T2T reference genome assembly and genome-wide association study reveal the genetic basis of Chinese bayberry fruit quality.

Chinese bayberry (Myrica rubra or Morella rubra; 2n = 16) produces fruit with a distinctive flavor, high nutritional, and economic value. However, previous versions of the bayberry genome lack sequence continuity. Moreover, to date, no large-scale germplasm resource association analysis has examined the allelic and genetic variations determining fruit quality traits. Therefore, in this study, we assembled a telomere-to-telomere (T2T) gap-free reference genome for the cultivar 'Zaojia' using PacBio HiFi long reads. The resulting 292.60 Mb T2T genome, revealed 8 centromeric regions, 15 telomeres, and 28 345 genes. This represents a substantial improvement in the genome continuity and integrity of Chinese bayberry. Subsequently, we re-sequenced 173 accessions, identifying 6 649 674 single nucleotide polymorphisms (SNPs). Further, the phenotypic analyses of 29 fruit quality-related traits enabled a genome-wide association study (GWAS), which identified 1937 SNPs and 1039 genes significantly associated with 28 traits. An SNP cluster pertinent to fruit color was identified on Chr6: 3407532 to 5 153 151 bp region, harboring two MYB genes (MrChr6G07650 and MrChr6G07660), exhibiting differential expression in extreme phenotype transcriptomes, linked to anthocyanin synthesis. An adjacent, closely linked gene, MrChr6G07670 (MLP-like protein), harbored an exonic missense variant and was shown to increase anthocyanin production in tobacco leaves tenfold. This SNP cluster, potentially a quantitative trait locus (QTL), collectively regulates bayberry fruit color. In conclusion, our study presented a complete reference genome, uncovered a suite of allelic variations related to fruit-quality traits, and identified functional genes that could be harnessed to enhance fruit quality and breeding efficiency of bayberries.

Toxicologic effect and transcriptome analysis for sub-chronic exposure to carbendazim, prochloraz, and their combination on the liver of mice.

Carbendazim (CBZ) and prochloraz (PCZ) are broad-spectrum fungicides used in agricultural peat control. Both fungicides leave large amounts of residues in fruits and are toxic to non-target organisms. However, the combined toxicity of the fungicides to non-target organisms is still unknown. Therefore, we characterized the toxic effects of dietary supplementation with CBZ, PCZ, and their combination for 90 days in 6-week-old male Institute of Cancer Research (ICR) mice. CBZ-H (100 mg/kg day), PCZ-H (10 mg/kg day), and their combination treatments increased the relative liver weights and caused liver injury. The serum total cholesterol (TC), triglyceride (TG), glucose (Glu), pyruvate (PYR), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were reduced, and synergistic toxicity was observed. Hepatic transcriptome revealed that 326 differentially expressed genes (DEGs) of liver were observed in the CBZ treatment group, 149 DEGs in the PCZ treatment group, and 272 DEGs in the combination treatment group. According to KEGG enrichment analysis, the fungicides and their combination affected lipid metabolism, amino acid metabolism, and ferroptosis. In addition, the relative mRNA levels of key genes involved in lipid metabolism were also examined. Compared with individual exposure, combined exposure to CBZ and PCZ caused a more obvious decrease in the expression of some genes related to glycolipid metabolism. Furthermore, the relative mRNA levels of some key genes in the combination treatment group were lower than those in the CBZ and PCZ treated groups. In summary, CBZ, PCZ, and their combination generally caused hepatotoxicity and glycolipid metabolism disorders, which could provide new insights for investigating the combined toxicity of multiple fungicides to animals.

Identifying the active sites in unequal iron-nitrogen single-atom catalysts.

Single-atom catalysts (SACs) have become one of the most attractive frontier research fields in catalysis and energy conversion. However, due to the atomic heterogeneity of SACs and limitations of ensemble-averaged measurements, the essential active sites responsible for governing specific catalytic properties and mechanisms remain largely concealed. In this study, we develop a quantitative method of single-atom catalysis-fluorescence correlation spectroscopy (SAC-FCS), leveraging the atomic structure-dependent catalysis kinetics and single-turnover resolution of single-molecule fluorescence microscopy. This method enables us to investigate the oxidase-like single-molecule catalysis on unidentical iron-nitrogen (Fe-N) coordinated SACs, quantifying the active sites and their kinetic parameters. The findings reveal the significant differences of single sites from the average behaviors and corroborate the oxidase-like catalytic mechanism of the Fe-N active sites. We anticipate that the method will give essential insights into the rational design and application of SACs.

Molecular cloning and functional analysis of Chinese bayberry MrSPL4 that enhances growth and flowering in transgenic tobacco.

Chinese bayberry (Myrica rubra) is an important tree in South China, with its fruit being of nutritional and high economic value. In this study, early ripening (ZJ), medium ripening (BQ) and late ripening (DK) varieties were used as test materials. Young leaves of ZJ, BQ and DK in the floral bud morphological differentiation periods were selected for transcriptome sequencing to excavate earliness related genes. A total of 4,538 differentially expressed genes were detected. Based on clustering analysis and comparisons with genes reportedly related to flowering in Arabidopsis thaliana, 25 homologous genes were identified. Of these, one gene named MrSPL4 was determined, with its expression down-regulated in DK but up-regulated in ZJ and BQ. MrSPL4 contained SBP domain and the target site of miR156, and its total and CDS length were 1,664 bp and 555 bp respectively. The overexpression vector of MrSPL4 (35S::35S::MrSPL4-pCambia2301-KY) was further constructed and successfully transfected into tobacco to obtain MrSPL4-positive plants. Based on the results of qRT-PCR, the relative expression of MrSPL4 was up regulated by 3,862.0-5,938.4 times. Additionally, the height of MrSPL4-positive plants was also significantly higher than that of wild-type (WT), with the bud stage occurring 12 days earlier. Altogether, this study identified an important gene -MrSPL4 in Chinese bayberry, which enhanced growth and flowering, which provided important theoretical basis for early-mature breeding of Chinese bayberry.

Alcohol extracts of Chinese bayberry branch induce S-phase arrest and apoptosis in HepG2 cells.

The alcohol extracts of Chinese bayberry (Myrica rubra) branches (MRBE) are rich in flavonoids which have a variety of medicinal benefits, but their effects on human HepG2 were unknown. In this study, the effects of MRBE on HepG2 cell growth and its potential for inhibiting cancer were explored. The results displayed that MRBE inhibited HepG2 proliferation both by arresting cells in S phase and promoting apoptosis. Quantitative reverse-transcription PCR (qRT-PCR), western blotting, and immunofluorescence showed that MRBE induced S-phase arrest by upregulating p21, which in turn downregulated cyclin and cyclin-dependent kinase messenger RNA (mRNA) and protein. Apoptosis was induced by blocking the expression of BCL-2 and suppression of the Raf/ERK1 signaling pathways. These results indicated that MRBE may have the potential for treatment of human liver cancer, highlighting novel approaches for developing new pharmacological tools for the treatment of this deadly type cancer. Meanwhile, it provides a new direction for the medicinal added values of Chinese bayberry, which helped to broaden the diversified development of its industry chain.

Bubble-templated synthesis of nanocatalyst Co/C as NADH oxidase mimic.

Designing highly active nanozymes for various enzymatic reactions remains a challenge in practical applications and fundamental research. In this work, by studying the catalytic functions of natural NADH oxidase (NOX), we devised and synthesized a porous carbon-supported cobalt catalyst (Co/C) to mimic NOX. The Co/C can catalyze dehydrogenation of NADH and transfers electrons to O2 to produce H2O2. Density functional theory calculations reveal that the Co/C can catalyze O2 reduction to H2O2 or H2O considerably. The Co/C can also mediate electron transfer from NADH to heme protein cytochrome c, thereby exhibiting cytochrome c reductase-like activity. The Co/C nanoparticles can deplete NADH in cancer cells, induce increase of the reactive oxygen species, lead to impairment of oxidative phosphorylation and decrease in mitochondrial membrane potential, and cause ATP production to be damaged. This 'domino effect' facilitates the cell to approach apoptosis.

An overview of the nutritional value, health properties, and future challenges of Chinese bayberry.

Chinese bayberry (CB) is among the most popular and valuable fruits in China owing to its attractive color and unique sweet/sour taste. Recent studies have highlighted the nutritional value and health-related benefits of CB. CB has special biological characteristics of evergreen, special aroma, dioecious, nodulation, nitrogen fixation. Moreover, the fruits, leaves, and bark of CB plants harbor a number of bioactive compounds including proanthocyanidins, flavonoids, vitamin C, phenolic acids, and anthocyanins that have been linked to the anti-cancer, anti-oxidant, anti-inflammatory, anti-obesity, anti-diabetic, and neuroprotective properties and to the treatment of cardiovascular and cerebrovascular diseases. The CB fruits have been used to produce a range of products: beverages, foods, and washing supplies. Future CB-related product development is thus expected to further leverage the health-promoting potential of this valuable ecological resource. The present review provides an overview of the botanical characteristics, processing, nutritional value, health-related properties, and applications of CB in order to provide a foundation for further research and development.

Discovery of Small Molecule NSC290956 as a Therapeutic Agent for KRas Mutant Non-Small-Cell Lung Cancer.

HRas-GTP has a transient intermediate state with a "non-signaling open conformation" in GTP hydrolysis and nucleotide exchange. Due to the same hydrolysis process and the structural homology, it can be speculated that the active KRas adopts the same characteristics with the "open conformation." This implies that agents locking this "open conformation" may theoretically block KRas-dependent signaling. Applying our specificity-affinity drug screening approach, NSC290956 was chosen by high affinity and specificity interaction with the "open conformation" structure HRasG60A-GppNp. In mutant KRas-driven non-small-cell lung cancer (NSCLC) model system, NSC290956 effectively suppresses the KRas-GTP state and gives pharmacological KRas inhibition with concomitant blockages of both the MAPK-ERK and AKT-mTOR pathways. The dual inhibitory effects lead to the metabolic phenotype switching from glycolysis to mitochondrial metabolism, which promotes the cancer cell death. In the xenograft model, NSC290956 significantly reduces H358 tumor growth in nude mice by mechanisms similar to those observed in the cells. Our work indicates that NSC290956 can be a promising agent for the mutant KRas-driven NSCLC therapy.

Synthesis of adenosine analogues with indole moiety as human adenosine A3 receptor ligands.

Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3, which belong to the G-protein-coupled receptor superfamily. The human A3AR (hA3AR) subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anti-cancer and cardioprotective agents. Here, we prepared novel adenosine derivatives with indole moiety as hA3AR ligands. According to the biological assay, we found that 2-substituents 11 were critical structural determinants for A3AR ligands (Ki = 111 nM). The observed structure-affinity relationships of this class of ligands were also exhaustively rationalized using the molecular modelling approach. This allows the investigation on the binding mode of the potential compound in the ligand-binding pocket of the human A3 receptor. The results demonstrated that 11 can interact with the ASN250, GLN167, PHE168 and VAL178 through hydrogen bonding, which are shown to be important for ligand-receptor interaction.

In silico and crystallographic studies identify key structural features of biliverdin IXß reductase inhibitors having nanomolar potency.

Heme cytotoxicity is minimized by a two-step catabolic reaction that generates biliverdin (BV) and bilirubin (BR) tetrapyrroles. The second step is regulated by two non-redundant biliverdin reductases (IXα (BLVRA) and IXß (BLVRB)), which retain isomeric specificity and NAD(P)H-dependent redox coupling linked to BR's antioxidant function. Defective BLVRB enzymatic activity with antioxidant mishandling has been implicated in metabolic consequences of hematopoietic lineage fate and enhanced platelet counts in humans. We now outline an integrated platform of in silico and crystallographic studies for the identification of an initial class of compounds inhibiting BLVRB with potencies in the nanomolar range. We found that the most potent BLVRB inhibitors contain a tricyclic hydrocarbon core structure similar to the isoalloxazine ring of flavin mononucleotide and that both xanthene- and acridine-based compounds inhibit BLVRB's flavin and dichlorophenolindophenol (DCPIP) reductase functions. Crystallographic studies of ternary complexes with BLVRB-NADP+-xanthene-based compounds confirmed inhibitor binding adjacent to the cofactor nicotinamide and interactions with the Ser-111 side chain. This residue previously has been identified as critical for maintaining the enzymatic active site and cellular reductase functions in hematopoietic cells. Both acridine- and xanthene-based compounds caused selective and concentration-dependent loss of redox coupling in BLVRB-overexpressing promyelocytic HL-60 cells. These results provide promising chemical scaffolds for the development of enhanced BLVRB inhibitors and identify chemical probes to better dissect the role of biliverdins, alternative substrates, and BLVRB function in physiologically relevant cellular contexts.

Computational discovery and experimental verification of tyrosine kinase inhibitor pazopanib for the reversal of memory and cognitive deficits in rat model neurodegeneration.

Cognition and memory impairment are hallmarks of the pathological cascade of various neurodegenerative disorders. Herein, we developed a novel computational strategy with two-dimensional virtual screening for not only affinity but also specificity. We integrated the two-dimensional virtual screening with ligand screening for 3D shape, electrostatic similarity and local binding site similarity to find existing drugs that may reduce the signs of cognitive deficits. For the first time, we found that pazopanib, a tyrosine kinase inhibitor marketed for cancer treatment, inhibits acetylcholinesterase (AchE) activities at sub-micromolar concentration. We evaluated and compared the effects of intragastrically-administered pazopanib with donepezil, a marketed AchE inhibitor, in cognitive and behavioral assays including the novel object recognition test, Y maze and Morris water maze test. Surprisingly, we found that pazopanib can restore memory loss and cognitive dysfunction to a similar extent as donepezil in a dosage of 15 mg kg-1, only one fifth of the equivalent clinical dosage for cancer treatment. Furthermore, we demonstrated that pazopanib dramatically enhances the hippocampal Ach levels and increases the expression of the synaptic marker SYP. These findings suggest that pazopanib may become a viable treatment option for memory and cognitive deficits with a good safety profile in humans.

Spiclomazine induces apoptosis associated with the suppression of cell viability, migration and invasion in pancreatic carcinoma cells.

The effective treatment for pancreatic carcinoma remains critically needed. Herein, this current study showed that spiclomazine treatment caused a reduction in viability in pancreatic carcinoma cell lines CFPAC-1 and MIA PaCa-2 in vitro. It was notable in this regard that, compared with pancreatic carcinoma cells, normal human embryonic kidney (HEK-293) and liver (HL-7702) cells were more resistant to the antigrowth effect of spiclomazine. Biochemically, spiclomazine treatment regulated the expression of protein levels in the apoptosis related pathways. Consistent with this effect, spiclomazine reduced the mitochondria membrane potential, elevated reactive oxygen species, and activated caspase-3/9. In addition, a key finding from this study was that spiclomazine suppressed migration and invasion of cancer cells through down-regulation of MMP-2/9. Collectively, the proposed studies did shed light on the antiproliferation effect of spiclomazine on pancreatic carcinoma cell lines, and further clarified the mechanisms that spiclomazine induced apoptosis associated with the suppression of migration and invasion.

A small-molecule induces apoptosis and suppresses metastasis in pancreatic cancer cells.

Pancreatic cancer is one of the most malignant tumor diseases with the characters of aggressive growth and metastasis. With the inefficiency of the current therapeutics, new potential targets and new therapeutic agents for healing of pancreatic cancer are critically needed. We have previously found a small molecule, named 4-tert-butyl-2-[(cyclohexylamino) methyl]-6-methylphenol (TBMMP, NSC number: 48160), which can freeze the intermediate of Ras-GTP hydrolysis in the open non-signaling conformation with high affinity and high specificity in silico. In this work, we studied the effect and mechanism of TBMMP on two pancreatic cancer cell lines, CFPAC-1 and BxPC-3. The results showed that TBMMP could restrain the growth of the pancreatic cancer cells with IC(50) value 84.3 µM for CPFAC-1 and 94.5 µM for BxPC-3, respectively. Additionally, TBMMP increased cytochrome c release, reduced mitochondrial membrane potential, activated caspase-3, -9, elevated ROS and increased expression of the Bax in the pancreatic cancer cell lines. The results indicated that TBMMP induced the apoptosis of pancreatic cancer cells through the mitochondrial pathway. Further, we also found that TBMMP could suppress the metastasis of both pancreatic cancer cells in vitro. Taken together, we proposed that TBMMP might be a therapeutic potential lead for treating patients with pancreatic cancer.

Rational drug design: the search for Ras protein hydrolysis intermediate conformation inhibitors with both affinity and specificity.

Computer-aided drug design (CADD) plays significant roles in all stages of today's drug discovery. Many CADD technologies and methods were employed in finding promising hits against different targets during the past several decades. In this review, the most common molecular modeling methods applied to computer-aided drug design are discussed. However, how to effectively integrate these computational methods and then combine them with experiments to improve the hit rate is still a challenge. In addition, the present study reviews the ISR (intrinsic specificity ratio) as a novel concept and quantitative criterion for binding specificity to be applied as a complement in addition to binding affinity for finding new leads. Using Ras protein as a case study, Molecular modeling calculations and the subsequent biological testings for the hits are performed, the specificity of these hits is also studies against the normal and cancer cells aiming at discovering the novel chemical compounds with minimal side effects. Herein, the case study also includes the evaluations for tumor-specific cytotoxicity on different cell lines. The current results suggest that ISR is useful for quantitative assessment of specificity of small molecules.

Selective induction of apoptosis: promising therapy in pancreatic cancer.

Pancreatic cancer is one of lethal and poor prognostic malignancies. Due to the absence of effective detecting methods, quite a number of efforts have been made to improve a survival advantage for treatment in patients with pancreatic cancer. Over the past decade, single-agent gemcitabine and gemcitabine-containing combinations were considered standard first-line therapies for advanced pancreatic cancer. Although these routine uses of chemotherapy failed to significantly improve survival benefit for most therapies, these trials provided insights into the molecular mechanisms involved in the development of pancreatic cancer and therefore opened up new therapeutic avenues. Apoptotic inducer as a therapeutic concept has been widely proposed and experimentally identified in some works. Some reviews have revealed that apoptosis-inducing was a promising therapy in cancers with the least side effects and more effectiveness. Apoptosis is a highly controlled physiological mechanism and proceeds through two major pathways for apoptosis-inducing. Some anticancer drugs kill cancer cells by inducing apoptosis via death receptor pathway; however, other chemotherapeutic drugs trigger apoptosis via mitochondrial pathway. In this review, we summarize briefly current chemotherapy in pancreatic cancer, describe the apoptotic mechanisms, and provide a novel therapeutic strategy by targeting Ras intermediate.

Pocket-based drug design: exploring pocket space.

The identification and application of druggable pockets of targets play a key role in in silico drug design, which is a fundamental step in structure-based drug design. Herein, some recent progresses and developments of the computational analysis of pockets have been covered. Also, the pockets at the protein-protein interfaces (PPI) have been considered to further explore the pocket space for drug discovery. We have presented two case studies targeting the kinetic pockets generated by normal mode analysis and molecular dynamics method, respectively, in which we focus upon incorporating the pocket flexibility into the two-dimensional virtual screening with both affinity and specificity. We applied the specificity and affinity (SPA) score to quantitatively estimate affinity and evaluate specificity using the intrinsic specificity ratio (ISR) as a quantitative criterion. In one of two cases, we also included some applications of pockets located at the dimer interfaces to emphasize the role of PPI in drug discovery. This review will attempt to summarize the current status of this pocket issue and will present some prospective avenues of further inquiry.

A potent lead induces apoptosis in pancreatic cancer cells.

Pancreatic cancer is considered a lethal and treatment-refractory disease. To obtain a potent anticancer drug, the cytotoxic effect of 2-(benzo[d]oxazol-3(2H)-ylmethyl)-5-((cyclohexylamino)methyl)benzene-1,4-diol, dihydrochloride (NSC48693) on human pancreatic cancer cells CFPAC-1, MiaPaCa-2, and BxPC-3 was assessed in vitro. The proliferation of CFPAC-1, MiaPaCa-2, and BxPC-3 is inhibited with IC(50) value of 12.9±0.2, 20.6±0.3, and 6.2±0.6 µM at 48 h, respectively. This discovery is followed with additional analysis to demonstrate that NSC48693 inhibition is due to induction of apoptosis, including Annexin V staining, chromatins staining, and colony forming assays. It is further revealed that NSC48693 induces the release of cytochrome c, reduces mitochondrial membrane potential, generates reactive oxygen species, and activates caspase. These results collectively indicate that NSC48693 mainly induces apoptosis of CFPAC-1, MiaPaCa-2, and BxPC-3 cells by the mitochondrial-mediated apoptotic pathway. Excitingly, the study highlights an encouraging inhibition effect that human embryonic kidney (HEK-293) and liver (HL-7702) cells are more resistant to the antigrowth effect of NSC48693 compared to the three cancer cell lines. From this perspective, NSC48693 should help to open up a new opportunity for the treatment of patients with pancreatic cancer.

'Non-destructive' biocomputing security system based on gas-controlled biofuel cell and potentially used for intelligent medical diagnostics.

MOTIVATION: Biofuel cells (BFCs) based on enzymes and microbes are the promising future alternative sources of sustainable electrical energy under mild conditions (i.e. ambient temperature and neutral pH). By combining the adaptive behavior of BFCs self-regulating energy release with the versatility of biocomputing, we construct a novel gas-controlled biocomputing security system, which could be used as the potential implantable self-powered and 'smart' medical system with the logic diagnosis aim. RESULTS: We have demonstrated a biocomputing security system based on BFCs. Due to the unique 'RESET' reagent of N(2) applied in this work, the prepared biocomputing security system can be reset and cycled for a large number of times with no 'RESET' reagent-based 'waste'. This would be advantageous for the potential practical applications of such keypad lock as well as the development of biocomputing security devices. In order to validate the universality of the system and also to harvest energy directly from biofuels with enhanced power output, we replace the glucose with orange juice as the biofuel to operate BFCs-based biocomputing system, which also possesses the function of keypad lock. In addition, by introducing BFCs into the biocomputing security system, the adaptive behavior of the BFCs self-regulating the power release would be an immense advantage of such security keypad lock devices in potential self-powered implantable medical systems. The designed sequence gives the maximum power output and discriminate itself from the rest of the sequences. From this, we find that maximizing the dimensionless ratio of gap versus SD of the power output spectrum (a funnel in power outputs) gives the quantitative optimal design criterion. Therefore, our construction here may also provide a practical example and microscopic structural basis for mimicking the real biological network systems and bridge the gaps between the theoretical concepts and experiments important for biomolecular systems and synthetic biology.

Molecular analysis of thymopentin binding to HLA-DR molecules.

Thymopentin (TP5) triggers an immune response by contacting with T cells; however the molecular basis of how TP5 achieves this process remains incompletely understood. According to the main idea of immunomodulation, we suppose that it would be necessary for TP5 to form complex with human class II major histocompatibility complex DR molecules (HLA-DR) before TP5 interacts with T cells. The uptake of TP5 by EBV-transformed B cells expressing HLA-DR molecules and the histogram of fluorescence intensities were observed by using fluorescent- labeled TP5, testifying the direct binding of TP5 to HLA-DR. The binding specificity was confirmed by the inhibition with unlabeled TP5, suggesting the recognition of TP5 by HLA-DR. To confirm the interaction between TP5 and HLA-DR, the complex formation was predicted by using various modeling strategies including six groups of trials with different parameters, alanine substitutions of TP5, and the mutants of HLA-DR. The results demonstrated that TP5 and its alanine substitutions assumed distinct conformations when they bound to HLA-DR. The observation further showed that there was flexibility in how the peptide bound within the binding cleft. Also, the molecular analysis supplemented a newly important discovery to the effect of Val anchor on TP5 binding HLA-DR, and revealed the important effects of Glu11 and Asn62 on the recognition of TP5. These results demonstrated the capability of TP5 to associate with HLA-DR in living antigen presenting cells (APC), thereby providing a new and promising strategy to understand the immunomodulation mechanism induced by TP5 and to design potential immunoregulatory polypeptides.