (PDA) at 28 °C in the dark for 7 d. A total of seven isolates were obtained through single-spore isolation, and one representative isolate, N2-3, was selected for further analysis. After 7 d of incubation, colonies displayed flat, white, and extensively branched aerial hyphae. Over time, the reverse side of the colony changed from white to yellowish-white. The pycnidia were black with conidial droplets ranging from cream to pale yellow exuding from their ostioles. The α-conidia were one-celled, hyaline, ovoid to cylindrical, typically with one or two droplets, 2.6 to 5.9 ×1.4 to 3.9 µm (n=50). These morphological traits align with those of the genus Diaporthe, as reported by Li et al. (2022) and Crous et al. (2015). To identify the species, isolate N2-3 underwent sequencing of the internal transcribed spacer (ITS), ß-tubulin (BT), and translation elongation factor 1 alpha (EF1-α) sections (Huang et al. 2021). Obtained sequences of ITS, BT and EF1-α (Genebank accessions nos. OR600532, OR662169 and OR662168) displayed a 99% similarity to Diaporthe tulliensis (Genebank accessions nos. OP219651, ON932382, OL412437, respectively). Based on the concatenated ITS, BT and EF1-α, a neighbor-joining phylogenetic analyses using MEGA7.0 clustered with D. tulliensis. Therefore, the fungus was identified as D. tulliensis (teleomorph name) based on morphological and molecular features. A pathogenicity test was conducted on 1-year-old M. speciosa seedlings by gently abrading healthy leaves with sterilized toothpicks to create superficial wounds. Wounded leaves were then inoculated with 5 mm diameter mycelial plugs, while control seedlings received PDA plugs. Three leaves per plant and five plants per treatment were selected for assessment. All seedlings were kept in a controlled greenhouse (12/12h light/dark, 25 ± 2 °C, 90% humidity). After 7 d, the inoculated leaves showed symptoms like those in the field, while control plants remained healthy. The fungus was consistently reisolated from the infected leaves, satisfying Koch's postulates. Notably, D. tulliensis has caused Boston ivy leaf spot, bodhi tree leaf spot, cacao pod rot, and jasmine stem canker (Huang et al. 2021; Li et al. 2022; Serrato-Diaz et al. 2022; Hsu et al. 2023). This discovery is significant as it marks the first report of Diaporthe tulliensis causing leaf spot on Millettia speciossa in China, which has direct implications for the development of diagnostic tools and research into potential disease management strategies.

Metformin promotes the survival of random skin flaps via the activation of Nrf2/HO-1 signaling.

The random flap is one of the commonly used techniques for tissue defect repair in surgery and orthopaedics, however the risk of ischaemic necrosis at the distal end of the flap limits its size and clinical application. Metformin (Met) is a first-line medication in the treatment of type 2 diabetes, with additional effects such as anti-tumor, anti-aging, and neuroprotective properties. In this study, we aimed to investigate the biological effects and potential mechanisms of Met in improving the survival of random skin flaps. Twenty-four male Sprague-Dawley rats and 12 male C57BL/6J mice underwent McFarlane flap surgery and divided into control (Ctrl) and Met groups (100 mg/kg). The survival rate of the flap were evaluated on day 7. Angiography, Laser doppler blood flow imaging, and H&E staining were used to assess blood flow supply and the levels of microvascular density. Then, reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured by test kits. Immunohistochemistry analysis was conducted to evaluate the expression of Vascular Endothelial Growth Factor A (VEGFA), Vascular endothelial cadherin (VE-cadherin) and CD31. Rats and mice in the Met group exhibited higher flap survival rate, microcirculatory flow, and higher expression levels of VEGFA and VE-cadherin compared with the Ctrl group. In addition, the level of oxidative stress was significantly lower in the met group. And then we demonstrated that the human umbilical vein endothelial cells (HUVECs) treated with Met can alleviate tert-butyl hydroperoxide (TBHP)-stimulated cellular dysfunction and oxidative stress injury. Mechanistically, Met markedly stimulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), and promoted Nrf2 nuclear translocation. Silencing of Nrf2 partially abolished the antioxidant and therapeutic effects of Met. In summary, our data have confirmed that Met has a positive effect on flap survival and reduces necrosis. The mechanism of action involves the regulation of the Nrf2/HO-1 signaling pathway to combat oxidative stress and reduce damage.

Identification, fermentation optimization, and biocontrol efficacy of actinomycete YG-5 for the prevention of Alternaria leaf spot disease in star anise.

Star anise (Illicium verum), a valuable spice tree, faces significant threats from fungal diseases, particularly Alternaria leaf spot. This study investigates the potential of a soil-derived actinomycete strain, YG-5, as a biocontrol agent against Alternaria tenuissima, the causative pathogen on Alternaria leaf spot in star anise. Through comprehensive morphology, physiology, biochemistry, and genetic analyses, we identified the isolate as Streptomyces sp. YG-5. The strain exhibited broad-spectrum antimicrobial activity against several plant pathogens, with inhibition rates ranging between 36.47 to 80.34%. We systematically optimized the fermentation conditions for YG-5, including medium composition and cultivation parameters. The optimized process resulted in an 89.56% inhibition rate against A. tenuissima, a 14.72% improvement over non-optimized conditions. Notably, the antimicrobial compounds produced by YG-5 demonstrated stability across various temperatures, pH levels, and UV irradiation. In vivo efficacy trials showed promising results, with YG-5 fermentation broth reducing Alternaria leaf spot incidence on star anise leaves by 56.95%. These findings suggest that Streptomyces sp. YG-5 holds significant potential as a biocontrol agent against Alternaria leaf spot in star anise cultivation, offering a sustainable approach to disease management in this valuable crop.

Embedded periodically ABHs and distributed DVAs for passive low-frequency broadband vibration attenuation in thin-walled structures.

The acoustic black hole (ABH) structure exhibits remarkable energy focalization above a given cut-on frequency, offering potential for broadband vibration suppression in structures. However, its energy focusing properties diminish significantly below this cut-on frequency. Therefore, it is crucial to enhance the vibration attenuation capabilities of ABH structures within the low frequency range. This study presents a numerical investigation into the impact of thin-walled structures with embedded ABHs and distributed dynamic vibration absorbers (DVAs) on low frequency broadband vibration reduction. Initially, the focusing characteristics of the ABH thin-walled structure is analyzed, aiding in the attached position of DVAs. Furthermore, the influence of the design parameters and attached position of DVA on the broadband damping effect of the structure is explored. The findings indicate that DVAs designed for low frequencies can achieve significant vibration attenuation across the entire frequency spectrum, including low frequencies, when installed at specific focusing positions. When compared to the position with the maximum vibration response, while the attenuation of the low frequency common amplitude value is slightly reduced, greater vibration attenuation across the entire frequency band is achieved. This research offers valuable insights into optimizing the integration of DVAs with ABHs in thin-walled structures for enhanced broadband vibration attenuation.

[Genetic diversity of SP1 population of space mutagenized Pueraria montana].

To explore the mutagenic effect of the space environment on Pueraria montana and select the elite germplasm with good growth conditions and high isoflavone content, this study observed the agronomic traits, determined the flower isoflavone content, and labeled amplified fragment length polymorphism(AFLP) fluorescent molecular markers of 79 P. montana plants exposed to space mutagenesis(SP1 group) and 10 control plants of P. montana(CK group). Excel 2019, SPSS 25.0, NTSYSpc-2.11F, and Popgen 32 were employed to analyze the genetic diversity and perform the cluster analysis. The results showed that the SP1 group presented changed leaf hairy attitude and flower structure and higher CV and H' of quantitative traits than the CK group. The cluster analysis screened out five plants in the SP1 group. Ten P. montana plants in the SP1 group had higher content of 6″-O-xylosyl-tectoridin and tectoridin in the flowers than the control group, with the total content of both exceeding 11%. After clustering, 9 plants in the SP1 group were separated. Nine pairs of polymorphic primers were screened out frrom 64 pairs of primers. A total of 1 620 polymorphic loci were detected, with the average percentage of polymorphic loci(PPL) of 83.33%. The average Nei's gene diversity index(H) and Shannon's information index(I) were 0.192 2 and 0.305 2, respectively. After clustering, 4 plants in the SP1 group were screened out. According to the above results, plants No. 30, No. 66, and No. 89 in the SP1 group were subjected to greater mutagenic effect by the space environment and presented better growth and higher flower isoflavone content. Moreover, plant No. 30 showed the flower structure variation and flower weight two times of that in the CK group. These plants can be used as key materials for the subsequent experiments.

Hippocampal HDAC5-mediated histone acetylation underlies stress susceptibility in mice exposed to chronic social defeat stress.

Chronic stress leads to social avoidance and anhedonia in susceptible individuals, a phenomenon that has been observed in both human and animal models. Nevertheless, the underlying molecular mechanisms underpinning stress susceptibility and resilience remain largely unclear. There is growing evidence that epigenetic histone deacetylase (HDAC) mediated histone acetylation is involved in the modulation of depressive-related behaviors. We hypothesized that histone deacetylase 5 (HDAC5), which is associated with stress-related behaviors and antidepressant response, may play a vital role in the susceptibility to chronic stress. In the current study, we detected the levels of HDAC5 and acetylation of histone 4 (H4) in the hippocampus subsequent to chronic social defeat stress (CSDS) in C57BL/6J mice. We found that CSDS induces a notable increase in HDAC5 expression, concomitant with a reduction in the acetylation of histone H4 at lysine 12 (H4K12) in the hippocampus of susceptible mice. Meanwhile, intrahippocampal infusion of HDAC5 shRNA or HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) both reversed the depression susceptibility in susceptible mice that subjected to CSDS. Furthermore, HDAC5 overexpression was sufficient to induce depression susceptibility following microdefeat stress, accompanied by a significant reduction in H4K12 level within the hippocampus of mice. Additionally, the Morris water maze (MWM) results indicated that neither CSDS nor HDAC5 exerted significant effects on spatial memory function in mice. Taken together, these investigations indicated that HDAC5-modulated histone acetylation is implicated in regulating the depression susceptibility, and may be serve as potential preventive targets for susceptible individuals.

[Preparation and intestinal absorption mechanism of self-assembled nanoparticles of Herpetospermum caudigerum].

In this experiment, the micro-precipitation method was used to prepare self-assembled nanoparticles of Herpetospermum caudigerum Wall.(MP-SAN). The process was optimized using average particle size and polydispersity index(PDI)as evaluation indexes. The mean particle size, PDI,zeta potential, and microstructure of MP-SAN were characterized. The intestinal absorption mechanism of dehydrodiconiferyl alcohol(DA)and herpetrione(Her)in MP-SAN was investigated through single-pass intestinal perfusion in rats. The optimized process parameters for producing MP-SAN were a stirring speed of 800 r·min~(-1),stirring time of 5 min, and rotary evaporation temperature of 40℃. The resulting MP-SAN exhibited a spherical-like structure and uniform morphology, with a mean particle size of(267.63±13.27) nm, a PDI of 0.062 0±0.043 9,and a zeta potential of(-46.18±3.66) mV. The absorption rate constant(K_a)and apparent permeability coefficient(P_(app))of DA in the ileal segment were significantly higher than those in the jejunal segment(P<0.05). However, there was no significant difference in the absorption of Her between the ileal and jejunal segments. Intestinal absorption parameters of DA and Her tended to increase with increasing drug concentration. Specifically, the K_a and P_(app) of DA in MP-SAN in the high-concentration group were significantly higher than those in the low-concentration group(P<0.01). The addition of verapamil, a P-glycoprotein inhibitor, did not significantly affect the intestinal absorption of DA and Her. However, the absorption of both DA and Her in MP-SAN was significantly increased by the addition of indomethacin(P<0.05),suggesting that DA and Her may be substrates for multidrug resistance-associated protein 2.

Intraspecific Differentiation of Styrax japonicus (Styracaceae) as Revealed by Comparative Chloroplast and Evolutionary Analyses.

Styrax japonicus is a medicinal and ornamental shrub belonging to the Styracaceae family. To explore the diversity and characteristics of the chloroplast genome of S. japonicus, we conducted sequencing and comparison of the chloroplast genomes of four naturally distributed S. japonicus. The results demonstrated that the four chloroplast genomes (157,914-157,962 bp) exhibited a typical quadripartite structure consisting of a large single copy (LSC) region, a small single copy (SSC) region, and a pair of reverse repeats (IRa and IRb), and the structure was highly conserved. DNA polymorphism analysis revealed that three coding genes (infA, psbK, and rpl33) and five intergene regions (petA-psbJ, trnC-petN, trnD-trnY, trnE-trnT, and trnY-trnE) were identified as mutation hotspots. These genetic fragments have the potential to be utilized as DNA barcodes for future identification purposes. When comparing the boundary genes, a small contraction was observed in the IR region of four S. japonicus. Selection pressure analysis indicated positive selection for ycf1 and ndhD. These findings collectively suggest the adaptive evolution of S. japonicus. The phylogenetic structure revealed conflicting relationships among several S. japonicus, indicating divergent evolutionary paths within this species. Our study concludes by uncovering the genetic traits of the chloroplast genome in the differentiation of S. japonicus variety, offering fresh perspectives on the evolutionary lineage of this species.

A Monte Carlo simulation-based health risk assessment of heavy metals in soils of the tropical region in southern China.

The disturbance of ecological stability may take place in tropical regions due to the elevated biomass density resulting from heavy metal and other contaminant pollution. In this study, 62 valid soil samples were collected from Sanya. Source analysis of heavy metals in the area was carried out using absolute principal component-multiple linear regression receptor modelling (APCS-MLR); the comprehensive ecological risk of the study area was assessed based on pollution sources; the Monte-Carlo model was used to accurately predict the health risk of pollution sources in the study area. The results showed that: The average contents of soil heavy metals Cu, Ni and Cd in Sanya were 5.53, 6.56 and 11.66 times higher than the background values of heavy metals. The results of soil geo-accumulation index (Igeo) showed that Cr, Mo, Mn and Zn were unpolluted to moderately polluted, Cu and Ni were moderately polluted, and Cd was moderately polluted to strongly polluted. The main sources of heavy metal pollution were natural sources (57.99%), agricultural sources (38.44%) and traffic sources (3.57%). Natural and agricultural sources were jointly identified as priority control pollution sources and Cd was the priority control pollution element for soil ecological risk. Heavy metal content in Sanya did not pose a non-carcinogenic risk to the population, but there was a carcinogenic risk to children. The element Zn had a high carcinogenic risk to children, and was a priority controlling pollutant element for the risk of human health, with agricultural sources as the priority controlling pollutant source.

A CD36-dependent non-canonical lipid metabolism program promotes immune escape and resistance to hypomethylating agent therapy in AML.

Environmental lipids are essential for fueling tumor energetics, but whether these exogenous lipids transported into cancer cells facilitate immune escape remains unclear. Here, we find that CD36, a transporter for exogenous lipids, promotes acute myeloid leukemia (AML) immune evasion. We show that, separately from its established role in lipid oxidation, CD36 on AML cells senses oxidized low-density lipoprotein (OxLDL) to prime the TLR4-LYN-MYD88-nuclear factor κB (NF-κB) pathway, and exogenous palmitate transfer via CD36 further potentiates this innate immune pathway by supporting ZDHHC6-mediated MYD88 palmitoylation. Subsequently, NF-κB drives the expression of immunosuppressive genes that inhibit anti-tumor T cell responses. Notably, high-fat-diet or hypomethylating agent decitabine treatment boosts the immunosuppressive potential of AML cells by hijacking CD36-dependent innate immune signaling, leading to a dampened therapeutic effect. This work is of translational interest because lipid restriction by US Food and Drug Administration (FDA)-approved lipid-lowering statin drugs improves the efficacy of decitabine therapy by weakening leukemic CD36-mediated immunosuppression.

Association of Human Whole-blood NAD + Levels with Nabothian Cyst.

Objective: Little is known about the association between whole-blood nicotinamide adenine dinucleotide (NAD +) levels and nabothian cysts. This study aimed to assess the association between NAD + levels and nabothian cysts in healthy Chinese women. Methods: Multivariate logistic regression analysis was performed to analyze the association between NAD + levels and nabothian cysts. Results: The mean age was 43.0 ± 11.5 years, and the mean level of NAD + was 31.3 ± 5.3 µmol/L. Nabothian cysts occurred in 184 (27.7%) participants, with single and multiple cysts in 100 (15.0%) and 84 (12.6%) participants, respectively. The total nabothian cyst prevalence gradually decreased from 37.4% to 21.6% from Q1 to Q4 of NAD + and the prevalence of single and multiple nabothian cysts also decreased across the NAD + quartiles. As compared with the highest NAD + quartile (≥ 34.4 µmol/L), the adjusted odds ratios with 95% confidence interval of the NAD + Q1 was 1.89 (1.14-3.14) for total nabothian cysts. The risk of total and single nabothian cysts linearly decreased with increasing NAD + levels, while the risk of multiple nabothian cysts decreased more rapidly at NAD + levels of 28.0 to 35.0 µmol/L. Conclusion: Low NAD + levels were associated with an increased risk of total and multiple nabothian cysts.

First report of Corynespora cassiicola causing leaf spot on Nanhaia speciosa in China.

Nanhaia speciosa, commonly known as Niudali, is a medicinal woody vine belonging to the Leguminosae family. Valued for its culinary and medicinal properties, it is extensively cultivated, covering approximately 5,973 hm2 in the Guangxi Zhuang Autonomous Region of China. The edible tubers of this plant are reported to possess antibacterial and antioxidant effects (Luo et al., 2023; Shu et al., 2020). In July 2021, a Niudali plantation in Yulin, Guangxi, China (22°64'N; 110°29'E) exhibited leaf spot symptoms, with an incidence rate exceeding 40% across a 46,690 m2 area. Initially, small circular, pale yellow spots appeared on the leaves, which subsequently evolved into dark brown lesions surrounded by yellow halos, ultimately leading to foliage wilting. Leaves exhibiting typical symptoms were collected for pathogen investigation. The leaves were thoroughly washed with sterile water and small tissue fragments (5×5 mm) were excised from the lesion periphery. These fragments were surface-sterilized with 75% ethanol and 1% NaClO, rinsed three times with sterile water, and subsequently cultured on potato dextrose agar (PDA) at 28 °C in darkness for 7 days. Through single-spore isolation, seven isolates with similar morphological traits were obtained. After 7 days of incubation on PDA at 28 °C in dark, the colonies exhibited a white to grey coloration on the upper surface with abundant aerial hyphae, while the underside appeared dark black. The conidia, cylindrical or obclavate in shape, were straight, pale brown, and measured 30.1-128.9 µm × 4.8-15.0 µm (n=50). The morphological characteristics matched those of Corynespora sp.(Wang et al. 2021). For molecular identification, the isolate N5-2 underwent DNA sequence analysis using genomic DNA and primers ITS1/ITS4 and EF1-688F/EF1-1251R. The sequences (ITS: OP550425; TEF1-α: OQ117118) were deposited in GenBank, exhibiting 98% identity to C. cassiicola (OP981637) for TEF1-α and 99% homology to C. cassiicola (OP957070) for ITS. Based on the concatenated ITS and TEF1-α, a maximum likelihood phylogenetic analyses using MEGA7.0 clustered the isolate with C. cassiicola. Consequently, the fungus was identified as C. cassiicola based on its morphological and molecular features. In the pathogenicity test on 1-year-old Nanhaia speciosa seedlings, leaves were gently scratched and inoculated with mycelial plugs (5 mm). Control seedlings received PDA plugs. Five leaves per plant and five plants per treatment were selected for assessment. All seedling were maintained in a greenhouse (12/12h light/dark cycle, 25 ± 2°C, 90% humidity). After a 7-day incubation period, all leaves subjected to fungal inoculation exhibited symptoms consistent with those observed in the field, while control plants remained symptom-free. The fungus was successfully reisolated from the infected leaves in three successive trials, fulfilling Koch's postulates. While C. cassiicola is well-documented for inducing leaf spots on various plant species, including Jasminum nudiflorum, Strobilanthes cusia, Acanthus ilicifolius, Syringa species (Hu et al., 2023; Liu et al., 2023; Xie et al., 2021; Wang et al., 2021), this study represents the first report of C. cassiicola causing leaf spots on Nanhaia speciosa in China. The identification of this pathogen in Nanhaia speciosa has significant implications for future epidemiological investigations and serves as a valuable reference for controlling leaf spot disease in Nanhaia speciosa.

Impact of Various Ration Energy Levels on the Slaughtering Performance, Carcass Characteristics, and Meat Qualities of Honghe Yellow Cattle.

Consumers are increasing their daily demand for beef and are becoming more discerning about its nutritional quality and flavor. The present objective was to evaluate how the ration energy content (combined net energy, Nemf) impacts the slaughter performance, carcass characteristics, and meat qualities of Honghe yellow cattle raised in confinement. Fifteen male Honghe yellow cattle were divided into three groups based on a one-way design: a low-energy group (LEG, 3.72 MJ/kg), a medium-energy group (MEG, 4.52 MJ/kg), and a high-energy group (HEG, 5.32 MJ/kg). After a period of 70 days on these treatments, the animals were slaughtered and their slaughter performance was determined, and the longissimus dorsi muscle (LD) and biceps femoris (BF) muscles were gathered to evaluate meat quality and composition. Increasing the dietary energy concentration led to marked improvements (p < 0.05) in the live weight before slaughter (LWBS), weight of carcass, backfat thickness, and loin muscle area. HEG also improved the yield of high-grade commercial cuts (13.47% vs. 10.39%) (p < 0.05). However, meat quality traits were not affected by treatment except for shear force, which was affected by dietary energy. A significant improvement (p < 0.05) in the intramuscular fat (IMF) content was observed in the HEG. Little effect on the amino acid profile was observed (p > 0.05), except for a tendency (p = 0.06) to increase the histidine concentration in the BF muscle. Increasing dietary energy also reduced C22:6n-3 and saturated fatty acids (SFAs) and enhanced C18:1 cis-9 and monounsaturated fatty acids (MUFAs, p < 0.05). Those results revealed that increasing energy levels of diets could enhance slaughter traits and affect the meat quality and fatty acid composition of different muscle tissues of Honghe yellow cattle. These results contribute to the theoretical foundation to formulate nutritional standards and design feed formulas for the Honghe yellow cattle.

GASZ is indispensable for gametogenesis in the silkworm, Bombyx mori.

The prominent role of the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway in animals is to silence transposable elements and maintain genome stability, ensuring proper gametogenesis in gonads. GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) is an evolutionarily conserved protein located on the outer mitochondrial membrane of germ cells and plays vital roles in the piRNA pathway and spermatogenesis in mammals. In the model insect Drosophila melanogaster, GASZ is essential for piRNA biogenesis and oogenesis, whereas its biological functions in non-drosophilid insects are still unknown. Here, we describe a comprehensive investigation of GASZ functions in the silkworm, Bombyx mori, a lepidopteran model insect, by using a binary transgenic CRISPR/Cas9 system. The BmGASZ mutation did not affect growth and development, but led to sterility in both males and females. Eupyrene sperm bundles of mutant males exhibited developmental defects, while the apyrene sperm bundles were normal, which were further confirmed through double copulation experiments with sex-lethal mutants, which males possess functional eupyrene sperm and abnormal apyrene sperm. In female mutant moths, ovarioles were severely degenerated and the eggs in ovarioles were deformed compared with that of wild type (WT). Further RNA-seq and RT-qPCR analysis revealed that amounts of piRNAs and transposon expression were dysregulated in gonads of mutants. In summary, this study has demonstrated vital roles of BmGASZ in gametogenesis through regulating the piRNA pathway in B. mori.

Correlation analysis of phosphorylation of myofibrillar protein and muscle quality of tilapia during storage in ice.

In this study, the correlation between protein phosphorylation and deterioration in the quality of tilapia during storage in ice was examined by assessing changes in texture, water-holding capacity (WHC), and biochemical characteristics of myofibrillar protein throughout 7 days of storage. The hardness significantly decreased from 471.50 to 252.17 g, whereas cooking and drip losses significantly increased from 26.5% to 32.6% and 2.9% to 9.1%, respectively (P < 0.05). Myofibril fragmentation increased, while myofibrillar protein sulfhydryl content and Ca2+-ATPase activity decreased from 119.33 to 89.29 µmol/g prot and 0.85 to 0.46 µmolPi/mg prot/h, respectively (P < 0.05). Correlation analysis revealed that the myofibrillar protein phosphorylation level was positively correlated with hardness and Ca2+-ATPase activity but negatively correlated with WHC. Myofibrillar protein phosphorylation affects muscle contraction by influencing the dissociation of actomyosin, thereby regulating hardness and WHC. This study provides novel insights for the establishment of quality control strategies for tilapia storage based on protein phosphorylation.

The mechanoreceptor Piezo is required for spermatogenesis in Bombyx mori.

BACKGROUND: The animal sperm shows high diversity in morphology, components, and motility. In the lepidopteran model insect, the silkworm Bombyx mori, two types of sperm, including nucleate fertile eupyrene sperm and anucleate unfertile apyrene sperm, are generated. Apyrene sperm assists fertilization by facilitating the migration of eupyrene spermatozoa from the bursa copulatrix to the spermatheca. During spermatogenesis, eupyrene sperm bundles extrude the cytoplasm by peristaltic squeezing, while the nuclei of the apyrene sperm bundles are discarded with the same process, forming matured sperm. RESULTS: In this study, we describe that a mechanoreceptor BmPiezo, the sole Piezo ortholog in B. mori, plays key roles in larval feeding behavior and, more importantly, is essential for eupyrene spermatogenesis and male fertility. CRISPR/Cas9-mediated loss of BmPiezo function decreases larval appetite and subsequent body size and weight. Immunofluorescence analyses reveal that BmPiezo is intensely localized in the inflatable point of eupyrene sperm bundle induced by peristaltic squeezing. BmPiezo is also enriched in the middle region of apyrene sperm bundle before peristaltic squeezing. Cytological analyses of dimorphic sperm reveal developmental arrest of eupyrene sperm bundles in BmPiezo mutants, while the apyrene spermatogenesis is not affected. RNA-seq analysis and q-RT-PCR analyses demonstrate that eupyrene spermatogenic arrest is associated with the dysregulation of the actin cytoskeleton. Moreover, we show that the deformed eupyrene sperm bundles fail to migrate from the testes, resulting in male infertility due to the absence of eupyrene sperm in the bursa copulatrix and spermatheca. CONCLUSIONS: In conclusion, our studies thus uncover a new role for Piezo in regulating spermatogenesis and male fertility in insects.

Custom-designed, mass silk production in genetically engineered silkworms.

Genetically engineered silkworms have been widely used to obtain silk with modified characteristics especially by introducing spider silk genes. However, these attempts are still challenging due to limitations in transformation strategies and difficulties in integration of the large DNA fragments. Here, we describe three different transformation strategies in genetically engineered silkworms, including transcription-activator-like effector nuclease (TALEN)-mediated fibroin light chain (FibL) fusion (BmFibL-F), TALEN-mediated FibH replacement (BmFibH-R), and transposon-mediated genetic transformation with the silk gland-specific fibroin heavy chain (FibH) promoter (BmFibH-T). As the result, the yields of exogenous silk proteins, a 160 kDa major ampullate spidroin 2 (MaSp2) from the orb-weaving spider Nephila clavipes and a 226 kDa fibroin heavy chain protein (EvFibH) from the bagworm Eumeta variegate, reach 51.02 and 64.13% in BmFibH-R transformed cocoon shells, respectively. Moreover, the presence of MaSp2 or EvFibH significantly enhances the toughness of genetically engineered silk fibers by ∼86% in BmFibH-T and ∼80% in BmFibH-R silkworms, respectively. Structural analysis reveals a substantial ∼40% increase in fiber crystallinity, primarily attributed to the presence of unique polyalanines in the repetitive sequences of MaSp2 or EvFibH. In addition, RNA-seq analysis reveals that BmFibH-R system only causes minor impact on the expression of endogenous genes. Our study thus provides insights into developing custom-designed silk production using the genetically engineered silkworm as the bioreactor.

Structural insights into the orthosteric inhibition of P2X receptors by non-ATP analog antagonists.

P2X receptors are extracellular ATP-gated ion channels that form homo- or heterotrimers and consist of seven subtypes. They are expressed in various tissues, including neuronal and nonneuronal cells, and play critical roles in physiological processes such as neurotransmission, inflammation, pain, and cancer. As a result, P2X receptors have attracted considerable interest as drug targets, and various competitive inhibitors have been developed. However, although several P2X receptor structures from different subtypes have been reported, the limited structural information of P2X receptors in complex with competitive antagonists hampers the understanding of orthosteric inhibition, hindering the further design and optimization of those antagonists for drug discovery. We determined the cryogenic electron microscopy (cryo-EM) structures of the mammalian P2X7 receptor in complex with two classical competitive antagonists of pyridoxal-5'-phosphate derivatives, pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) and pyridoxal phosphate-6-azophenyl-2',5'-disulfonic acid (PPADS), and performed structure-based mutational analysis by patch-clamp recording as well as molecular dynamics (MD) simulations. Our structures revealed the orthosteric site for PPADS/PPNDS, and structural comparison with the previously reported apo- and ATP-bound structures showed how PPADS/PPNDS binding inhibits the conformational changes associated with channel activation. In addition, structure-based mutational analysis identified key residues involved in the PPNDS sensitivity of P2X1 and P2X3, which are known to have higher affinity for PPADS/PPNDS than other P2X subtypes.

Protective effects of sodium butyrate on fluorosis in rats by regulating bone homeostasis and serum metabolism.

Fluorosis due to high fluoride levels in drinking water profoundly affects the development of human skeletal and dental structures. Sodium butyrate (NaB) has been found to regulate overall bone mass and prevent pathological bone loss. However, the mechanism of NaB action on fluorosis remains unclear. In this study, a rat model of fluorosis induced by 100 mg/L sodium fluoride was used to investigate the impact of NaB on bone homeostasis and serum metabolomics. It was found that NaB significantly reduced the levels of bone resorption markers CTX-Ⅰ and TRACP-5B in fluorosis rats. Moreover, NaB increased calcium and magnesium levels in bone, while decreasing phosphorus levels. In addition, NaB improved various bone microstructure parameters, including bone mineral density (BMD), trabecular thickness (Tb. Th), trabecular bone separation (Tb. SP), and structural model index (SMI) in the femur. Notably, NaB intervention also enhanced the antioxidant capacity of plasma in fluorosis rats. Furthermore, a comprehensive analysis of serum metabolomics by LC-MS revealed a significant reversal trend of seven biomarkers after the intervention of NaB. Finally, pathway enrichment analysis based on differential metabolites indicated that NaB exerted protective effects on fluorosis by modulating arginine and proline metabolic pathways. These findings suggest that NaB has a beneficial effect on fluorosis and can regulate bone homeostasis by ameliorating metabolic disorders.