Influence of the community assemblage on sulfur distributions in the South China sea.

Marine distribution of dimethylsulfoniopropionate (DMSP) and its cleavage product dimethyl sulfide (DMS) is greatly affected by the community structures of bacteria, phytoplankton, and zooplankton. Spatial distributions of dissolved and particulate DMSP (DMSPd,p), and DMS were measured and their relationships with DMSP lyase activity (DLA), abundance of DMSP-consuming bacteria (DCB), and the community structures of phytoplankton, zooplankton, and bacteria were determined during summer in the South China Sea (SCS). The depth distributions of DMSPd,p exhibited a similar trend with Chl a, reaching their maxima in the mixing layer. The DMS concentration was positively correlated with DCB abundance and DLA, indicating that DCB and DMSP lyase had a significant effect on DMS production. High DMS concentrations in the horizontal distribution coincided with high DCB abundance and DLA and may be due to the rapid growth of phytoplankton resulting from the high dissolved inorganic nitrogen concentration brought by the cold vortices. Moreover, the highest copepod abundance at station G3 coincided with the highest DMS concentrations there among stations B4, F2, and G3. These results suggest that copepod may play an important role in DMS production. The bacterial SAR11 clade was positively correlated with DLA, indicating its significant contribution to DMSP degradation in the SCS. These findings contribute to the understanding of the effect of the community assemblage on DMSP/DMS distributions in the SCS dominated by mesoscale vortices.

Effects of nanoplastics exposure on ingestion, life history traits, and dimethyl sulfide production in rotifer Brachionus plicatilis.

Microplastics (MPs) and nanoplastics (NPs) have gained global concern due to their detrimental effects on marine organisms. We investigated the effects of 80 nm polystyrene (PS) NPs on life history traits, ingestion, and dimethyl sulfide (DMS) and dimethylsulfoniopropionate (DMSP) production in the rotifer Brachionus plicatilis. Fluorescently labeled 80 nm PS NPs were ingested by the rotifer B. plicatilis and accumulated in the digestive tract. The lethal rates of B. plicatilis exposed to NPs were dose-dependent. High concentrations of PS NPs exposure had negative effects on developmental duration, leading to prolonged embryonic development and pre-reproductive periods, shortened reproductive period, post-reproductive period, and lifespan in B. plicatilis. High concentrations of PS NPs exposure inhibited life table demographic parameters such as age-specific survivorship and fecundity, generation time, net reproductive rate, and life expectancy. Consequently, the population of B. plicatilis was adversely impacted. Furthermore, exposure to PS NPs resulted in a reduced ingestion rate in B. plicatilis, as well as a decreased in DMS, particulate DMSP (DMSPp) concentration, and DMSP lyase activity (DLA), which exhibited a dose-response relationship. B. plicatilis grazing promoted DLA and therefore increased DMS production. PS NPs exposure caused a decline in the increased DMS induced by rotifer grazing. Our results help to understand the ecotoxicity of NPs on rotifer and their impact on the biogeochemical cycle of dimethylated sulfur compounds.

Molecular Classification of Endometrial Endometrioid Carcinoma With Microcystic Elongated and Fragmented Pattern.

The studies on the molecular classification of endometrioid carcinoma (EC) with microcystic, elongated, and fragmented (MELF) pattern invasion are limited. In this study, 77 cases of ECs with MELF patterns in Chinese women were collected. The molecular classification of the fifth edition of the World Health Organization was used to classify the molecular subtypes using immunohistochemistry staining (mismatch repair [MMR]-immunohistochemistry: MSH2, MSH6, MLH1, and PMS2; p53) and Sanger sequencing targeted POLE . The results showed that the prevalence of the 4 molecular subtypes in EC with MELF pattern was 6.5% (5/77) for POLE mutation, 20.8% (16/77) for MMR deficient, 11.7% (9/77) for p53-mutant, and 61.0% (47/77) for no specific molecular profile. The clinicopathological characteristics of each subtype were compared. The p53-mutant and no specific molecular profile subgroups were associated with higher International Federation of Gynecology and Obstetrics stage and International Federation of Gynecology and Obstetrics grade, deeper myometrial invasion, lymphovascular space invasion, lymph node metastasis, and absence of tumor-infiltrating lymphocytes, whereas the POLE mutation and MMR deficient subgroups were associated with lower aggressive features and prominent tumor-infiltrating lymphocytes. Progression-free survival showed that the p53-mutant and no specific molecular profile subgroups had a poorer prognosis than the POLE mutation and MMR deficient subgroups. However, lymph node metastasis was an independent factor associated with a higher risk of disease recurrence in multivariate analysis. In conclusion, ECs with MELF patterns can be divided into 4 molecular subtypes with discrepancies in aggressive clinicopathological characteristics and tumor-infiltrating lymphocytes. Molecular classification has clinical significance in a morpho-molecular approach for ECs with MELF patterns.

Identification of SIRT3 as an eraser of H4K16la.

Lysine lactylation (Kla) is a novel histone post-translational modification discovered in late 2019. Later, HDAC1-3, were identified as the robust Kla erasers. While the Sirtuin family proteins showed weak eraser activities toward Kla, as reported. However, the catalytic mechanisms and physiological functions of HDACs and Sirtuins are not identical. In this study, we observed that SIRT3 exhibits a higher eraser activity against the H4K16la site than the other human Sirtuins. Crystal structures revealed the detailed binding mechanisms between lactyl-lysine peptides and SIRT3. Furthermore, a chemical probe, p-H4K16laAlk, was developed to capture potential Kla erasers from cell lysates. SIRT3 was captured by this probe and detected via proteomic analysis. And another chemical probe, p-H4K16la-NBD, was developed to detect the eraser-Kla delactylation processes directly via fluorescence indication. Our findings and chemical probes provide new directions for further investigating Kla and its roles in gene transcription regulation.

Mutational spectrum for guiding the decision of adjuvant treatment in patients with resected biliary tract carcinoma.

BACKGROUND: Systemic chemotherapy or chemoradiation therapy has proven to be effective in treating advanced biliary tract carcinoma (BTC). However, its efficacy in the adjuvant setting remains controversial. Therefore, this study aimed to determine the prognostic significance of genomic biomarkers in resected BTC and their potential role in stratifying patients for adjuvant treatment. METHODS: We retrospectively reviewed 113 BTC patients who underwent curative-intent surgery and had available tumor sequencing data. Disease-free survival (DFS) was the primary outcome examined and univariate analysis was used to identify gene mutations with prognostic value. Favorable and unfavoratble gene subsets were distinguished from the selected genes through grouping, respectively. Multivariate Cox regression was used to identify independent prognostic factors of DFS. RESULTS: Our results indicated that mutations in ACVR1B, AR, CTNNB1, ERBB3, and LRP2 were favorable mutations, while mutations in ARID1A, CDKN2A, FGFR2, NF1, NF2, PBRM1, PIK3CA, and TGFBR1 were unfavorable mutations. In addition to age, sex, and node positive, favorable genes (HR = 0.15, 95% CI = 0.04-0.48, p = 0.001) and unfavorable genes (HR = 2.86, 95% CI = 1.51-5.29, p = 0.001) were identified as independent prognostic factors for DFS. Out of the 113 patients, only 35 received adjuvant treatment whereas the majority (78) did not. For patients with both favorable and unfavorable mutations undetected, adjuvant treatment showed negative effect on DFS (median DFS: S441 vs. 956 days, p = 0.010), but there was no significant difference in DFS among those in other mutational subgroups. CONCLUSIONS: Genomic testing might be useful in guiding the decisions regarding adjuvant treatment in BTC.

Regioselective stilbene O-methylations in Saccharinae grasses.

O-Methylated stilbenes are prominent nutraceuticals but rarely produced by crops. Here, the inherent ability of two Saccharinae grasses to produce regioselectively O-methylated stilbenes is reported. A stilbene O-methyltransferase, SbSOMT, is first shown to be indispensable for pathogen-inducible pterostilbene (3,5-bis-O-methylated) biosynthesis in sorghum (Sorghum bicolor). Phylogenetic analysis indicates the recruitment of genus-specific SOMTs from canonical caffeic acid O-methyltransferases (COMTs) after the divergence of Sorghum spp. from Saccharum spp. In recombinant enzyme assays, SbSOMT and COMTs regioselectively catalyze O-methylation of stilbene A-ring and B-ring respectively. Subsequently, SOMT-stilbene crystal structures are presented. Whilst SbSOMT shows global structural resemblance to SbCOMT, molecular characterizations illustrate two hydrophobic residues (Ile144/Phe337) crucial for substrate binding orientation leading to 3,5-bis-O-methylations in the A-ring. In contrast, the equivalent residues (Asn128/Asn323) in SbCOMT facilitate an opposite orientation that favors 3'-O-methylation in the B-ring. Consistently, a highly-conserved COMT is likely involved in isorhapontigenin (3'-O-methylated) formation in wounded wild sugarcane (Saccharum spontaneum). Altogether, our work reveals the potential of Saccharinae grasses as a source of O-methylated stilbenes, and rationalize the regioselectivity of SOMT activities for bioengineering of O-methylated stilbenes.

Effectiveness of lenvatinib plus immune checkpoint inhibitors in primary advanced hepatocellular carcinoma beyond oligometastasis.

BACKGROUND: Targeted therapy combined with immune checkpoint inhibitors is considered a promising treatment for primary advanced hepatocellular carcinoma (HCC). Nevertheless, the difference between synchronous and asynchronous treatment of lenvatinib with programmed death receptor-1 (PD-1) inhibitor in advanced HCC is still unclear. The aim of this investigation is to evaluate the effectiveness of synchronous and asynchronous of lenvatinib and PD-1 inhibitor on the advanced HCC beyond oligometastasis. METHODS: In this study, 213 patients from four institutions in China were involved. Patients were split into two collections: (1) lenvatinib plus PD-1 inhibitor were used synchronously (synchronous treatment group); (2) patients in asynchronous treatment group received PD-1 inhibitor after 3 months of lenvatinib treatment prior to tumour progression. To analyse progression-free survival (PFS), overall survival (OS), efficacy and safety of patients in both groups, we employed propensity score matching (PSM). RESULTS: The 6-, 12- and 24-month OS rates were 100%, 93.4% and 58.1% in the synchronous treatment group and 100%, 71.5% and 25.3% in the asynchronous treatment group, respectively. In contrast to the asynchronous treatment group, the group treated synchronously exhibited a substantially enhanced OS (hazard ratio [HR], 0.45; 95% confidence interval [CI], 0.30-0.66; p < .001). The 6-, 12- and 18-month PFS rates were 82.6%, 42.6% and 10.8% in the synchronous treatment group and 63.3%, 14.2% and 0% in the asynchronous treatment group, respectively. A significant difference was observed in the PFS rate (HR, 0.46; 95% CI, 0.33-0.63; p < .001) between the two collections. CONCLUSIONS: Patients with advanced HCC beyond oligometastasis, simultaneous administration of lenvatinib and PD-1 inhibitor led to significant improvements in survival.

The Role of Purine Metabolism-Related Genes PPAT and IMPDH1 in the Carcinogenesis of Intrahepatic Cholangiocarcinoma Based on Metabonomic and Bioinformatic Analyses.

In this study, we investigated the role of tumor microenvironment and serum differential metabolites in intrahepatic cholangiocarcinoma (ICC) carcinogenesis, providing new evidence for ICC treatment. Serum samples from healthy individuals and ICC patients were collected for metabolomic analysis. The purine metabolites such as inosine, guanosine, hypoxanthine, and xanthine were increased in patient serum. TCGA database samples were collected, and the correlation between purine metabolism-related genes and ICC clinical features was analyzed using R language to obtain the differential genes including PPAT, PFAS, ATIC, and IMPDH2. High PPAT expression was associated with poor ICC prognosis. A PPAT silencing model in HCCC-9810 cells was constructed. The cell phenotype was examined by qRT-PCR, CCK-8, transwell, and flow cytometry, showing a decrease in IMPDH1 expression, colony and invasive cells numbers, and an increase in apoptosis. Guanosine reversed IMPDH1 expression in HCCC-9810 cells, promoting the secretion of inflammatory factors IL-6, IL-8, OPN, VEGF, and VCAM-1 and intensifying epithelial-mesenchymal transition (EMT) progression in the cells. In nude mice, the IMPDH1 inhibitory drug MMF inhibited tumor growth and reduced the expression of tumor stem cell characteristic markers CD133 and SOX2. Guanosine accelerated the malignant progression of ICC inhibition of purine metabolism-related genes, PPAT and IMPDH2, suppressed the malignant phenotype in HCCC-9810 cells, and inhibited tumor growth.

Universal Strategy to Develop Fluorogenic Probes for Lysine Deacylase/Demethylase Activity and Application in Discriminating Demethylation States.

Dynamically controlling the post-translational modification of the ε-amino groups of lysine residues is critical for regulating many cellular events. Increasing studies have revealed that many important diseases, including cancer and neurological disorders, are associated with the malfunction of lysine deacylases and demethylases. Developing fluorescent probes that are capable of detecting lysine deacylase and demethylase activity is highly useful for interrogating their roles in epigenetic regulation and diseases. Due to the distinct substrate recognition of these epigenetic eraser enzymes, designing a universal strategy for detecting their activity poses substantial difficulty. Moreover, designing activity-based probes for differentiating their demethylation states is even more challenging and still remains largely unexplored. Herein, we report a universal strategy to construct probes that can detect the enzymatic activity of epigenetic "erasers" through NBD-based long-distance intramolecular reactions. The probes can be easily prepared by installing the O-NBD group at the C-terminal residue of specific peptide substrates by click chemistry. Based on this strategy, detecting the activity of lysine deacetylase, desuccinylase, or demethylase with superior sensitivity and selectivity has been successfully achieved through single-step probe development. Furthermore, the demethylase probe based on this strategy is capable of distinguishing different demethylation states by both absorption and fluorescence lifetime readout. We envision that these newly developed probes will provide powerful tools to facilitate drug discovery in epigenetics in the future.

linc00958/miR-185-5p/RSF-1 modulates cisplatin resistance and angiogenesis through AKT1/GSK3ß/VEGFA pathway in cervical cancer.

BACKGROUND: Chemoresistance is one of the major obstacles that lead to poor prognosis in cervical cancer. linc00958 was reported to be an oncogene in cervical cancer. However, its role in mediating chemoresistance remains to be revealed. PURPOSE: To explore the regulatory mechanisms of linc00958 in cisplatin-resistant cervical cancer cells and further validate in xenograft mice. METHODS: Online bioinformatic tools were used to conduct the pre-investigation of linc00958/miR-185-5p/RSF-1 and predict the associations between RSF-1 and AKT1/GSK3ß/VEGFA in cervical cancer. RT-qPCR measured the RNA expression levels of linc00958/miR-185-5p/RSF-1 in SiHa and SiHa/DDP. Cell survival rates were evaluated by CCK8 methods after cells were exposed to differential concentrations of DDP. Dual-luciferase reporter methods were used to measure luciferase activity. Western blot measured RSF-1 protein and phosphorylated changes of AKT1/GSK3ß. Immunofluorescence was employed to observe VEGFA secretion in vitro. Tube formation was applied to evaluate the in-vitro changes of angiogenesis. The SiHa/DDP cells stably transfected with pLKO-sh-NC or pLKO-sh-linc00958 plasmids, were injected into mice, establishing xenograft models. The changes in mice weight and tumor volumes were recorded. H&E staining and Immunohistochemistry (IHC) method was further performed. RESULTS: linc00958 expression was higher in SiHa/DDP cells. High linc00958 expression was associated with low overall survival. In SiHa/DDP cells linc00958/miR-185-5p/RSF-1 axis inhibited the cellular resistance to cisplatin and suppressed VEGFA and the tube formation through AKT1/GSK3ß/VEGFA pathway. The knockdown of linc00958 inhibited RSF-1 and Ki67, curbing tumor growth; it also inhibited VEGFA and CD34, decreasing angiogenesis in mice. CONCLUSION: linc00958/miR-185-5p/RSF-1 modulates cisplatin resistance and angiogenesis through AKT1/GSK3ß/VEGFA pathway in cervical cancer.

Downregulation of SOX21-AS1 Alleviated Cisplatin Resistance in Cervical Cancer Through Epithelial-Mesenchymal Transition Inhibition.

Cisplatin is widely used in chemotherapies in cervical cancer (CC). Nevertheless, drug resistance in cancer patients poses a major threat to efficacy of treatment. To explore the underlying modulatory mechanism of SOX21-AS1 in cisplatin resistance in CC cell and mice models, Gepia database was referred for SOX21-AS1 expression in cancer tissues and normal ones. Reverse transcription quantitative real-time polymerase chain reaction was used to measure the differential expression of SOX21-AS1 in parental Siha cells and cisplatin-resistant Siha/DDP cells. Luciferase reporter gene assays were conducted to verify putative bindings between SOX21-AS1 and miR-9-3p. Western blot method was employed to evaluate the changes in cleaved-caspase 7 protein expression. Cisplatin resistance was evaluated in each transfected group using cell counting kit 8 method after cells were exposed to cisplatin (0, 7.5, 15, 30, 60, 120, and 240 µg/mL) for 24 hours. Flow cytometry method was used to measure the apoptosis rates. Cell migration and invasion were measured using Transwell assays. Immunofluorescence method was applied to observe epithelial to mesenchymal transition (EMT) markers, including E-cadherin, Snail, matrix metalloproteinase (MMP)3, and MMP9. Siha/DDP cell groups stably transfected with sh-NC and sh-SOX21-AS1 were injected through tail vein of Balb/C mice. Lung tissue sections were used for hematoxylin and eosin staining and immunohistochemistry analysis. SOX1-AS1 expression was higher in cancer tissues than normal ones and was also higher in Siha/DDP rather than Siha cells. SOX21-AS1 was targeted by miR-9-3p in CC cells. Downregulation of SOX21-AS1 or overexpression of miR-9-3p inhibited cisplatin resistance in Siha/DDP cells and reduced cell invasion and migration and attenuated EMT progression. In vivo, the SOX21-AS1 knockdown led to less severe lung metastasis. Downregulation of SOX21-AS1 alleviated cisplatin resistance in CC through EMT inhibition.

Binding of ruthenium and osmium at non­iron sites of transferrin accounts for their iron-independent cellular uptake.

Being identified with less toxic and generally showing selective effects for solid tumor metastases, ruthenium and osmium compounds are promising drug candidates for clinical uses. Human serum proteins, such as albumin and transferrin, play vital roles in the transportation and accumulation of ruthenium and osmium agents into target tissues. However, the molecular mechanism of how transferrin transport ruthenium and their osmium analogues at atomic level remains obscure. In this study, we uncovered that the cellular uptake of Os3+ or Ru3+ are not competed by Fe3+. To unveil the molecular mechanism behind the phenomena, we report the first crystal structures of human serum transferrin (hTF) in complex with ruthenium and osmium compounds bound to the non-conserved residues on the surface of hTF without altering its overall conformation. As for Ru3+ and Os3+, these binding sites by descending affinity are: His14/His289, His349-350 ~ His578/Arg581. Ruthenium drugs and their osmium analogues preferentially bind to His14/His289 with bipyridine or imidazole ligands leaving. These binding sites on hTF surface are also available in human lactoferrin and some transferrin family member of other species. The presence of these binding sites makes the cellular uptake of Ru3+ and Os3+ less affected by Fe3+, compare to Zr4+ or Hf4+. Collectively, these findings are critical for our understanding of the role of serum transferrin in cellular delivery of ruthenium and osmium anticancer agents.

First-line or second-line PD-1 inhibition in advanced oesophageal squamous cell carcinoma: A prospective, multicentre, registry study.

WHAT IS KNOWN AND OBJECTIVE: First-line and second-line immunotherapy with programmed death-1 (PD-1) inhibitors both improve overall survival in patients with advanced oesophageal squamous cell cancer (ESCC). This study explored survival differences between first-line and second-line PD-1 inhibition in advanced ESCC. METHODS: This registry study included 167 patients with advanced ESCC who were exposed to PD-1 inhibitors in either a first-line or a second-line setting between 15 January 2019 and 31 October 2020. The primary endpoint was overall survival, and secondary endpoints included overall tumour response, progression-free survival (PFS) and PFS2. A propensity score-matching (PSM) analysis was performed using the nearest-neighbour method. RESULTS AND DISCUSSION: Sixty-one patients started first-line treatment with chemotherapy and a PD-1 inhibitor (Group 1), while 106 started chemotherapy as the first-line choice and received a PD-1 inhibitor as the second-line choice (Group 2). The median PFS was 7.1 months in Group 1 and 4.1 months in Group 2 (log-rank p = 0.001). The median PFS2 was 7.1 months in Group 1 and 7.4 months in Group 2 (log-rank p = 0.4). Before PSM, the median overall survival was 13.5 months in Group 1 and 14.1 months in Group 2 (log-rank p = 0.9), and the sensitivity analysis showed consistent results (14.0 vs. 14.1 months). After PSM, the median overall survival rates for Group 1 (n = 61) and Group 2 (n = 61) were 13.5 and 13.1 months (log-rank p = 0.7) respectively. WHAT IS NEW AND CONCLUSION: In this study, patients with advanced ESCC who received first-line or second-line PD-1 inhibitors seemed to have comparable overall survival.

Effects of Three Different Doses of Dexmedetomidine and Ropivacaine on Analgesia and the Stress Response in Hypospadias Surgery: A Randomized Trial.

Objective: This study was designed to investigate the effects of three different doses of dexmedetomidine in caudal blocks on postoperative stress and pain after pediatric urethroplasty. Methods: A total of 160 children who underwent elective urethroplasty were enrolled in this study. They were randomly divided into four groups: groups D1, D2, and D3, in which the patients were injected respectively with a mixed solution of 1, 1.5, or 2 µg kg-1 of dexmedetomidine and 0.25% ropivacaine into the sacral canal; and group R, in which the patients were injected with 0.25% ropivacaine into the sacral canal. Cortisol and interleukin-6 (IL-6) levels within 24 h, the incidence of adverse events in the circulatory system during surgery, onset time of the caudal block, duration of postoperative analgesia, the incidence of agitation during recovery, and other anesthetic adverse reactions were observed and recorded. Results: Compared with group R, cortisol and IL-6 levels in groups D1, D2, and D3 decreased within 24 h after the operation (T2-T6). The incidence of intraoperative hypertension, tachycardia, and shivering during the recovery period decreased, the onset time of the caudal block decreased, and the duration of postoperative analgesia increased (p < 0.01). Compared with group D1, the duration of postoperative analgesia increased in groups D2 and D3 (p < 0.01). Compared with groups D1 and D2, the incidence of excessive sedation and bradycardia in group D3 increased (p < 0.05). Conclusion: The administration of 1.5 µg kg-1 of dexmedetomidine appears to be most feasible in accelerating the onset of the caudal block, reducing stress and inflammation, stabilizing the circulation, increasing the duration of postoperative analgesia, and reducing anesthesia- and operation-associated adverse events.

Discovery of a Novel Specific Inhibitor Targeting Influenza A Virus Nucleoprotein with Pleiotropic Inhibitory Effects on Various Steps of the Viral Life Cycle.

Influenza A viruses (IAVs) continue to pose an imminent threat to humans due to annual influenza epidemic outbreaks and episodic pandemics with high mortality rates. In this context, the suboptimal vaccine coverage and efficacy, coupled with recurrent events of viral resistance against a very limited antiviral portfolio, emphasize an urgent need for new additional prophylactic and therapeutic options, including new antiviral targets and drugs with new mechanisms of action to prevent and treat influenza virus infection. Here, we characterized a novel influenza A virus nucleoprotein (NP) inhibitor, FA-6005, that inhibited a broad spectrum of human pandemic and seasonal influenza A and B viruses in vitro and protects mice against lethal influenza A virus challenge. The small molecule FA-6005 targeted a conserved NP I41 domain and acted as a potentially broad, multimechanistic anti-influenza virus therapeutic since FA-6005 suppressed influenza virus replication and perturbed intracellular trafficking of viral ribonucleoproteins (vRNPs) from early to late stages. Cocrystal structures of the NP/FA-6005 complex reconciled well with concurrent mutational studies. This study provides the first line of direct evidence suggesting that the newly identified NP I41 pocket is an attractive target for drug development that inhibits multiple functions of NP. Our results also highlight FA-6005 as a promising candidate for further development as an antiviral drug for the treatment of IAV infection and provide chemical-level details for inhibitor optimization.IMPORTANCE Current influenza antivirals have limitations with regard to their effectiveness and the potential emergence of resistance. Therefore, there is an urgent need for broad-spectrum inhibitors to address the considerable challenges posed by the rapid evolution of influenza viruses that limit the effectiveness of vaccines and lead to the emergence of antiviral drug resistance. Here, we identified a novel influenza A virus NP antagonist, FA-6005, with broad-spectrum efficacy against influenza viruses, and our study presents a comprehensive study of the mode of action of FA-6005 with the crystal structure of the compound in complex with NP. The influenza virus inhibitor holds promise as an urgently sought-after therapeutic option offering a mechanism of action complementary to existing antiviral drugs for the treatment of influenza virus infection and should further aid in the development of universal therapeutics.

Single-step fluorescent probes to detect decrotonylation activity of HDACs through intramolecular reactions.

Lysine crotonylation plays vital roles in gene transcription and cellular metabolism. Nevertheless, methods for dissecting the molecular mechanisms of decrotonyaltion remains limited. So far, there is no single-step fluorescent method developed for enzymatic decrotonylation activity detection. The major difficulty is that the aliphatic crotonylated lysine doesn't allow π-conjugation to a fluorophore and decrotonylation can not modulate the electronic state directly. Herein, we have designed and synthesized two activity-based single-step fluorogenic probes KTcr-I and KTcr-II for detecting enzymatic decrotonylation activity. These two probes can be recognized by histone deacetylases and undergo intramolecular nucleophilic exchange reaction to generate fluorescence signal. Notably, peptide sequence-dependent effect was observed. KTcr-I can be recognized by Sirt2 more effectively, while KTcr-II with LGKcr peptide sequence preferentially reacted with HDAC3. Compared to other methods of studying enzymatic decrotonylation activity, our single-step fluorescent method has a number of advantages, such as facileness, high sensitivity, cheap facility and little material consumed. We envision that the probes developed in this study will provide useful tools to screen inhibitors which suppress the decrotonylation activity of HDACs. Such probes will be useful for further delineating the roles of decrotonylation enzyme and aid in biomarker identification and drug discovery.

Overexpression and Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA Synthase Affect Central Metabolic Pathways in Tobacco.

Little has been established on the relationship between the mevalonate (MVA) pathway and other metabolic pathways except for the sterol and glucosinolate biosynthesis pathways. In the MVA pathway, 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to form 3-hydroxy-3-methylglutaryl-coenzyme A. Our previous studies had shown that, while the recombinant Brassica juncea HMGS1 (BjHMGS1) mutant S359A displayed 10-fold higher enzyme activity than wild-type (wt) BjHMGS1, transgenic tobacco overexpressing S359A (OE-S359A) exhibited higher sterol content, growth rate and seed yield than OE-wtBjHMGS1. Herein, untargeted proteomics and targeted metabolomics were employed to understand the phenotypic effects of HMGS overexpression in tobacco by examining which other metabolic pathways were affected. Sequential window acquisition of all theoretical mass spectra quantitative proteomics analysis on OE-wtBjHMGS1 and OE-S359A identified the misregulation of proteins in primary metabolism and cell wall modification, while some proteins related to photosynthesis and the tricarboxylic acid cycle were upregulated in OE-S359A. Metabolomic analysis indicated corresponding changes in carbohydrate, amino acid and fatty acid contents in HMGS-OEs, and F-244, a specific inhibitor of HMGS, was applied successfully on tobacco to confirm these observations. Finally, the crystal structure of acetyl-CoA-liganded S359A revealed that improved activity of S359A likely resulted from a loss in hydrogen bonding between Ser359 and acyl-CoA, which is evident in wtBjHMGS1. This work suggests that regulation of plant growth by HMGS can influence the central metabolic pathways. Furthermore, this study demonstrates that the application of the HMGS-specific inhibitor (F-244) in tobacco represents an effective approach for studying the HMGS/MVA pathway.

Structure-guided optimization of D-captopril for discovery of potent NDM-1 inhibitors.

ß-lactam antibiotics have long been the mainstay for the treatment of bacterial infections. New Delhi metallo-ß-lactamase 1 (NDM-1) is able to hydrolyze nearly all ß-lactam antibiotics and even clinically used serine-ß-lactamase inhibitors. The wide and rapid spreading of NDM-1 gene among pathogenic bacteria has attracted extensive attention, therefore high potency NDM-1 inhibitors are urgently needed. Here we report a series of structure-guided design of D-captopril derivatives that can inhibit the activity of NDM-1 in vitro and at cellular levels. Structural comparison indicates the mechanisms of inhibition enhancement and provides insights for further inhibitor optimization.

Crystal Structure of Human APPL BAR-PH Heterodimer Reveals a Flexible Dimeric BAR curve: Implication in Mutual Regulation of Endosomal Targeting.

The APPL (adaptor proteins containing pleckstrin homology domain, phosphotyrosine binding domain and a leucine zipper motif) family consists of two isoforms, APPL1 and APPL2. By binding to curved plasma membrane, these adaptor proteins associate with multiple transmembrane receptors and recruit various downstream signaling components. They are involved in the regulation of signaling pathways evoked by a variety of extracellular stimuli, such as adiponectin, insulin, FSH (follicle stimulating hormone), EGF (epidermal growth factor). And they play important roles in cell proliferation, apoptosis, glucose uptake, insulin secretion and sensitivity. However, emerging evidence suggests that APPL1 and APPL2 perform different or even opposite functions and the underlying mechanism remains unclear. As APPL proteins can either homodimerize or heterodimerize in vivo, we hypothesized that heterodimerization of APPL proteins might account for the mechanism. By solving the crystal structure of APPL1-APPL2 BAR-PH heterodimer, we find that the overall structure is crescent-shaped with a longer curvature radius of 76 Å, compared to 55 Å of the APPL1 BAR-PH homodimer. However, there is no significant difference of the curvature between APPL BAR-PH heterodimer and APPL2 homodimer. The data suggest that the APPL1 BAR-PH homodimer, APPL2 BAR-PH homodimer and APPL1/APPL2 BAR-PH heterodimer may bind to endosomes of different sizes.   Different positive charge distribution is observed on the concave surface of APPL BAR-PH heterodimer than the homodimers, which may change the affinity of membrane association and subcellular localization. Collectively, APPL2 may regulate APPL1 function through altering the preference of endosome binding by heterodimerization.

Resensitizing carbapenem- and colistin-resistant bacteria to antibiotics using auranofin.

Global emergence of Gram-negative bacteria carrying the plasmid-borne resistance genes, blaMBL and mcr, raises a significant challenge to the treatment of life-threatening infections by the antibiotics, carbapenem and colistin (COL). Here, we identify an antirheumatic drug, auranofin (AUR) as a dual inhibitor of metallo-ß-lactamases (MBLs) and mobilized colistin resistance (MCRs), two resistance enzymes that have distinct structures and substrates. We demonstrate that AUR irreversibly abrogates both enzyme activity via the displacement of Zn(II) cofactors from their active sites. We further show that AUR synergizes with antibiotics on killing a broad spectrum of carbapenem and/or COL resistant bacterial strains, and slows down the development of ß-lactam and COL resistance. Combination of AUR and COL rescues all mice infected by Escherichia coli co-expressing MCR-1 and New Delhi metallo-ß-lactamase 5 (NDM-5). Our findings provide potential therapeutic strategy to combine AUR with antibiotics for combating superbugs co-producing MBLs and MCRs.