SKF96365 Inhibits Tumor Proliferation by Inducing Apoptosis and Autophagy in Human Esophageal Squamous Cell Carcinoma.

Calcium channel blockers are emerging as a new generation of attractive anticancer drugs. SKF96365, originally thought to be a store-operated calcium entry (SOCE) inhibitor, is now often used as a TRPC channel blocker and is widely used in medical diagnostics. SKF96365 has shown antitumor effects on a variety of cancer cell lines. The objective of this study was to investigate the anticancer effect of SKF96365 on esophageal cancer in vivo and in vitro. Cell Counting Kit-8 (CCK-8) and colony formation were used to test the proliferation inhibition of SKF96365 on cell lines. Western blot and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to detect cell apoptosis rates. In addition, we demonstrated the antitumor effect of SKF96365 in vivo in xenografted mice. As a result, SKF96365 significantly inhibited the proliferation of K510, K30, and EC9706 in vitro. SKF96365 induces apoptosis in three cell lines through the poly(adenosine diphosphate-ribose) polymerase (PARP), caspase-9, and BCL-2 pathways in a dose-dependent and time-dependent manner. Moreover, SKF96365 treatment also induced apoptosis and inhibited tumor growth in nude mice. The calcium channel TRPC1 was significantly downregulated by SKF96365. Autophagy was also induced during the treatment of SKF96365. In summary, SKF96365 induces apoptosis (PARP, caspase-9, and BCL-2) and autophagy (LC3-A/B) by inhibiting TRPC1 in esophageal cancer cells, thereby inhibiting tumor growth.

Insights into response of seagrass (Zostera marina) to sulfide exposure at morphological, physiochemical and molecular levels in context of coastal eutrophication and warming.

Sulfide in sediment porewaters, is toxic to rooted macrophytes in both marine and freshwater environments. Current research on sulfide stress in seagrasses primarily focuses on morphological and physiological aspects, with little known about the molecular response and resistance mechanisms. This study first investigated the damage caused by sulfide to eelgrass (Zostera marina L.) using transcriptomic, metabolomic, and other physiological and biochemical indicators and explored the potential resistance of eelgrass at molecular level through laboratory simulated and in-situ sulfide stress experiments. Comprehensive results showed that sulfide stress severely inhibited the growth, photosynthesis, and antioxidant enzyme activities of eelgrass. Importantly, transcriptome analysis revealed significant activation of pathways related to carbohydrate and sulfur metabolism. This activation served a dual purpose: providing an energy source for eelgrass stress response and achieving detoxification through accelerated sulfur metabolism-a potential resistance mechanism. The toxicity of sulfide increased with rising temperature as evidenced by a decrease in EC50. Results from recovery experiments indicated that when Fv/Fm reduced to about 0 under sulfide stress, the growth and photosynthesis of eelgrass recovered to normal level after timely removal of sulfide. However, prolonged exposure to sulfide resulted in failure to recover, leading ultimately to plant death. This study not only enhances our understanding of the molecular-level impacts of sulfide on seagrasses but also provides guidance for the management and ecological restoration of seagrass meadows under sulfide stress.

CFAP58 is involved in the sperm head shaping and flagellogenesis of cattle and mice.

CFAP58 is a testis-enriched gene that plays an important role in the sperm flagellogenesis of humans and mice. However, the effect of CFAP58 on bull semen quality and the underlying molecular mechanisms involved in spermatogenesis remain unknown. Here, we identified two single-nucleotide polymorphisms (rs110610797, A>G and rs133760846, G>T) and one indel (g.-1811_ g.-1810 ins147bp) in the promoter of CFAP58 that were significantly associated with semen quality of bulls, including sperm deformity rate and ejaculate volume. Moreover, by generating gene knockout mice, we found for the first time that the loss of Cfap58 not only causes severe defects in the sperm tail, but also affects the manchette structure, resulting in abnormal sperm head shaping. Cfap58 deficiency causes an increase in spermatozoa apoptosis. Further experiments confirmed that CFAP58 interacts with IFT88 and CCDC42. Moreover, it may be a transported cargo protein that plays a role in stabilizing other cargo proteins, such as CCDC42, in the intra-manchette transport/intra-flagellar transport pathway. Collectively, our findings reveal that CFAP58 is required for spermatogenesis and provide genetic markers for evaluating semen quality in cattle.

The Construction of the Self-Induced Sal System and Its Application in Salicylic Acid Production.

The design and construction of more complex and delicate genetic control circuits suffer from poor orthogonality in quorum sensing (QS) systems. The Sal system, which relies on salicylic acid as a signaling molecule, is an artificially engineered regulatory system with a structure that differs significantly from that of natural QS signaling molecules. Salicylic acid is an important drug precursor, mainly used in the production of drugs such as aspirin and anti-HIV drugs. However, there have been no reports on the construction of a self-induced Sal system in single cells. In this study, a high-copy plasmid backbone was used to construct the regulatory proteins and a self-induced promoter of salicylic acid in E. coli by adjusting the precise regulation of key gene expression; the sensitivity and induction range of this system were improved. Subsequently, the exogenous gene pchBA was introduced in E. coli to extend the shikimate pathway and synthesize salicylic acid, resulting in the construction of the first complete self-induced Sal system. Finally, the self-induced Sal System was combined with artificial trans-encoded sRNAs (atsRNAs) to repress the growth-essential gene ppc and accumulate the precursor substance PEP, thereby increasing the titer of salicylic acid by 151%. This construction of a self-induced artificial system introduces a new tool for selecting communication tools and induction systems in synthetic biology and metabolic engineering, but also demonstrates a self-inducible pathway design strategy for salicylic acid biosynthesis.

Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform.

In Phi29-α-hemolysin (α-HL) nanopore sequencing systems, a strong electrochemical signal is dependent on a high concentration of salt. However, high salt concentrations adversely affect polymerase activity. Sequencing by synthesis (SBS) requires the use of phi29 polymerase without exonuclease activity to prevent the degradation of modified nucleotide tags; however, the lack of exonuclease activity also affects polymerase processivity. This study aimed to optimize phi29 polymerase for improved salt tolerance and processivity while maintaining its lack of exonuclease activity to meet the requirements of nanopore sequencing. Using salt tolerance compartmentalized self-replication (stCSR) and a microfluidic platform, we obtained 11 mutant sites with enhanced salt tolerance attributes. Sequencing and biochemical analyses revealed that the substitution of conserved amino acids such as G197D, Y369E, T372N, and I378R plays a critical role in maintaining the processivity of exonuclease-deficient phi29 polymerase under high salt conditions. Furthermore, Y369E and T372N have been identified as important determinants of DNA polymerase binding affinity. This study provides insights into optimizing polymerase processability under high-salt conditions for real-time polymerase nanopore sequencing, paving the way for improved performance and applications in nanopore sequencing technologies.

Identifying mitophagy-related genes as prognostic biomarkers and therapeutic targets of gastric carcinoma by integrated analysis of single-cell and bulk-RNA sequencing data.

Gastric carcinoma (GC) is the fourth leading cause of cancer-related mortality worldwide. Patients with advanced GC tend to have poor prognoses and shortened survival. Finding novel predictive biomarkers for GC prognosis is an urgent need. Mitophagy is the selection degradation of damaged mitochondria to maintain cellular homeostasis, which has been shown to play both pro- and anti-tumor effects. This study combined single-cell sequencing data and transcriptomics to screen mitophagy-related genes (MRGs) associated with GC progression and analyze their clinical values. Reverse transcription-quantitative PCR (RT-qPCR) and immunochemistry (IHC) further verified gene expression profiles. A total of 18 DE-MRGs were identified after taking an intersection of single-cell sequencing data and MRGs. Cells with a higher MRG score were mainly distributed in the epithelial cell cluster. Cell-to-cell communications among epithelial cells with other cell types were significantly upregulated. We established and validated a reliable nomogram model based on DE-MRGs (GABARAPL2 and CDC37) and traditional clinicopathological parameters. GABARAPL2 and CDC37 displayed different immune infiltration states. Given the significant correlation between hub genes and immune checkpoints, targeting MRGs in GC may supplement more benefits to patients who received immunotherapy. In conclusion, GABARAPL2 and CDC37 may be prognostic biomarkers and candidate therapeutic targets of GC.

Coordinated alternation of DNA methylation and alternative splicing of PBRM1 affect bovine sperm structure and motility.

DNA methylation and gene alternative splicing drive spermatogenesis. In screening DNA methylation markers and transcripts related to sperm motility, semen from three pairs of full-sibling Holstein bulls with high and low motility was subjected to reduced representation bisulphite sequencing. A total of 948 DMRs were found in 874 genes (gDMRs). Approximately 89% of gDMR-related genes harboured alternative splicing events, including SMAD2, KIF17, and PBRM1. One DMR in exon 29 of PBRM1 with the highest 5mC ratio was found, and hypermethylation in this region was related to bull sperm motility. Furthermore, alternative splicing events at exon 29 of PBRM1 were found in bull testis, including PBRM1-complete, PBRM1-SV1 (exon 28 deletion), and PBRM1-SV2 (exons 28-29 deletion). PBRM1-SV2 exhibited significantly higher expression in adult bull testes than in newborn bull testes. In addition, PBRM1 was localized to the redundant nuclear membrane of bull sperm, which might be related to sperm motility caused by sperm tail breakage. Therefore, the hypermethylation of exon 29 may be associated with the production of PBRM1-SV2 in spermatogenesis. These findings indicated that DNA methylation alteration at specific loci could regulate gene splicing and expression and synergistically alter sperm structure and motility.

Atomically Dispersed Fe-N4 Sites and NiFe-LDH Sub-Nanoclusters as an Excellent Air Cathode for Rechargeable Zinc-Air Batteries.

The sluggish four-electron processes of the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) limit the development of rechargeable Zn-air batteries (RZABs). Highly efficient ORR/OER bifunctional electrocatalysts are therefore highly desired for the commercialization of RZABs in large scale. Herein, the Fe-N4-C (ORR active sites) and NiFe-LDH clusters (OER active sites) are successfully integrated within a NiFe-LDH/Fe,N-CB electrocatalyst. The NiFe-LDH/Fe,N-CB electrocatalyst is first prepared by the introduction of Fe-N4 into carbon black (CB), followed by the growth of NiFe-LDH clusters. The cluster nature of NiFe-LDH effectively avoids the blocking of Fe-N4-C ORR active centers and affords excellent OER activity. The NiFe-LDH/Fe,N-CB electrocatalyst thus exhibits an excellent bifunctional ORR and OER performance, with a potential gap of only 0.71 V. The NiFe-LDH/Fe,N-CB-based RZAB exhibits an open-circuit voltage of 1.565 V and a specific capacity of 731 mAh gZn-1, which is much better than the RZAB composed of Pt/C and IrO2. Particularly, the NiFe-LDH/Fe,N-CB-based RZAB displays excellent long-term charging/discharging cyclic stability and rechargeability. Even at a large charging/discharging current density (20 mA cm-2), the charging/discharging voltage gap is only ∼1.33 V and exhibits an increase smaller than 5% after 140 cycles. This work provides a new low-cost bifunctional ORR/OER electrocatalyst with high activity and superior long-term stability and will be helpful to the commercialization of RZAB in large scale.

Signatures of selection in indigenous Chinese cattle genomes reveal adaptive genes and genetic variations to cold climate.

Cold climate shapes the genome of animals and drives them to carry sufficient genetic variations to adapt to changes in temperature. However, limited information is available about the genome-wide pattern of adaptations to cold environments in cattle. In the present study, we used 777K SNP genotyping (15 Chinese cattle breeds, 198 individuals) and whole genome resequencing data (54 cattle breeds of the world, 432 individuals) to disentangle divergent selection signatures, especially between the cold-adapted (annual average temperature of habitat, 6.24 °C to 10.3 °C) and heat-adapted (20.2 °C to 24.73 °C) Chinese native cattle breeds. Genomic analyses revealed a set of candidate genes (e.g., UQCR11, DNAJC18, EGR1, and STING1) were functionally associated with thermogenesis and energy metabolism. We also characterized the adaptive loci of cattle exposed to cold temperatures. Our study finds new candidate genes and provides new insights into adaptations to cold climates in cattle.

Cold climates can affect cattle performance, survival, and health. Local cattle breeds have been adapted to the local environments including extremely cold temperatures after a long period of natural and artificial selection. Selection and local adaptation are shaping populations. However, identifying loci associated with cold adaptation has been a major challenge. We used high-density SNP arrays and resequencing data to comprehensively analyze and compare the genomic selection signatures of Chinese northern and southern cattle, and elucidated several adaptive genes and alleles involved in cold adaptation. The complexity of genetic adaptation mechanism among different low-temperature adapted cattle breeds was also emphasized.

Notopterygium incisum Root Extract (NRE) Alleviates Epileptiform Symptoms in PTZ-Induced Acute Seizure Mice.

BACKGROUND: Epilepsy is a common neurological disorder affecting more than 70 million people worldwide. Despite numerous efforts on new antiepileptic drugs, approximately one-third of epilepsy patients suffer from uncontrolled seizures. It leads to serious psychosocial consequences, cognitive problems, and decreased quality of life. OBJECTIVE: Our previous studies have shown that N. incisum root extract (NRE) can improve cognitive dysfunction in Alzheimer's disease (AD) mice. In addition, our research shows that AD and epilepsy have pathological mechanisms overlapping. Therefore, we tried to investigate whether NRE can ameliorate the seizures of epileptic mice in this study. METHODS: NRE-treated mice group was given an oral administration with 1 g/kg/d for 7 days. On the 8th day, mice were exposed to PTZ (i.p. injection) to induce epilepsy. Then the cognitive tests of mice in the water maze were carried out, and the biochemical indexes and pathological tests were carried out after the mice were sacrificed. RESULTS: SOD level in the NRE group was significantly higher than that in the PTZ group, while MDA, TNF-α, and IL-1ß levels were decreased. The cognitive ability of NRE-treated mice was significantly improved compared with the PTZ group. Immunohistochemistry (IHC) results showed that the activation of microglia and astrocytes in the hippocampus and cortex of NRE mice were inhibited. CONCLUSION: This study suggests that NRE can alleviate epilepsy and improve cognitive function in mice with epilepsy, and its mechanism may be through reducing inflammation and enhancing antioxidant defense.

Novel characterization discoveries of ferroptosis-associated molecules in COAD microenvironment based TCGA data.

Background and Objective: One of the most recent forms of programmed cell death, ferroptosis, is crucial in tumorigenesis. Ferroptosis is characterized by iron-dependent oxidative destruction of cellular membranes following the antioxidant system's failure. However, it is unknown whether ferroptosis-related genes (FRGs) are associated with colon adenocarcinoma (COAD) metastasis, immune cell infiltration, and oxidative stress in COAD. The current study concentrated on FRGs expression in colon cancer metastasis, their relationship to immune cell infiltration (ICI), and potential pathological pathways in COAD. Methods and Results: Clinical information and mRNA expression patterns for patients with COAD metastasis were obtained from the public TCGA database. Patients with low mRNA levels showed good overall survival than patients with high mRNA levels. The genomic-clinicopathologic nomogram was subsequently created by combining risk score and clinicopathological features. Absolute Shrinkage and Selection Operator have shown a 4 gene signature that can stratify cancer patients into high-risk versus low-risk. These four FRGs were found to be significantly linked to the overall survival of COAD patients and predicted high risk score. Next, age, stage, and PTNM were combined in univariate and multivariate cox regression models to perform a filtering procedure. The receiver operating characteristic (ROC) and calibration curves indicated that constructed signature model exhibited high prediction accuracy and clinical relevance in COAD. ARID3A showed a strong negative correlation with a wide range of immune tumour-infiltrating cells in COAD microenvironment. According to the single sample gene set enrichment analysis (ssGSEA) results, FRGs are involved in variety of pathological pathways including PI3K-AKT-mTOR pathway, reactive oxygen species (ROS) pathway, response to hypoxia pathway, and other inflammation related pathways. Moreover, dysregulation of FRGs in COAD patients showed a significance correlation with wide range of miRNAs and transcription factors (TFs). Conclusion: We identified new diagnostic biomarkers and established prognostic models for ferroptosis related programmed cell death in COAD metastasis. FRGs may improve tumor cell survival by activating the TGFB pathway, which can stimulate ROS production, accelerates ECM breakdown, and promote tumor progression and invasion. Genes implicated in ferroptosis, as revealed by the Kaplan Meier and a genomic-clinicopathologic nomogram, are potential therapeutic targets and prognosis indications for metastasis COAD patients.

Selection Signature and CRISPR/Cas9-Mediated Gene Knockout Analyses Reveal ZC3H10 Involved in Cold Adaptation in Chinese Native Cattle.

Cold stress is an important factor affecting cattle health, production performance, and reproductive efficiency. Understanding of the potential mechanism underlying genetic adaptation to local environments, particularly extreme cold environment, is limited. Here, by using FLK and hapFLK methods, we found that the Zinc finger CCCH-type containing 10 (ZC3H10) gene underwent positive selection in the Menggu, Fuzhou, Anxi, and Shigatse humped cattle breeds that are distributed in the cold areas of China. Furthermore, ZC3H10 expression significantly increased in bovine fetal fibroblast (BFF) cells at 28 °C for 4 h. ZC3H10 knockout BFFs were generated using CRISPR/Cas9. Wild and ZC3H10-deleted BFFs were treated at two temperatures and were divided into four groups (WT, wild and cultured at 38 °C; KO, ZC3H10-/- and 38 °C; WT_LT, wild, and 28 °C for 4 h; and KO_LT, ZC3H10-/- and 28 °C for 4 h. A total of 466, 598, 519, and 650 differently expressed genes (two-fold or more than two-fold changes) were identified by determining transcriptomic difference (KO_LT vs. KO, WT_LT vs. WT, KO vs. WT, and KO_LT vs. WT_LT, respectively). Loss of ZC3H10 dysregulated pathways involved in thermogenesis and immunity, and ZC3H10 participated in immunity-related pathways induced by cold stress and regulated genes involved in glucose and lipid metabolism and lipid transport (PLTP and APOA1), thereby facilitating adaptability to cold stress. Our findings provide a foundation for further studies on the function of ZC3H10 in cold stress and development of bovine breeding strategies for combatting the influences of cold climate.

Aberrantly hypermethylated ARID1B is a novel biomarker and potential therapeutic target of colon adenocarcinoma.

Background and Objective: Understanding the tumor microenvironment (TME) and immune cell infiltration (ICI) may help guide immunotherapy efforts for colon cancer (COAD). However, whether ARID1B is truly regulated by hypermethylation or linked to immune infiltration remains unknown. The current work focused on the ARID1B gene expression and methylation in COAD, as well as its relation with ICI. Methods and Results: Multiple tools based on TCGA were used to analyze the differences in the expression of the ARID1B gene, DNA methylation, and its association with various clinicopathological features, somatic mutations, copy number variation, and the prognosis of patients with COAD. According to the analysis results, patients with high mRNA, low methylation levels showed better overall survival than patients with low mRNA, high methylation levels. The correlation analysis of immune cell infiltration and immune checkpoint gene expression showed that the infiltration rates of the main ICI subtypes, cancer-associated fibroblast, and myeloid cells were significantly enriched and correlated with ARID1B in COAD. An association between ARID1B expression and immune infiltration in COAD was found by correlating ICI indicators with ARID1B expression in the immune cell composition of the COAD microenvironment. Notably, M2 chemokines were related to ARID1B expression, while M1 chemokines were not. Conclusion: This study provided evidence that ARID1B may have a role in the pathogenesis of COAD. The specific underlying mechanisms that could be responsible for ARID1B's downregulation in COAD will need to be investigated in the future.

Discovery of novel ß-carboline derivatives as selective AChE inhibitors with GSK-3ß inhibitory property for the treatment of Alzheimer's disease.

The natural product harmine, a representative ß-carboline alkaloid from the seeds of Peganum harmala L. (Zygophyllaceae), possesses a broad spectrum of biological activities. In this study, a novel series of harmine derivatives containing N-benzylpiperidine moiety were identified for the treatment of Alzheimer's disease (AD). The results showed that all the derivatives possessed significant anti-acetylcholinesterase (AChE) activity and good selectivity over butyrylcholinesterase (BChE). In particular, compound ZLWH-23 exhibited potent anti-AChE activity (IC50 = 0.27 µM) and selective BChE inhibition (IC50 = 20.82 µM), as well as acceptable glycogen synthase kinase-3 (GSK-3ß) inhibition (IC50 = 6.78 µM). Molecular docking studies and molecular dynamics simulations indicated that ZLWH-23 could form stable interaction with AChE and GSK-3ß. Gratifyingly, ZLWH-23 exhibited good selectivity for GSK-3ß over multi-kinases and very low cytotoxicity towards SH-SY5Y, HEK-293T, HL-7702, and HepG2 cell lines. Importantly, ZLWH-23 displayed efficient reduction against tau hyperphosphorylation on Ser-396 site in Tau (P301L) 293T cell model. Collectively, harmine-based derivatives could be considered as possible drug leads for the development of AD therapies.

EGR1-CCL2 Feedback Loop Maintains Epithelial-Mesenchymal Transition of Cisplatin-Resistant Gastric Cancer Cells and Promotes Tumor Angiogenesis.

BACKGROUND: The mechanism of cisplatin resistance in gastric cancer (GC) is still elusive; several recent evidences proposed that chemoresistant tumor cells acquired aggressive behaviors. AIMS: This study was aimed to investigate the mechanism of epithelial-mesenchymal transition (EMT) and angiogenesis in chemoresistant GC. METHODS: Bioinformatics analysis and function or mechanism experiments including RT-qPCR, immunofluorescence, Western blot, luciferase reporter assay, Chromatin immunoprecipitation, Chicken chorioallantoic membrane assay and animal experiments were applied to evaluate the role of EGR1-CCL2 feedback loop. RESULTS: Compared with the parental cell line SGC7901, cisplatin resistant SGC7901R cells underwent EMT and showed increased angiogenic capabilities. Mechanistically, SGC7901R cells showed increased levels of EGR1, which could transcriptionally activate the angiogenic factor CCL2 and EMT regulator ZEB2. Reciprocally, CCL2 activated the CCR2-ERK-ELK1-EGR1 pathway, thus forming a positive feed-forward loop. Moreover, CCL2 in culture medium of SGC7901R cells promoted angiogenesis of Human Umbilical Vein Endothelial Cells (HUVECs). EGR1 expression was positively correlated with CCL2 and ZEB2 in clinical GC tissues, and the depletion of ERG1 could also decrease microvessel density and ZEB2 expression in metastatic nodules of nude mice. CONCLUSIONS: EGR1-CCL2 feedback loop might exert critical roles on EMT and angiogenesis of chemoresistant GC.

Microplastics influence physiological processes, growth and reproduction in the Manila clam, Ruditapes philippinarum.

Microplastics (<5 mm) are widely distributed in marine environments and pose a serious threat to bivalves. Here, the ingestion and accumulation of polystyrene microplastics (PS microplastics, diameters 5 and 10 µm) by the Manila clam, Ruditapes philippinarum, and their impacts on physiological processes, growth and reproduction were studied. The results showed that both PS microplastics were ingested by the Manila clam and accumulated in their gills, hepatopancreases and intestines. Furthermore, the accumulation of 5 and 10 µm PS microplastics significantly increased the rates of respiration and excretion while significantly decreasing feeding and absorption efficiency (AE), leading to a dramatically reduced amount of energy available for growth (SfG) and ultimately led to slower growth. The dynamic energy budget (DEB) model predicts that PS microplastic exposure for 200 days would cause lower shell/flesh growth rates and reproductive potentiality. Transcriptomic profiles support these results, as carbon and protein metabolism and oxytocin and insulin-related signaling pathways were significantly altered in clams in response to PS microplastics. This study provides evidence that microplastics strongly affect the physiological activities, energy allocation, growth and reproduction of filter-feeding bivalves.

Identification of a Human Anti-Alpha-Toxin Monoclonal Antibody Against Staphylococcus aureus Infection.

Staphylococcus aureus is a major pathogenic bacterium that causes a variety of clinical infections. The emergence of multi-drug resistant mechanisms requires novel strategies to mitigate S. aureus infection. Alpha-hemolysin (Hla) is a key virulence factor that is believed to play a significant role in the pathogenesis of S. aureus infections. In this study, we screened a naïve human Fab library for identification of monoclonal antibodies targeting Hla by phage display technology. We found that the monoclonal antibody YG1 blocked the Hla-mediated lysis of rabbit red blood cells and inhibited Hla binding to A549 cells in a concentration-dependent manner. YG1 also provided protection against acute peritoneal infection, bacteremia, and pneumonia in murine models. We further characterized its epitope using different Hla variants and found that the amino acids N209 and F210 of Hla were functionally and structurally important for YG1 binding. Overall, these results indicated that targeting Hla with YG1 could serve as a promising protective strategy against S. aureus infection.

Design, synthesis and biological evaluation of harmine derivatives as potent GSK-3ß/DYRK1A dual inhibitors for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disease, characterized by irreversible cognitive impairment, memory loss and behavioral disturbances, ultimately leading to death. Glycogen synthase kinase 3ß (GSK-3ß) and dual-specificity tyrosine phosphorylation regulated kinase1A (DYRK1A) have gained a lot of attention for its role in tau pathology. To search for potential dual GSK-3ß/DYRK1A inhibitors, we focused on harmine, a natural ß-carboline alkaloid, which has been extensively studied for its various biological effects on the prevention of AD. In this study, a new series of harmine derivatives were designed, synthesized and evaluated as dual GSK-3ß/DYRK1A inhibitors for their multiple biological activities. The in vitro results indicated that most of them displayed promising activity against GSK-3ß and DYRK1A. Among them, compound ZDWX-25 showed potent inhibitory effects on GSK-3ß and DYRK1A with IC50 values of 71 and 103 nM, respectively. Molecular modelling and kinetic studies verified that ZDWX-25 could interact with the ATP binding pocket of GSK-3ß and DYRK1A. Western blot analysis revealed that ZDWX-25 inhibited hyperphosphorylation of tau protein in okadaic acid (OKA)-induced SH-SY5Y cells. In addition, ZDWX-25 showed good blood-brain barrier penetrability in vitro. More importantly, ZDWX-25 could ameliorate the impaired learning and memory in APP/PS1/Tau transgenic mice. These results indicated that the harmine-based compounds could be served as promising dual-targeted candidates for AD.

Study on the experimental verification and regulatory mechanism of Rougui-Ganjiang herb-pair for the actions of thermogenesis in brown adipose tissue based on network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia Presl (Rougui) has character of xin、gan、wen, belongs to Jing of heart、lung、bladder, and has the effect of dispersing cold and relieving pain. It is widely used to resolve the exterior and dissipate cold in Treatise on Febrile Diseases (Shang Han Lun), such as Chaihu Guizhi Ganjiang Tang and Guizhi Renshen Tang. Both these two prescriptions contain Cinnamomum cassia Presl and Zingiber officinale Rosc (Ganjiang). Rougui-Ganjiang herb-pair (RGHP) can warm viscera and remove cold, which is widely used in Shang Han Lun. And in modern times, recent studies have showed that cinnamon and ginger also have the effect of thermogenesis and regulating the body temperature, respectively. AIM OF THE STUDY: To maintain the body thermal homeostasis and prevent cold invasion of main organs, in this study, we assessed the underlying physiological changes induced by RGHP in mice exposed to -20 °C and explored the mechanisms for the thermogenic actions of RGHP in brown adipose tissue (BAT) by network pharmacology and molecular docking. MATERIALS AND METHODS: Male Kunming (KM) mice were fed normal diet with orally administration of distilled water or ethanol RGHP extract (three doses: 375,750 and 1500 mg/kg) for 21 days, once per day and then exposed to -20 °C for 2 h. The core temperature, activity ability and the degree of frostbite in mice, morphological and ATP content of adipocytes were measured. In addition, the network pharmacology was employed to predict the targets of RGHP' s thermogenesis effect on BAT. Pathway analysis and biological process with key genes was carried out through KEGG and GO analysis, respectively. Furthermore, the core ingredients and targets obtained by network pharmacology were verified by molecular docking and Western blot assays. RESULTS: RGHP can significantly increase the core body temperature, reduce the degree of frostbite and enhance the activity ability of mice after cold exposure. Meanwhile, it can also improve the lipid morphology and decrease ATP production in BAT. A network pharmacology-based analysis identified 246 ingredients from RGHP (two herbs), which related to 222 target genes. There were 8 common genes between 222 compounds target genes and 62 thermogenesis associated target genes, which linked to 49 potential compounds. There are 24 ingredients which degree are greater than the average. Among them, we found that oleic acid, EIC, 6-gingerol, eugenol, isohomogenol and sitogluside could be detected in mice plasma. The cAMP-PPAR signaling pathway was enriched for thermogenesis after KEGG analysis with 8 genes. Molecular docking analysis and Western blot assay further confirmed that oleic acid, 6-gingerol, eugenol and isohomogenol were potential active ingredients for RGHP's heat production effect. And UCP1, PGC-1α, PPARα and PPARγ are key thermogenesis proteins. CONCLUSIONS: RGHP treatment can significantly maintain the rectal temperature of mice by enhancing the BAT heat production. RGHP exhibited the heat production effect, which might be mainly attributed to increasing thermogenesis through the cAMP-PPAR signaling pathway in cold exposure mice. Oleic acid, 6-gingerol, eugenol and isohomogenol might be considered the potential therapeutic ingredients which affect the key targets of thermogenesis effect.

Chimeric Phi29 DNA polymerase with helix-hairpin-helix motifs shows enhanced salt tolerance and replication performance.

Phi29 DNA polymerase (Phi29 Pol) has been successfully applied in DNA nanoball-based sequencing, real-time DNA sequencing from single polymerase molecules and nanopore sequencing employing the sequencing by synthesis (SBS) method. Among these, polymerase-assisted nanopore sequencing technology analyses nucleotide sequences as a function of changes in electrical current. This ionic, current-based sequencing technology requires polymerases to perform replication at high salt concentrations, for example 0.3 M KCl. Nonetheless, the salt tolerance of wild-type Phi29 Pol is relatively low. Here, we fused helix-hairpin-helix (HhH)2 domains E-L (eight repeats in total) of topoisomerase V (Topo V) from the hyperthermophile Methanopyrus kandleri to the Phi29 Pol COOH terminus, designated Phi29EL DNA polymerase (Phi29EL Pol). Domain fusion increased the overall enzyme replication efficiency by fourfold. Phi29EL Pol catalysed rolling circle replication in a broader range of salt concentrations than did Phi29 Pol, extending the KCl concentration range for activity up to 0.3 M. In addition, the mutation of Glu375 to Ser or Gln increased Phi29EL Pol activity in the presence of KCl. In this work, we produced a salt-tolerant Phi29 Pol derivative by means of (HhH)2 domain insertion. The multiple advantages of this insertion make it a good substitute for Phi29 Pol, especially for use in nanopore sequencing or other circumstances that require high salt concentrations.