TAK1 is an essential kinase for STING trafficking.

The translocation of stimulator of interferon genes (STING) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) enables its activation. However, the mechanism underlying the regulation of STING exit from the ER remains elusive. Here, we found that STING induces the activation of transforming growth factor beta-activated kinase 1 (TAK1) prior to STING trafficking in a TAK1 binding protein 1 (TAB1)-dependent manner. Intriguingly, activated TAK1 directly mediates STING phosphorylation on serine 355, which facilitates its interaction with STING ER exit protein (STEEP) and thereby promotes its oligomerization and translocation to the ERGIC for subsequent activation. Importantly, activation of TAK1 by monophosphoryl lipid A, a TLR4 agonist, boosts cGAMP-induced antitumor immunity dependent on STING phosphorylation in a mouse allograft tumor model. Taken together, TAK1 was identified as a checkpoint for STING activation by promoting its trafficking, providing a basis for combinatory tumor immunotherapy and intervention in STING-related diseases.

From genetic associations to genes: methods, applications, and challenges.

Genome-wide association studies (GWASs) have identified numerous genetic loci associated with human traits and diseases. However, pinpointing the causal genes remains a challenge, which impedes the translation of GWAS findings into biological insights and medical applications. In this review, we provide an in-depth overview of the methods and technologies used for prioritizing genes from GWAS loci, including gene-based association tests, integrative analysis of GWAS and molecular quantitative trait loci (xQTL) data, linking GWAS variants to target genes through enhancer-gene connection maps, and network-based prioritization. We also outline strategies for generating context-dependent xQTL data and their applications in gene prioritization. We further highlight the potential of gene prioritization in drug repurposing. Lastly, we discuss future challenges and opportunities in this field.

MPEK: a multitask deep learning framework based on pretrained language models for enzymatic reaction kinetic parameters prediction.

Enzymatic reaction kinetics are central in analyzing enzymatic reaction mechanisms and target-enzyme optimization, and thus in biomanufacturing and other industries. The enzyme turnover number (kcat) and Michaelis constant (Km), key kinetic parameters for measuring enzyme catalytic efficiency, are crucial for analyzing enzymatic reaction mechanisms and the directed evolution of target enzymes. Experimental determination of kcat and Km is costly in terms of time, labor, and cost. To consider the intrinsic connection between kcat and Km and further improve the prediction performance, we propose a universal pretrained multitask deep learning model, MPEK, to predict these parameters simultaneously while considering pH, temperature, and organismal information. Through testing on the same kcat and Km test datasets, MPEK demonstrated superior prediction performance over the previous models. Specifically, MPEK achieved the Pearson coefficient of 0.808 for predicting kcat, improving ca. 14.6% and 7.6% compared to the DLKcat and UniKP models, and it achieved the Pearson coefficient of 0.777 for predicting Km, improving ca. 34.9% and 53.3% compared to the Kroll_model and UniKP models. More importantly, MPEK was able to reveal enzyme promiscuity and was sensitive to slight changes in the mutant enzyme sequence. In addition, in three case studies, it was shown that MPEK has the potential for assisted enzyme mining and directed evolution. To facilitate in silico evaluation of enzyme catalytic efficiency, we have established a web server implementing this model, which can be accessed at http://mathtc.nscc-tj.cn/mpek.

Historic dog Furs Unravel the Origin and Artificial Selection of Modern Nordic Lapphund and Elkhound dog Breeds.

The origins and extreme morphological evolution of the modern dog breeds are poorly studied because the founder populations are extinct. Here, we analyse eight 100 to 200 years old dog fur samples obtained from traditional North Swedish clothing, to explore the origin and artificial selection of the modern Nordic Lapphund and Elkhound dog breeds. Population genomic analysis confirmed the Lapphund and Elkhound breeds to originate from the local dog population, and showed a distinct decrease in genetic diversity in agreement with intense breeding. We identified eleven genes under positive selection during the breed development. In particular, the MSRB3 gene, associated with breed-related ear morphology, was selected in all Lapphund and Elkhound breeds, and functional assays showed that a SNP mutation in the 3'UTR region suppresses its expression through miRNA regulation. Our findings demonstrate analysis of near-modern dog artifacts as an effective tool for interpreting the origin and artificial selection of the modern dog breeds.

Lycium barbarum polysaccharide increases thermogenesis and energy metabolism through modulation of the gut microbiota to confer resistance to cold temperatures.

Traditional Chinese medical literature contains numerous records of many traditional Chinese herbal medicines that exhibit efficacy in enhancing resistance to cold, yet there is a lack of scientific explanation. Lycium barbarum is among the herbal medicines that are explicitly documented to enhance resistance to cold in the "Ben Cao Gang Mu (Compendium of Materia Medica)". Herein, we investigated L. barbarum polysaccharide (LBP)-induced browning of inguinal white adipose tissue (iWAT), energy expenditure and thermogenic function in a long-term (4 months) treatment mouse model. LBP supplementation resulted in a significant reduction in weight and adipocyte size in iWAT, along with increased gut microbiota diversity. Specifically, the levels of Lachnospiraceae, Ruminococcaceae and Bacteroidaceae (short-chain fatty acid-producing bacteria) were elevated, leading to a higher level of short-chain fatty acids (SCFAs) in the caecal content. These effects subsequently triggered the release of glucagon-like peptide-1 (GLP-1) and activated the CREB/PGC1α signaling pathway in iWAT, thereby increasing energy expenditure and enhancing thermogenic function. The antibiotic treatment experiments confirmed that the LBP-mediated gut microbiota participated in the process of iWAT browning. In summary, our findings provide the first scientific explanation and mechanistic insights into the cold resistance of L. barbarum and identify potentially safe natural product supplements for individuals in alpine areas.

Accurate quantification of DNA using on-site PCR (osPCR) by characterizing DNA amplification at single-molecule resolution.

Despite the need in various applications, accurate quantification of nucleic acids still remains a challenge. The widely-used qPCR has reduced accuracy at ultralow template concentration and is susceptible to nonspecific amplifications. The more recently developed dPCR is costly and cannot handle high-concentration samples. We combine the strengths of qPCR and dPCR by performing PCR in silicon-based microfluidic chips and demonstrate high quantification accuracy in a large concentration range. Importantly, at low template concentration, we observe on-site PCR (osPCR), where only certain sites of the channel show amplification. The sites have almost identical ct values, showing osPCR is a quasi-single molecule phenomenon. Using osPCR, we can measure both the ct values and the absolute concentration of templates in the same reaction. Additionally, osPCR enables identification of each template molecule, allowing removal of nonspecific amplification during quantification and greatly improving quantification accuracy. We develop sectioning algorithm that improves the signal amplitude and demonstrate improved detection of COVID in patient samples.

Pharmaceutical pollution of the world's rivers.

Environmental exposure to active pharmaceutical ingredients (APIs) can have negative effects on the health of ecosystems and humans. While numerous studies have monitored APIs in rivers, these employ different analytical methods, measure different APIs, and have ignored many of the countries of the world. This makes it difficult to quantify the scale of the problem from a global perspective. Furthermore, comparison of the existing data, generated for different studies/regions/continents, is challenging due to the vast differences between the analytical methodologies employed. Here, we present a global-scale study of API pollution in 258 of the world's rivers, representing the environmental influence of 471.4 million people across 137 geographic regions. Samples were obtained from 1,052 locations in 104 countries (representing all continents and 36 countries not previously studied for API contamination) and analyzed for 61 APIs. Highest cumulative API concentrations were observed in sub-Saharan Africa, south Asia, and South America. The most contaminated sites were in low- to middle-income countries and were associated with areas with poor wastewater and waste management infrastructure and pharmaceutical manufacturing. The most frequently detected APIs were carbamazepine, metformin, and caffeine (a compound also arising from lifestyle use), which were detected at over half of the sites monitored. Concentrations of at least one API at 25.7% of the sampling sites were greater than concentrations considered safe for aquatic organisms, or which are of concern in terms of selection for antimicrobial resistance. Therefore, pharmaceutical pollution poses a global threat to environmental and human health, as well as to delivery of the United Nations Sustainable Development Goals.

High-throughput DNA synthesis for data storage.

With the explosion of digital world, the dramatically increasing data volume is expected to reach 175 ZB (1 ZB = 1012 GB) in 2025. Storing such huge global data would consume tons of resources. Fortunately, it has been found that the deoxyribonucleic acid (DNA) molecule is the most compact and durable information storage medium in the world so far. Its high coding density and long-term preservation properties make itself one of the best data storage carriers for the future. High-throughput DNA synthesis is a key technology for "DNA data storage", which encodes binary data stream (0/1) into quaternary long DNA sequences consisting of four bases (A/G/C/T). In this review, the workflow of DNA data storage and the basic methods of artificial DNA synthesis technology are outlined first. Then, the technical characteristics of different synthesis methods and the state-of-the-art of representative commercial companies, with a primary focus on silicon chip microarray-based synthesis and novel enzymatic DNA synthesis are presented. Finally, the recent status of DNA storage and new opportunities for future development in the field of high-throughput, large-scale DNA synthesis technology are summarized.

The Sec1-Munc18 protein VPS33B forms a uniquely bidirectional complex with VPS16B.

Loss-of-function variants of vacuolar protein sorting proteins VPS33B and VPS16B (VIPAS39) are causative for arthrogryposis, renal dysfunction, and cholestasis syndrome, where early lethality of patients indicates that VPS33B and VPS16B play essential cellular roles. VPS33B is a member of the Sec1-Munc18 protein family and thought to facilitate vesicular fusion via interaction with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, like its paralog VPS33A in the homotypic fusion and vacuole sorting complex. VPS33B and VPS16B are known to associate, but little is known about the composition, structure, or function of the VPS33B-VPS16B complex. We show here that human VPS33B-VPS16B is a high molecular weight complex, which we expressed in yeast to perform structural, composition, and stability analysis. Circular dichroism data indicate VPS33B-VPS16B has a well-folded α-helical secondary structure, and size-exclusion chromatography-multiangle light scattering revealed a molecular weight of ∼315 kDa. Quantitative immunoblotting indicated a VPS33B:VPS16B ratio of 2:3. Expression of arthrogryposis, renal dysfunction, and cholestasis syndrome-causing VPS33B missense variants showed L30P disrupts complex formation but not S243F or H344D. Truncated VPS16B (amino acids 143 to 316) was sufficient to form a complex with VPS33B. Small-angle X-ray scattering and negative-staining EM revealed a two-lobed shape for VPS33B-VPS16B. Avidin tagging indicated that each lobe contains a VPS33B molecule, and they are oriented in opposite directions. We propose a structure for VPS33B-VPS16B that allows the VPS33B at each end to interact with separate SNARE bundles and/or SNAREpins, plus associated membrane components. These observations reveal the only known potentially bidirectional Sec1-Munc18 protein complex.

Constructing Asymmetric Fe-Nb Diatomic Sites to Enhance ORR Activity and Durability.

Iron-nitrogen-carbon (Fe-N-C) materials have been identified as a promising class of platinum (Pt)-free catalysts for the oxygen reduction reaction (ORR). However, the dissolution and oxidation of Fe atoms severely restrict their long-term stability and performance. Modulating the active microstructure of Fe-N-C is a feasible strategy to enhance the ORR activity and stability. Compared with common 3d transition metals (Co, Ni, etc.), the 4d transition metal atom Nb has fewer d electrons and more unoccupied orbitals, which could potentially forge a more robust interaction with the Fe site to optimize the binding energy of the oxygen-containing intermediates while maintaining stability. Herein, an asymmetric Fe-Nb diatomic site catalyst (FeNb/c-SNC) was synthesized, which exhibited superior ORR performance and stability compared with those of Fe single-atom catalysts (SACs). The strong interaction within the Fe-Nb diatomic sites optimized the desorption energy of key intermediates (*OH), so that the adsorption energy of Fe-*OH approaches the apex of the volcano plot, thus exhibiting optimal ORR activity. More importantly, introducing Nb atoms could effectively strengthen the Fe-N bonding and suppress Fe demetalation, causing an outstanding stability. The zinc-air battery (ZAB) and hydroxide exchange membrane fuel cell (HEMFC) equipped with our FeNb/c-SNC could deliver high peak power densities of 314 mW cm-2 and 1.18 W cm-2, respectively. Notably, the stable operation time for ZAB and HEMFC increased by 9.1 and 5.8 times compared to Fe SACs, respectively. This research offers further insights into developing stable Fe-based atomic-level catalytic materials for the energy conversion process.

CD204-positive M2-like tumor-associated macrophages increase migration of gastric cancer cells by upregulating miR-210 to reduce NTN4 expression.

BACKGROUND: Tumor-associated macrophages (TAMs) are the predominant immune cells in the tumor microenvironment and portend poor prognosis. However, the molecular mechanisms underlying the tumor promotion of TAMs have not been fully elucidated. METHODS: Coculture of gastric cancer cells with U937 cells was performed to investigate the impact of TAMs on cancer cell behavior. MicroRNA (miRNA) microarray and bioinformatics were applied to identify the involved miRNAs and the functional target genes. The regulation of the miRNA on its target gene was studied using anti-miRNA and miRNA mimic. RESULTS: Coculture with CD204+ M2-like TAMs increased proliferation, migration, and epithelial-mesenchymal transition of gastric cancer cells. MiR-210 was the most upregulated miRNA in cancer cells identified by miRNA microarray after coculture. In gastric cancer tissues, miR-210 expression was positively correlated with CD204+ M2-like TAM infiltration. Inactivation of miR-210 by antimir attenuated CD204+ M2-like TAMs-induced cancer cell migration. Using pharmacological inhibitors and neutralizing antibodies, CD204+ M2-like TAMs-secreted TNFα was found to upregulate miR-210 through NF-κB/HIF-1α signaling. Bioinformatics analysis showed netrin-4 (NTN4) as a potential target of miR-210 to suppress gastric cancer cell migration. We also found an inverse expression between miR-210 and NTN4 in cancer cells after coculture or in tumor xenografts. Anti-miR-210 increased NTN4 expression, while miR-210 mimics downregulated NTN4 in cancer cells. Reporter luciferase assays showed that MiR-210 mimics suppressed NTN4 3' untranslated region-driven luciferase activity in cancer cells, but this effect was blocked after mutating miR-210 binding site. CONCLUSIONS: CD204+ M2-like TAMs can utilize the TNF-α/NF-κB/HIF-1α/miR-210/NTN4 pathway to facilitate gastric cancer progression.

Pedigree analysis exploring the inconsistency between diverse phenotypes and testing criteria for germline TP53 mutations in Chinese women with breast cancer.

PURPOSE: In the present study, we addressed the inconsistency between the testing criteria and diverse phenotypes for germline TP53 mutation in patients with breast cancer in the Chinese population. METHOD: We proposed a new added item (synchronous or metachronous bilateral breast cancer) as one of the testing criteria (aimed at high-penetrance breast cancer susceptibility genes) and applied it for determining TP53 germline mutation status in 420 female patients with breast cancer using multigene panel-based next-generation sequencing, Sanger sequencing, and mass spectrometry. RESULTS: We found that 1.4% of patients carried a pathogenic or likely pathogenic germline TP53 mutation. Compared with BRCA mutation carriers (8.0%) and non-carriers (7.1%), TP53 mutation carriers (33.3%) developed breast cancer earlier. The majority of TP53 mutation carriers (66.7%) developed breast cancer after age 30 and had bilateral breast cancer (33.3%). Pedigree investigation of four TP53 carriers and a patient with a TP53 variant of unknown significance revealed that neither of their parents harbored the same mutations as the probands, indicating that the mutations might occur de novo. CONCLUSION: Our study revealed distinguishing features of TP53 carriers among Chinese women with breast cancer, which is inconsistent with the currently used testing criteria; therefore, the newly proposed testing criteria may be more appropriate.

Multifunctionally Reusing Waste Solder to Prepare Highly Efficient Sn-Pb Perovskite Solar Cells.

The preparation of perovskite components (PbI2 and SnI2) using waste materials is of great significance for the commercialization of perovskite solar cells (PSCs). However, this goal is difficult to achieve due to the purity of the recovered products and the easy oxidation of Sn2+. Here, a simple one-step synthetic process to convert waste Sn-Pb solder into SnI2/PbI2 and then applied as-prepared SnI2/PbI2 to PSCs for high additional value is adopted. During fabrication, Sn-Pb waste solder is also employed to serve as a reducing agent to reduce the Sn4+ in Sn-Pb mixed narrow perovskite precursor and hence remove the deep trap states in perovskite. The target PSCs achieved an efficiency of 21.04%, which is better than the efficiency of the device with commercial SnI2/PbI2 (20.10%). Meanwhile, the target PSC maintained an initial efficiency of 80% even after 800 h under continuous illumination, which is significantly better than commercial devices. In addition, the method achieved a recovery rate of 90.12% for Sn-Pb waste solder, with a lab-grade purity (over 99.8%) for SnI2/PbI2, and the cost of perovskite active layer reduced to 39.81% through this recycling strategy through calculation.

Work Function Tuning of Carbon Electrode to Boost the Charge Extraction in Hole Transport Layer-Free Perovskite Solar Cells.

Perovskite solar cell (PSC) is a promising photovoltaic technology that achieves over 26% power conversion efficiency (PCE). However, the high materials costs, complicated fabrication process, as well as poor long-term stability, are stumbling blocks for the commercialization of the PSCs in normal structures. The hole transport layer (HTL)-free carbon-based PSCs (C-PSCs) are expected to overcome these challenges. However, C-PSCs have suffered from relatively low PCE due to severe energy loss at the perovskite/carbon interface. Herein, the study proposes to boost the hole extraction capability of carbon electrode by incorporating functional manganese (II III) oxide (Mn3O4). It is found that the work function (WF) of the carbon electrode can be finely tuned with different amounts of Mn3O4 addition, thus the interfacial charge transfer efficiency can be maximized. Besides, the mechanical properties of carbon electrode can also be strengthened. Finally, a PCE of 19.03% is achieved. Moreover, the device retains 90% of its initial PCE after 2000 h of storage. This study offers a feasible strategy for fabricating efficient paintable HTL-free C-PSCs.

High-Performance Monolithic 3D Integrated Complementary Inverters Based on Monolayer n-MoS2 and p-WSe2.

Herein, the structure of integrated M3D inverters are successfully demonstrated where a chemical vapor deposition (CVD) synthesized monolayer WSe2 p-type nanosheet FET is vertically integrated on top of CVD synthesized monolayer MoS2 n-type film FET arrays (2.5 × 2.5 cm) by semiconductor industry techniques, such as transfer, e-beam evaporation (EBV), and plasma etching processes. A low temperature (below 250 °C) is employed to protect the WSe2 and MoS2 channel materials from thermal decomposition during the whole fabrication process. The MoS2 NMOS and WSe2 PMOS device fabricated show an on/off current ratio exceeding 106 and the integrated M3D inverters indicate an average voltage gain of ≈9 at VDD = 2 V. In addition, the integrated M3D inverter demonstrates an ultra-low power consumption of 0.112 nW at a VDD of 1 V. Statistical analysis of the fabricated inverters devices shows their high reliability, rendering them suitable for large-area applications. The successful demonstration of M3D inverters based on large-scale 2D monolayer TMDs indicate their high potential for advancing the application of 2D TMDs in future integrated circuits.

Distinct characteristics of the gut virome in patients with osteoarthritis and gouty arthritis.

BACKGROUND/PURPOSE(S): The gut microbiota and its metabolites play crucial roles in pathogenesis of arthritis, highlighting gut microbiota as a promising avenue for modulating autoimmunity. However, the characterization of the gut virome in arthritis patients, including osteoarthritis (OA) and gouty arthritis (GA), requires further investigation. METHODS: We employed virus-like particle (VLP)-based metagenomic sequencing to analyze gut viral community in 20 OA patients, 26 GA patients, and 31 healthy controls, encompassing a total of 77 fecal samples. RESULTS: Our analysis generated 6819 vOTUs, with a considerable proportion of viral genomes differing from existing catalogs. The gut virome in OA and GA patients differed significantly from healthy controls, showing variations in diversity and viral family abundances. We identified 157 OA-associated and 94 GA-associated vOTUs, achieving high accuracy in patient-control discrimination with random forest models. OA-associated viruses were predicted to infect pro-inflammatory bacteria or bacteria associated with immunoglobulin A production, while GA-associated viruses were linked to Bacteroidaceae or Lachnospiraceae phages. Furthermore, several viral functional orthologs displayed significant differences in frequency between OA-enriched and GA-enriched vOTUs, suggesting potential functional roles of these viruses. Additionally, we trained classification models based on gut viral signatures to effectively discriminate OA or GA patients from healthy controls, yielding AUC values up to 0.97, indicating the clinical utility of the gut virome in diagnosing OA or GA. CONCLUSION: Our study highlights distinctive alterations in viral diversity and taxonomy within gut virome of OA and GA patients, offering insights into arthritis etiology and potential treatment and prevention strategies.

Prospective Investigation of Optical Genome Mapping for Prenatal Genetic Diagnosis.

BACKGROUND: Optical genome mapping (OGM) is a novel assay for detecting structural variants (SVs) and has been retrospectively evaluated for its performance. However, its prospective evaluation in prenatal diagnosis remains unreported. This study aimed to prospectively assess the technical concordance of OGM with standard of care (SOC) testing in prenatal diagnosis. METHODS: A prospective cohort of 204 pregnant women was enrolled in this study. Amniotic fluid samples from these women were subjected to OGM and SOC testing, which included chromosomal microarray analysis (CMA) and karyotyping (KT) in parallel. The diagnostic yield of OGM was evaluated, and the technical concordance between OGM and SOC testing was assessed. RESULTS: OGM successfully analyzed 204 cultured amniocyte samples, even with a cell count as low as 0.24 million. In total, 60 reportable SVs were identified through combined OGM and SOC testing, with 22 SVs detected by all 3 techniques. The diagnostic yield for OGM, CMA, and KT was 25% (51/204), 22.06% (45/204), and 18.14% (37/204), respectively. The highest diagnostic yield (29.41%, 60/204) was achieved when OGM and KT were used together. OGM demonstrated a concordance of 95.56% with CMA and 75.68% with KT in this cohort study. CONCLUSIONS: Our findings suggest that OGM can be effectively applied in prenatal diagnosis using cultured amniocytes and exhibits high concordance with SOC testing. The combined use of OGM and KT appears to yield the most promising diagnostic outcomes.

T3SS protein EsrC binds to the lacI-like operator of type 1 fimbrial operon to suppress adhesion of Edwardsiella piscicida.

Type 1 fimbria, the short hair-like appendage assembled on the bacterial surface, plays a pivotal role in adhesion and invasion in Edwardsiella piscicida. The type III secretion system (T3SS), another bacterial surface appendage, facilitates E. piscicida's replication in vivo by delivering effectors into host cells. Our previous research demonstrated that E. piscicida T3SS protein EseJ inhibits adhesion and invasion of E. piscicida by suppressing type 1 fimbria. However, how EseJ suppresses type 1 fimbria remains elusive. In this study, a lacI-like operator (nt -245 to -1 of fimA) upstream of type 1 fimbrial operon in E. piscicida was identified, and EseJ inhibits type 1 fimbria through the lacI-like operator. Moreover, through DNA pull-down and electrophoretic mobility shift assay, an AraC-type T3SS regulator, EsrC, was screened and verified to bind to nt -145 to -126 and nt -50 to -1 of fimA, suppressing type 1 fimbria. EseJ is almost abolished upon the depletion of EsrC. EsrC and EseJ impede type 1 fimbria expression. Intriguingly, nutrition and microbiota-derived indole activate type 1 fimbria through downregulating T3SS, alleviating EsrC or EseJ's inhibitory effect on lacI-like operator of type 1 fimbrial operon. By this study, it is revealed that upon entering the gastrointestinal tract, rich nutrients and indole downregulate T3SS and thereof upregulate type 1 fimbria, stimulating efficient adhesion and invasion; upon being internalized into epithelium, the limit in indole and nutrition switches on T3SS and thereof switches off type 1 fimbria, facilitating effector delivery to guarantee E. piscicida's survival/replication in vivo.IMPORTANCEIn this work, we identified the lacI-like operator of type 1 fimbrial operon in E. piscicida, which was suppressed by the repressors-T3SS protein EseJ and EsrC. We unveiled that E. piscicida upregulates type 1 fimbria upon sensing rich nutrition and the microbiota-derived indole, thereof promoting the adhesion of E. piscicida. The increase of indole and nutrition promotes type 1 fimbria by downregulating T3SS. The decrease in EseJ and EsrC alleviates their suppression on type 1 fimbria, and vice versa.

MADS-box protein PpDAM6 regulates chilling requirement-mediated dormancy and bud break in peach.

Bud dormancy is crucial for winter survival and is characterized by the inability of the bud meristem to respond to growth-promotive signals before the chilling requirement (CR) is met. However, our understanding of the genetic mechanism regulating CR and bud dormancy remains limited. This study identified PpDAM6 (DORMANCY-ASSOCIATED MADS-box) as a key gene for CR using a genome-wide association study analysis based on structural variations in 345 peach (Prunus persica (L.) Batsch) accessions. The function of PpDAM6 in CR regulation was demonstrated by transiently silencing the gene in peach buds and stably overexpressing the gene in transgenic apple (Malus × domestica) plants. The results showed an evolutionarily conserved function of PpDAM6 in regulating bud dormancy release, followed by vegetative growth and flowering, in peach and apple. The 30-bp deletion in the PpDAM6 promoter was substantially associated with reducing PpDAM6 expression in low-CR accessions. A PCR marker based on the 30-bp indel was developed to distinguish peach plants with non-low and low CR. Modification of the H3K27me3 marker at the PpDAM6 locus showed no apparent change across the dormancy process in low- and non-low- CR cultivars. Additionally, H3K27me3 modification occurred earlier in low-CR cultivars on a genome-wide scale. PpDAM6 could mediate cell-cell communication by inducing the expression of the downstream genes PpNCED1 (9-cis-epoxycarotenoid dioxygenase 1), encoding a key enzyme for ABA biosynthesis, and CALS (CALLOSE SYNTHASE), encoding callose synthase. We shed light on a gene regulatory network formed by PpDAM6-containing complexes that mediate CR underlying dormancy and bud break in peach. A better understanding of the genetic basis for natural variations of CR can help breeders develop cultivars with different CR for growing in different geographical regions.

Bone Morphogenetic Protein-4 Promotes Phenotypic Modulation via SMAD-4/MCT-4 Axis in Vascular Smooth Muscle Cells.

INTRODUCTION: This study aimed to determine whether bone morphogenetic protein-4 (BMP-4), which increases in response to intimal hyperplasia, promotes phenotype transition in vascular smooth muscle cells (VSMCs). METHODS: Balloon injury was used to induce intimal hyperplasia in rats. Hematoxylin-eosin staining was used to detect the alteration of vascular structure. Serum levels of BMP-4 and lactate were detected by ELISA. Human aortic smooth muscle cells (HA-SMCs) were cultured. Protein and mRNA expression levels were detected through Western blot and real-time PCR. Cell migration was measured by transwell assay. RESULTS: Our data showed that serum concentration of BMP-4 was upregulated after balloon injury. Treatment with BMP-4 inhibitor DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo(1,5-a)pyrimidin-3-yl)quinoline) suppressed the abnormal expression of BMP-4 and inhibited the intimal hyperplasia induced by balloon injury. Compared to BMP-4-negative medium, BMP-4-positive medium was associated with higher synthetic VSMC marker expression levels and lower in contractile gene markers in cultured HA-SMCs. Transfection of monocarboxylic acid transporters-4 (MCT-4) siRNA inhibited the excretion of lactate induced by BMP-4. CONCLUSION: Our analyses provided evidence that BMP-4 and its regulator Smad-4 are key regulators in MCT-4-mediated lactate excretion. This indicates that BMP-4 stimulates the phenotypic transition of VSMCs via SMAD-4/MCT-4 signaling pathway.