Endogenous nuclear RNAi mediates behavioral adaptation to odor.

Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. Here, we show that the endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene whose downregulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4 kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior. This class of siRNA may act broadly as a rheostat allowing prolonged stimulation to dampen gene expression and promote cellular memory formation. PAPERFLICK:

Antibody targeting of E3 ubiquitin ligases for receptor degradation.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

Strategies and Lessons Learned from Total Synthesis of Taxol.

Taxol (paclitaxel), the most well-known taxane diterpenoid, is the best-selling natural-source anticancer drug ever produced and one of the most common prescriptions in the treatment of breast, lung, and ovarian cancers, saving countless lives around the world. Structurally, Taxol possesses a highly oxygenated [6-8-6-4] core bearing 11 stereocenters, seven of which are contiguous chiral centers. Moreover, the extremely strained bicyclo[5.3.1] undecane ring system with a bridgehead double bond is a unique structural feature. All these features make Taxol a highly challenging synthetic target. Tremendous synthetic efforts from more than 60 research groups around the world have already culminated in ten total syntheses and three formal syntheses, as well as more than 60 synthetic model studies of Taxol. This review is intended to provide a long-overdue appraisal of the great achievements in the total syntheses of Taxol reported in the last few decades. In doing so, we summarize the development of synthesis toward Taxol from 1994 to 2022, including the evolution of synthetic strategy for accessing this complex molecular scaffold and key lessons learned from such endeavors. Finally, we briefly discuss the future of the research in this area.

Carbon Nanotubes as a Solid-State Electron Mediator for Visible-Light-Driven Z-Scheme Overall Water Splitting.

So-called Z-scheme systems, which typically comprise an H2 evolution photocatalyst (HEP), an O2 evolution photocatalyst (OEP), and an electron mediator, represent a promising approach to solar hydrogen production via photocatalytic overall water splitting (OWS). The electron mediator transferring photogenerated charges between the HEP and OEP governs the performance of such systems. However, existing electron mediators suffer from low stability, corrosiveness to the photocatalysts, and parasitic light absorption. In the present work, carbon nanotubes (CNTs) were shown to function as an effective solid-state electron mediator in a Z-scheme OWS system. Based on the high stability and good charge transfer characteristics of CNTs, this system exhibited superior OWS performance compared with other systems using more common electron mediators. The as-constructed system evolved stoichiometric amounts of H2 and O2 at near-ambient pressure with a solar-to-hydrogen energy conversion efficiency of 0.15%. The OWS reaction was also promoted in the case that this CNT-based Z-scheme system was immobilized on a substrate. Hence, CNTs are a viable electron mediator material for large-scale Z-scheme OWS systems.

Hyperfractionation compared with standard fractionation in intensity-modulated radiotherapy for patients with locally advanced recurrent nasopharyngeal carcinoma: a multicentre, randomised, open-label, phase 3 trial.

BACKGROUND: Reirradiation in standard fractionation for locally advanced recurrent nasopharyngeal carcinoma after a previous course of high-dose radiotherapy is often associated with substantial late toxicity, negating its overall benefit. We therefore aimed to investigate the efficacy and safety of hyperfractionation compared with standard fractionation in intensity-modulated radiotherapy. METHODS: This multicentre, randomised, open-label, phase 3 trial was done in three centres in Guangzhou, China. Eligible patients were aged 18-65 years with histopathologically confirmed undifferentiated or differentiated, non-keratinising, advanced locally recurrent nasopharyngeal carcinoma. Participants were randomly assigned (1:1) to either receive hyperfractionation (65 Gy in 54 fractions, given twice daily with an interfractional time interval of at least 6 h) or standard fractionation (60 Gy in 27 fractions, given once a day). Intensity-modulated radiotherapy was used in both groups. A computer program generated the assignment sequence and randomisation was stratified by treatment centre, recurrent tumour stage (T2-T3 vs T4), and recurrent nodal stage (N0 vs N1-N2), determined at the time of randomisation. The two primary endpoints were the incidence of severe late complications defined as the incidence of grade 3 or worse late radiation-induced complications occurring 3 months after the completion of radiotherapy until the latest follow-up in the safety population, and overall survival defined as the time interval from randomisation to death due to any cause in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT02456506. FINDINGS: Between July 10, 2015, and Dec 23, 2019, 178 patients were screened for eligibility, 144 of whom were enrolled and randomly assigned to hyperfractionation or standard fractionation (n=72 in each group). 35 (24%) participants were women and 109 (76%) were men. After a median follow-up of 45·0 months (IQR 37·3-53·3), there was a significantly lower incidence of grade 3 or worse late radiation-induced toxicity in the hyperfractionation group (23 [34%] of 68 patients) versus the standard fractionation group (39 [57%] of 68 patients; between-group difference -23% [95% CI -39 to -7]; p=0·023). Patients in the hyperfractionation group had better 3-year overall survival than those in the standard fractionation group (74·6% [95% CI 64·4 to 84·8] vs 55·0% [43·4 to 66·6]; hazard ratio for death 0·54 [95% CI 0·33 to 0·88]; p=0·014). There were fewer grade 5 late complications in the hyperfractionation group (five [7%] nasal haemorrhage) than in the standard fractionation group (16 [24%], including two [3%] nasopharyngeal necrosis, 11 [16%] nasal haemorrhage, and three [4%] temporal lobe necrosis). INTERPRETATION: Hyperfractionated intensity-modulated radiotherapy could significantly decrease the rate of severe late complications and improve overall survival among patients with locally advanced recurrent nasopharyngeal carcinoma. Our findings suggest that hyperfractionated intensity-modulated radiotherapy could be used as the standard of care for these patients. FUNDING: Key-Area Research and Development of Guangdong Province, the National Natural Science Foundation of China, the Special Support Program for High-level Talents in Sun Yat-sen University Cancer Center, the Guangzhou Science and Technology Plan Project, and the National Ten Thousand Talents Program Science and Technology Innovation Leading Talents, Sun Yat-Sen University Clinical Research 5010 Program.

Impact of treatment interval between neoadjuvant immunochemotherapy and surgery in lung squamous cell carcinoma.

OBJECTIVE: The optimal timing for surgery following neoadjuvant immunochemotherapy for lung squamous cell carcinoma appears to be a topic of limited data. Many clinical studies lack stringent guidelines regarding this timing. The objective of this study is to explore the effect of the interval between neoadjuvant immunochemotherapy and surgery on survival outcomes in patients with lung squamous cell carcinoma. METHODS: This study conducted a retrospective analysis of patients with lung squamous cell carcinoma who underwent neoadjuvant immunochemotherapy between January 2019 and October 2022 at The First Affiliated Hospital, Zhejiang University School of Medicine. Patients were divided into two groups based on the treatment interval: ≤33 days and > 33 days. The primary observational endpoints of the study were Disease-Free Survival (DFS) and Overall Survival (OS). Secondary observational endpoints included Objective response rate (ORR), Major Pathological Response (MPR), and Pathological Complete Remission (pCR). RESULTS: Using the Kaplan-Meier methods, the ≤ 33d group demonstrated a superior DFS curve compared to the > 33d group (p = 0.0015). The median DFS for the two groups was 952 days and 590 days, respectively. There was no statistical difference in the OS curves between the groups (p = 0.66), and the median OS was not reached for either group. The treatment interval did not influence the pathologic response of the tumor or lymph nodes. CONCLUSIONS: The study observed that shorter treatment intervals were associated with improved DFS, without influencing OS, pathologic response, or surgical safety. Patients should avoid having a prolonged treatment interval between neoadjuvant immunochemotherapy and surgery.

A cytoskeleton-membrane interaction conserved in fast-spiking neurons controls movement, emotion, and memory.

The pathogenesis of schizophrenia is believed to involve combined dysfunctions of many proteins including microtubule-associated protein 6 (MAP6) and Kv3.1 voltage-gated K+ (Kv) channel, but their relationship and functions in behavioral regulation are often not known. Here we report that MAP6 stabilizes Kv3.1 channels in parvalbumin-positive (PV+ ) fast-spiking GABAergic interneurons, regulating behavior. MAP6-/- and Kv3.1-/- mice display similar hyperactivity and avoidance reduction. Their proteins colocalize in PV+ interneurons and MAP6 deletion markedly reduces Kv3.1 protein level. We further show that two microtubule-binding modules of MAP6 bind the Kv3.1 tetramerization domain with high affinity, maintaining the channel level in both neuronal soma and axons. MAP6 knockdown by AAV-shRNA in the amygdala or the hippocampus reduces avoidance or causes hyperactivity and recognition memory deficit, respectively, through elevating projection neuron activity. Finally, knocking down Kv3.1 or disrupting the MAP6-Kv3.1 binding in these brain regions causes avoidance reduction and hyperactivity, consistent with the effects of MAP6 knockdown. Thus, disrupting this conserved cytoskeleton-membrane interaction in fast-spiking neurons causes different degrees of functional vulnerability in various neural circuits.

Exploring the mechanism of lung injury induced by lunar dust simulant in rats based on metabolomic analysis.

Inflammatory response and oxidative stress are considered to be important mechanisms of lung injury induced by lunar dust. However, the pulmonary toxicological mechanism remains unclear. In the present study, Wistar rats were exposed to CLDS-i 7 days/week, 4 h/day, for 4 weeks in the mouth and nose. Lung tissue samples were collected for histopathological analysis and ultra-performance liquid chromatography-mass spectrometry analysis. Enzyme activities and expression levels of key metabolic enzymes were detected by biochemical analysis and real-time PCR. The pathological features of lung tissue showed that CLDS-i caused congestion and inflammation in the lungs, and the lung structure was severely damaged. Metabolomics analysis showed that 141 metabolites were significantly changed in the lung tissue of the CLDS-i group compared with the control group. Combined with Kegg pathway analysis, it was found that the changes of amino acid metabolites were involved in these pathways, indicating that the simulated lunar dust exposure had the most obvious effect on amino acid metabolism in the lung tissue of rats. Real-time PCR analysis showed that the mRNA expression of six key enzymes related to amino acid metabolism was changed, and the enzyme activities of these key enzymes were also changed, which were consistent with the results of qPCR. These results suggest that changes in amino acid metabolism may be closely related to the pathogenesis of lung injury induced by lunar dust, and amino acid metabolism may be a potential biomarker of lung diseases related to lunar dust exposure.

[Role of macrophage polarization in hypertension and traditional Chinese medicine intervention: a review].

Hypertension is known to be a chronic inflammatory state and a key risk factor for heart failure, coronary heart disease, and atherosclerosis. Macrophages in the circulatory system are the main cell group that constitutes the immune system and participates in the inflammatory response. Depending on the local microenvironment, macrophages can be polarized into pro-inflammatory(M1) and anti-inflammatory(M2) phenotypes. When blood pressure is elevated, M1 macrophages can release pro-inflammatory cytokines and chemokines to generate an immune response. However, an excessive immune response can lead to tissue damage, and M2 macrophages release anti-inflammatory cytokines to promote the repair of wounds and tissue damage. It is clear that the dynamic balance between M1 and M2 macrophages resembles the traditional Chinese medicine(TCM) theory of Yin and Yang. That is, when Yin and Yang are imbalanced, the human body will exhibit pathological states, e.g., altered blood pressure rhythms. Studies have confirmed that TCM can produce positive therapeutic effects on hypertension by regulating macrophage polarization. Therefore, this study reviews the studies about the TCM regulation of macrophage polarization and summarized the mechanisms of TCM intervention in hypertension, with the aim of providing evidence for clinical treatment and ideas for scientific research design.

Cytochalasans with Inhibitory Activity against NPC1L1 from the Endophytic Fungus Chaetomium nigricolor F5.

Mass spectrometry (MS)-based metabolic profiling of the endophytic fungus Chaetomium nigricolor F5 guided the isolation of five novel cytochalasans, chamisides B-F (1-5), and two known ones, chaetoconvosins C and D (6 and 7). Their structures including stereochemistry were unambiguously determined by MS, nuclear magnetic resonance, and single-crystal X-ray diffraction analyses. Compounds 1-3 share a new 5/6/5/5/7-fused pentacyclic skeleton in cytochalasans and are appropriately proposed to be the key biosynthetic precursors of co-isolated cytochalasans with a 6/6/5/7/5, 6/6/5/5/7, or 6/6/5 ring system. Remarkably, compound 5 with a relatively flexible side chain showed promising inhibition activity against the cholesterol transporter protein Niemann-Pick C1-like 1 (NPC1L1), expanding the function of cytochalasans.

Azobenzene-Functionalized UiO-66-NH2: Solid Base Catalysts with Photocontrollable Activity.

Heterogeneous solid base catalysts are highly expected due to their high activity and environmentally friendly nature in a variety of reactions. However, the catalytic activity of traditional solid base catalysts is controlled by external factors (such as temperature and pressure), and regulation of the activity by in situ changing their own properties has never been reported. Herein, we report a smart solid base catalyst by chemically anchoring the photoresponsive azobenzene derivative p-phenylazobenzoyl chloride (PAC) onto the metal-organic framework UiO-66-NH2 (UN) for the first time, which can regulate the catalytic activity through remote control of external light. The prepared catalysts have a regular crystal structure and photoresponsive properties. It is fascinating that the configuration of PAC can be isomerized easily during UV- and visible-light irradiation and resulted in regulation of the catalytic activity. In the Knoevenagel condensation of 1-naphthaldehyde and ethyl cyanoacetate to ethyl 2-cyano-3-(1-naphthalenyl)acrylate, the optimal catalyst shows up to 56.2% of change after trans/cis isomerization, while the change of the yield over UN is negligible. The regulated catalytic behavior can be assigned to the steric hindrance change of the catalysts under external light irradiation. This work may shed light on the design and construction of smart solid base catalysts with tailorable properties for various reactions.

The Safety and Efficacy of Preoperative Immunotherapy Combined with Chemotherapy in Patients with Stage IIIA-IIIB Lung Squamous Cell Carcinoma.

OBJECTIVE: Data on preoperative immunotherapy combined with chemotherapy in potentially resectable lung squamous cell carcinoma (LUSC) remain scarce. This study was designed to investigate the safety and efficacy of preoperative immunotherapy and chemotherapy for stage IIIA-IIIB LUSC. METHODS: This study consecutively enrolled stage IIIA-IIIB LUSC who received preoperative immunotherapy combined with chemotherapy between January 2019 and July 2021. Patients received two to four cycles of immunotherapy combined with platinum-based doublet chemotherapy (platinum + paclitaxel) before surgery. Patients were assessed radiographically every one to two cycles until surgery. Postoperative pathological evaluation was also performed. Follow-up was performed until at least 3 months after surgery. RESULTS: Sixty-five patients with stage IIIA-IIIB LUSC were enrolled. The objective response rate was 78.46% (51/65), and no patients had progressive disease. Fifty-seven patients underwent surgery, and 55 patients achieved R0 resection. There were no perioperative deaths. The rate of pathological complete response (pCR) was 31.58% (18/57) and major pathological response was 68.42% (39/57). The incidence of grade 3 and 4 adverse reactions was 21.21 and 1.54%, respectively. CONCLUSION: Perioperative immunotherapy combined with chemotherapy followed by surgical resection for male patients with stage IIIA-IIIB LUSC was effective with a tolerable toxicity profile.

Aerobic degradation of bisphenol A by Pseudomonas sp. LM-1: characteristic and pathway.

Bisphenol A (BPA) has been widely used in the manufacture of polymeric materials. BPA is regarded as an endocrine disrupting chemical, posing a great threat to the public health. In this study, a bacterial strain LM-1, capable of utilizing BPA as the sole carbon and energy source under aerobic conditions, was originally isolated from an activated sludge sample. The isolate was identified as Pseudomonas sp. based on 16S rRNA gene sequence analysis. Strain LM-1 was able to completely degrade 25-100 mg/L BPA within 14-24 h, and it also exhibited high capacity for BPA degradation at a range of pH (6.0-8.0). (NH4)2SO4 and NH4NO3 were the suitable nitrogen sources for its growth and BPA biodegradation, and the BPA degradation could be accelerated when exogenous carbon sources were introduced as the co-substrates. Metal ions such as Zn2+, Cu2+, and Ni2+ could considerably suppress the growth of strain LM-1 and BPA degradation. According to the analysis of liquid chromatography coupled to Q-Exactive high resolution mass spectrometry, hydroquinone, p-hydroxybenzaldehyde, and p-hydroxybenzoate were the predominate metabolites in the BPA biodegradation and the degradation pathways were proposed. This study is important for assessment of the fate of BPA in engineered and natural systems and possibly for designing bioremediation strategies.

Genomic Insights into Pangenome and Antimicrobial Resistance in Campylobacter spp. Isolated from Chickens at Specific Growth Stages.

Improved understanding of the genetic basis of Campylobacter spp. colonization of poultry at specific growth stage is the key to developing a farm-based strategy to prevent flock colonization. In this study, 39 Campylobacter spp. strains (chicken isolates, n = 29; environmental isolates, n = 10) were collected from six marked chickens at the growth stage from week 7 to week 13. Then, we use comparative genomics techniques to analyze the temporal genomic characteristics of Campylobacter spp. in individual chickens across a production cycle. Genotype, average nucleotide identity (ANI), and phylogenetic trees all indicated the evolutionary relationships between the strains from different sampling weeks. The clustering of isolates was not dependent on sampling time and sample source, indicating that strains could persist over several weeks in a flock. Notably, 10 antimicrobial resistance (AMR) genes were identified in the genome of Campylobacter coli isolates, and the genomes of isolates sampled at week 11 harbored fewer AMR genes and insertion sequences (IS) than the isolates from other weeks. Consistent with this, pangenome-wide association analysis demonstrated that gene acquisition and loss could happen at week 11 and week 13. These genes were mainly associated with cell membrane biogenesis, ion metabolism, and DNA replication, suggesting that genomic change may be related to Campylobacter adaptive response. This is a novel study focused on the genetic changes occurring in Campylobacter spp. isolates in a particular space and time; it highlights that accessory genes and AMR genes were overall stable at chicken farm, which will help us understand the survival and the transmission route of Campylobacter spp. better, and have the potential to inform the strategy on the safety control of market-ready chickens.

Induction of COX-2 by feline calicivirus via activation of the MEK1-ERK1/2 pathway, and attenuation of feline lung inflammation and injury by MEK1 inhibitor AZD6244 (selumetinib).

Feline calicivirus (FCV) is an important and highly prevalent pathogen of cats that causes acute infectious respiratory disease. Here it is shown in vitro that FCV induces the production of cyclooxygenase-2 (COX-2) through the MEK1-ERK1/2 signaling pathway. Screening of FCV proteins revealed that FCV non-structural protein VPg enhanced COX-2 mRNA expression and protein production in CRFK cells in a concentration-dependent manner. Regions 24-54aa and 84-111aa in FCV VPg were essential for up-regulation. In vivo, COX-2 and IL-6 production caused by FCV infection of kittens was significantly suppressed by the MEK1 inhibitor AZD6244 (selumetinib) and lung inflammation and injury were practically eliminated, with body temperature being returned to normal. AZD6244 may therefore find application as an effective therapeutic agent for the treatment of FCV infection.

Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage.

DNA damage and epigenetic marks are well established to have profound influences on genome stability and cell phenotype, yet there are few technologies to obtain high-resolution genomic maps of the many types of chemical modifications of DNA. Here we present Nick-seq for quantitative, sensitive, and accurate mapping of DNA modifications at single-nucleotide resolution across genomes. Pre-existing breaks are first blocked and DNA modifications are then converted enzymatically or chemically to strand-breaks for both 3'-extension by nick-translation to produce nuclease-resistant oligonucleotides and 3'-terminal transferase tailing. Following library preparation and next generation sequencing, the complementary datasets are mined with a custom workflow to increase sensitivity, specificity and accuracy of the map. The utility of Nick-seq is demonstrated with genomic maps of site-specific endonuclease strand-breaks in purified DNA from Eschericia coli, phosphorothioate epigenetics in Salmonella enterica Cerro 87, and oxidation-induced abasic sites in DNA from E. coli treated with a sublethal dose of hydrogen peroxide. Nick-seq applicability is demonstrated with strategies for >25 types of DNA modification and damage.

Effects of different growth patterns of Tamarix chinensis on saline-alkali soil: implications for coastal restoration and management.

OBJECTIVE: To better understand the wetland restoration, the physicochemical property and microbial community in rhizosphere and bulk soil of the living and death Tamarix chinensis covered soil zones were studied. RESULTS: There were differences between growth conditions in the levels of soil pH, salinity, SOM, and nutrient. The living status of T. chinensis exhibited higher capacity of decreasing saline-alkali soil than the death condition of plants, and the living T. chinensis showed higher uptake of N, P, and K as compared with the death samples. Proteobacteria, Bacteroidota, and Chloroflexi were the predominant bacterial communities as revealed via high-throughput sequencing. CONCLUSIONS: It is great potential for using halophytes such as T. chinensis to ecological restore the coastal saline-alkali soil. This study could contribute to a better understanding of halophyte growth during the coastal phytoremediation process, and guide theoretically for management of T. chinensis population.

Retinal vascular caliber in coronary heart disease and its risk factors.

BACKGROUND: Many clinical and experimental articles have suggested that the retinal vascular diameter can be used as a predictor of coronary heart disease (CHD). However, the results and reliability of the prediction are still controversial. OBJECTIVE: A meta-analysis of observational study was conducted to clarify the relationships of retinal vessel caliber with CHD and CHD risk factors Method: PubMed and EMBASE were searched for all observational studies on the relationship of retinal vessel caliber with CHD and CHD risk factors from 2001 to 2021. The meta-analysis has been registered in PROSPERO (registration number is: CRD42021267154) Result: A total of 14 articles were selected for the inclusion in this meta-analysis. In the primary outcome, smaller retinal arteriolar caliber was related to CHD, and the results achieved statistical significance (MD -5.55 95%CI -8.07 to -3.02 P < 0.0001, Fig. 2), while there was no significant difference in vein caliber between CHD and healthy people (MD 1.10 95%CI -3.55 to 5.76 P = 0.64 > 0.05, Fig. 3). Smaller retinal arteriolar caliber was related to increasing age, male sex, bigger BMI, and hypertension. Bigger retinal arteriolar caliber was related to current smoking. Smaller retinal venule caliber was related to increasing age and hypertension. Bigger retinal venule caliber was related to current smoking, bigger BMI, and diabetes. Conclusion Smaller retinal arteriolar caliber was related to CHD, while there was no significant difference in venule caliber between CHD and healthy people. Retinal vascular caliber also independently related to CHD risk factors (e.g., age, gender, smoke, BMI, hypertension, and diabetes).

Estimation of the Mixed Layer Depth in the Indian Ocean from Surface Parameters: A Clustering-Neural Network Method.

The effective estimation of mixed-layer depth (MLD) plays a significant role in the study of ocean dynamics and global climate change. However, the methods of estimating MLD still have limitations due to the sparse resolution of the observed data. In this study, a hybrid estimation method that combines the K-means clustering algorithm and an artificial neural network (ANN) model was developed using sea-surface parameter data in the Indian Ocean as a case study. The oceanic datasets from January 2012 to December 2019 were obtained via satellite observations, Argo in situ data, and reanalysis data. These datasets were unified to the same spatial and temporal resolution (1° × 1°, monthly). Based on the processed datasets, the K-means classifier was applied to divide the Indian Ocean into four regions with different characteristics. For ANN training and testing in each region, the gridded data of 84 months were used for training, and 12-month data were used for testing. The ANN results show that the optimized NN architecture comprises five input variables, one output variable, and four hidden layers, each of which has 40 neurons. Compared with the multiple linear regression model (MLR) with a root-mean-square error (RMSE) of 5.2248 m and the HYbrid-Coordinate Ocean Model (HYCOM) with an RMSE of 4.8422 m, the RMSE of the model proposed in this study was reduced by 27% and 22%, respectively. Three typical regions with high variability in their MLDs were selected to further evaluate the performance of the ANN model. Our results showed that the model could reveal the seasonal variation trend in each of the selected regions, but the estimation accuracy showed room for improvement. Furthermore, a correlation analysis between the MLD and input variables showed that the surface temperature and salinity were the main influencing factors of the model. The results of this study suggest that the pre-clustering ANN method proposed could be used to estimate and analyze the MLD in the Indian Ocean. Moreover, this method can be further expanded to estimate other internal parameters for typical ocean regions and to provide effective technical support for ocean researchers when studying the variability of these parameters.

Role of PsnWRKY70 in Regulatory Network Response to Infection with Alternaria alternata (Fr.) Keissl in Populus.

WRKY is an important complex family of transcription factors involved in plant immune responses. Among them, WRKY70 plays an important role in the process of the plant defense response to the invasion of pathogens. However, the defense mechanism of PsnWRKY70 is not clear in Populus nigra. In this study, we showed that PsnWRKY70-overexpression lines (OE) had fewer leaf blight symptoms than PsnWRKY70-repressing lines (RE). PsnWRKY70 activated MAP kinase cascade genes (PsnM2K4, PsnMPK3, PsnM3K18), calcium channel proteins-related genes (PsnCNG3, PsnCNGC1, PsnCNG4), and calcium-dependent protein kinases genes (PsnCDPKL, PsnCDPKW, PsnCDPKS, PsnCDPKQ). Furthermore, 129 genes of PsnWRKY70 putative genome-wide direct targets (DTGs) were identified by using transcriptome (RNA-seq) and DNA affinity purification sequencing (DAP-seq). PsnWRKY70 directly binds to the promoters of homologous genes and LRR domain proteins to promote the expression of WRKY6, WRKY18, WRKY22, and WRKY22-1, LRR domain proteins LRR8, LRR-RLK, ADR1-like 2, NB-ARC, etc. Our study suggests that PsnWRKY70 enhances the resistance of A. alternata in poplar by activating genes in both pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI).