An inhibitory mechanism of AasS, an exogenous fatty acid scavenger: Implications for re-sensitization of FAS II antimicrobials.

Antimicrobial resistance is an ongoing "one health" challenge of global concern. The acyl-ACP synthetase (termed AasS) of the zoonotic pathogen Vibrio harveyi recycles exogenous fatty acid (eFA), bypassing the requirement of type II fatty acid synthesis (FAS II), a druggable pathway. A growing body of bacterial AasS-type isoenzymes compromises the clinical efficacy of FAS II-directed antimicrobials, like cerulenin. Very recently, an acyl adenylate mimic, C10-AMS, was proposed as a lead compound against AasS activity. However, the underlying mechanism remains poorly understood. Here we present two high-resolution cryo-EM structures of AasS liganded with C10-AMS inhibitor (2.33 Å) and C10-AMP intermediate (2.19 Å) in addition to its apo form (2.53 Å). Apart from our measurements for C10-AMS' Ki value of around 0.6 µM, structural and functional analyses explained how this inhibitor interacts with AasS enzyme. Unlike an open state of AasS, ready for C10-AMP formation, a closed conformation is trapped by the C10-AMS inhibitor. Tight binding of C10-AMS blocks fatty acyl substrate entry, and therefore inhibits AasS action. Additionally, this intermediate analog C10-AMS appears to be a mixed-type AasS inhibitor. In summary, our results provide the proof of principle that inhibiting salvage of eFA by AasS reverses the FAS II bypass. This facilitates the development of next-generation anti-bacterial therapeutics, esp. the dual therapy consisting of C10-AMS scaffold derivatives combined with certain FAS II inhibitors.

CAV3 alleviates diabetic cardiomyopathy via inhibiting NDUFA10-mediated mitochondrial dysfunction.

BACKGROUND: The progression of diabetic cardiomyopathy (DCM) is noticeably influenced by mitochondrial dysfunction. Variants of caveolin 3 (CAV3) play important roles in cardiovascular diseases. However, the potential roles of CAV3 in mitochondrial function in DCM and the related mechanisms have not yet been elucidated. METHODS: Cardiomyocytes were cultured under high-glucose and high-fat (HGHF) conditions in vitro, and db/db mice were employed as a diabetes model in vivo. To investigate the role of CAV3 in DCM and to elucidate the molecular mechanisms underlying its involvement in mitochondrial function, we conducted Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis and functional experiments. RESULTS: Our findings demonstrated significant downregulation of CAV3 in the cardiac tissue of db/db mice, which was found to be associated with cardiomyocyte apoptosis in DCM. Importantly, cardiac-specific overexpression of CAV3 effectively inhibited the progression of DCM, as it protected against cardiac dysfunction and cardiac remodeling associated by alleviating cardiomyocyte mitochondrial dysfunction. Furthermore, mass spectrometry analysis and immunoprecipitation assays indicated that CAV3 interacted with NDUFA10, a subunit of mitochondrial complex I. CAV3 overexpression reduced the degradation of lysosomal pathway in NDUFA10, restored the activity of mitochondrial complex I and improved mitochondrial function. Finally, our study demonstrated that CAV3 overexpression restored mitochondrial function and subsequently alleviated DCM partially through NDUFA10. CONCLUSIONS: The current study provides evidence that CAV3 expression is significantly downregulated in DCM. Upregulation of CAV3 interacts with NDUFA10, inhibits the degradation of lysosomal pathway in NDUFA10, a subunit of mitochondrial complex I, restores the activity of mitochondrial complex I, ameliorates mitochondrial dysfunction, and thereby protects against DCM. These findings indicate that targeting CAV3 may be a promising approach for the treatment of DCM.

Three enigmatic BioH isoenzymes are programmed in the early stage of mycobacterial biotin synthesis, an attractive anti-TB drug target.

Tuberculosis (TB) is one of the leading infectious diseases of global concern, and one quarter of the world's population are TB carriers. Biotin metabolism appears to be an attractive anti-TB drug target. However, the first-stage of mycobacterial biotin synthesis is fragmentarily understood. Here we report that three evolutionarily-distinct BioH isoenzymes (BioH1 to BioH3) are programmed in biotin synthesis of Mycobacterium smegmatis. Expression of an individual bioH isoform is sufficient to allow the growth of an Escherichia coli ΔbioH mutant on the non-permissive condition lacking biotin. The enzymatic activity in vitro combined with biotin bioassay in vivo reveals that BioH2 and BioH3 are capable of removing methyl moiety from pimeloyl-ACP methyl ester to give pimeloyl-ACP, a cognate precursor for biotin synthesis. In particular, we determine the crystal structure of dimeric BioH3 at 2.27Å, featuring a unique lid domain. Apart from its catalytic triad, we also dissect the substrate recognition of BioH3 by pimeloyl-ACP methyl ester. The removal of triple bioH isoforms (ΔbioH1/2/3) renders M. smegmatis biotin auxotrophic. Along with the newly-identified Tam/BioC, the discovery of three unusual BioH isoforms defines an atypical 'BioC-BioH(3)' paradigm for the first-stage of mycobacterial biotin synthesis. This study solves a long-standing puzzle in mycobacterial nutritional immunity, providing an alternative anti-TB drug target.

Complete pathway elucidation and heterologous reconstitution of (+)-nootkatone biosynthesis from Alpinia oxyphylla.

(+)-Nootkatone is a natural sesquiterpene ketone widely used in food, cosmetics, pharmaceuticals, and agriculture. It is also regarded as one of the most valuable terpenes used commercially. However, plants contain trace amounts of (+)-nootkatone, and extraction from plants is insufficient to meet market demand. Alpinia oxyphylla is a well-known medicinal plant in China, and (+)-nootkatone is one of the main components within the fruits. By transcriptome mining and functional screening using a precursor-providing yeast chassis, the complete (+)-nootkatone biosynthetic pathway in Alpinia oxyphylla was identified. A (+)-valencene synthase (AoVS) was identified as a novel monocot-derived valencene synthase; three (+)-valencene oxidases AoCYP6 (CYP71BB2), AoCYP9 (CYP71CX8), and AoCYP18 (CYP701A170) were identified by constructing a valencene-providing yeast strain. With further characterisation of a cytochrome P450 reductase (AoCPR1) and three dehydrogenases (AoSDR1/2/3), we successfully reconstructed the (+)-nootkatone biosynthetic pathway in Saccharomyces cerevisiae, representing a basis for its biotechnological production. Identifying the biosynthetic pathway of (+)-nootkatone in A. oxyphylla unravelled the molecular mechanism underlying its formation in planta and also supported the bioengineering production of (+)-nootkatone. The highly efficient yeast chassis screening method could be used to elucidate the complete biosynthetic pathway of other valuable plant natural products in future.

SPP1 induces idiopathic pulmonary fibrosis and NSCLC progression via the PI3K/Akt/mTOR pathway.

BACKGROUND: The prevalence of non-small cell lung cancer (NSCLC) is notably elevated in individuals diagnosed with idiopathic pulmonary fibrosis (IPF). Secreted phosphoprotein 1 (SPP1), known for its involvement in diverse physiological processes, including oncogenesis and organ fibrosis, has an ambiguous role at the intersection of IPF and NSCLC. Our study sought to elucidate the function of SPP1 within the pathogenesis of IPF and its subsequent impact on NSCLC progression. METHODS: Four GEO datasets was analyzed for common differential genes and TCGA database was used to analyze the prognosis. The immune infiltration was analyzed by TIMER database. SPP1 expression was examined in human lung tissues, the IPF fibroblasts and the BLM-induced mouse lung fibrosis model. Combined with SPP1 gene gain- and loss-of-function, qRT-PCR, Western blot, EdU and CCK-8 experiments were performed to evaluate the effects and mechanisms of SPP1 in IPF progression. Effect of SPP1 on NSCLC was detected by co-cultured IPF fibroblasts and NSCLC cells. RESULTS: Through bioinformatics analysis, we observed a significant overexpression of SPP1 in both IPF and NSCLC patient datasets, correlating with enhanced immune infiltration of cancer-associated fibroblasts in NSCLC. Elevated levels of SPP1 were detected in lung tissue samples from IPF patients and bleomycin-induced mouse models, with partial colocalization observed with α-smooth muscle actin. Knockdown of SPP1 inhibits TGF-ß1-induced differentiation of fibroblasts to myofibroblasts and the proliferation of IPF fibroblasts. Conversely, SPP1 overexpression promoted IPF fibroblast proliferation via PI3K/Akt/mTOR pathway. Furthermore, IPF fibroblasts promoted NSCLC cell proliferation and activated the PI3K/Akt/mTOR pathway; these effects were attenuated by SPP1 knockdown in IPF fibroblasts. CONCLUSIONS: Our findings suggest that SPP1 functions as a molecule promoting both fibrosis and tumorigenesis, positioning it as a prospective therapeutic target for managing the co-occurrence of IPF and NSCLC.

A novel real-time model for predicting acute kidney injury in critically ill patients within 12 hours.

BACKGROUND: A major challenge in prevention and early treatment of acute kidney injury (AKI) is the lack of high-performance predictors in critically ill patients. Therefore, we innovatively constructed U-AKIpredTM for predicting AKI in critically ill patients within 12 h of panel measurement. METHODS: The prospective cohort study included 680 patients in the training set and 249 patients in the validation set. After performing inclusion and exclusion criteria, 417 patients were enrolled in the training set and 164 patients were enrolled in the validation set finally. AKI was diagnosed by Kidney Disease Improving Global Outcomes (KDIGO) criteria. RESULTS: Twelve urinary kidney injury biomarkers (mALB, IgG, TRF, α1MG, NAG, NGAL, KIM-1, L-FABP, TIMP2, IGFBP7, CAF22 and IL-18) exhibited good predictive performance for AKI within 12 h in critically ill patients. U-AKIpredTM, combined with three crucial biomarkers (α1MG, L-FABP and IGFBP7) by multivariate logistic regression analysis, exhibited better predictive performance for AKI in critically ill patients within 12 h than the other twelve kidney injury biomarkers. The area under the curve (AUC) of the U-AKIpredTM, as a predictor of AKI within 12 h, was 0.802 (95% CI: 0.771-0.833, P < 0.001) in the training set and 0.844 (95% CI: 0.792-0.896, P < 0.001) in validation cohort. A nomogram based on the results of the training and validation sets of U-AKIpredTM was developed which showed optimal predictive performance for AKI. The fitting effect and prediction accuracy of U-AKIpredTM was evaluated by multiple statistical indicators. To provide a more flexible predictive tool, the dynamic nomogram (https://www.xsmartanalysis.com/model/U-AKIpredTM) was constructed using a web-calculator. Decision curve analysis (DCA) and a clinical impact curve were used to reveal that U-AKIpredTM with the three crucial biomarkers had a higher net benefit than these twelve kidney injury biomarkers respectively. The net reclassification index (NRI) and integrated discrimination index (IDI) were used to improve the significant risk reclassification of AKI compared with the 12 kidney injury biomarkers. The predictive efficiency of U-AKIpredTM was better than the NephroCheck® when testing for AKI and severe AKI. CONCLUSION: U-AKIpredTM is an excellent predictive model of AKI in critically ill patients within 12 h and would assist clinicians in identifying those at high risk of AKI.

An Infant-Type Hemispheric Glioma With SOX5::ALK: A Novel Fusion.

Infant-type hemispheric glioma (IHG) is a rare pediatric brain tumor with variable response to chemotherapy and radiotherapy. Molecular insights into IHG can be useful in identifying potentially active targeted therapy. A male fetus was found to have congenital hydrocephalus at the gestational age of 37 weeks. Fetal MRI showed a 2.6 × 2.0-cm tumor located at the frontal horn of the left lateral ventricle, involving the left basal nuclei and thalamus. Tumor biopsy at the age of 2 days revealed an IHG consisting of spindle tumor cells with strong expression of GFAP and ALK. Targeted RNA sequencing detected a novel fusion gene of SOX5::ALK. After initial chemotherapy with cyclophosphamide, carboplatin, and etoposide for 2 cycles, the tumor size progressed markedly and the patient underwent a subtotal resection of brain tumor followed by treatment with lorlatinib, an ALK tyrosine kinase inhibitor with central nervous system (CNS) activity. After 3 months of treatment, reduction of tumor size was observed. After 14 months of treatment, partial response was achieved, and the infant had normal growth and development. In conclusion, we identified a case of congenital IHG with a novel SOX5::ALK fusion that had progressed after chemotherapy and showed partial response and clinical benefit after treatment with the CNS-active ALK inhibitor lorlatinib.

Prediction of carbapenem-resistant gram-negative bacterial bloodstream infection in intensive care unit based on machine learning.

BACKGROUND: Predicting whether Carbapenem-Resistant Gram-Negative Bacterial (CRGNB) cause bloodstream infection when giving advice may guide the use of antibiotics because it takes 2-5 days conventionally to return the results from doctor's order. METHODS: It is a regional multi-center retrospective study in which patients with suspected bloodstream infections were divided into a positive and negative culture group. According to the positive results, patients were divided into the CRGNB group and other groups. We used the machine learning algorithm to predict whether the blood culture was positive and whether the pathogen was CRGNB once giving the order of blood culture. RESULTS: There were 952 patients with positive blood cultures, 418 patients in the CRGNB group, 534 in the non-CRGNB group, and 1422 with negative blood cultures. Mechanical ventilation, invasive catheterization, and carbapenem use history were the main high-risk factors for CRGNB bloodstream infection. The random forest model has the best prediction ability, with AUROC being 0.86, followed by the XGBoost prediction model in bloodstream infection prediction. In the CRGNB prediction model analysis, the SVM and random forest model have higher area under the receiver operating characteristic curves, which are 0.88 and 0.87, respectively. CONCLUSIONS: The machine learning algorithm can accurately predict the occurrence of ICU-acquired bloodstream infection and identify whether CRGNB causes it once giving the order of blood culture.

Prediction of successful weaning from renal replacement therapy in critically ill patients based on machine learning.

BACKGROUND: Predicting the successful weaning of acute kidney injury (AKI) patients from renal replacement therapy (RRT) has emerged as a research focus, and we successfully built predictive models for RRT withdrawal in patients with severe AKI by machine learning. METHODS: This retrospective single-center study utilized data from our general intensive care unit (ICU) Database, focusing on patients diagnosed with severe AKI who underwent RRT. We evaluated RRT weaning success based on patients being free of RRT in the subsequent week and their overall survival. Multiple logistic regression (MLR) and machine learning algorithms were adopted to construct the prediction models. RESULTS: A total of 976 patients were included, with 349 patients successfully weaned off RRT. Longer RRT duration (7.0 vs. 9.6 d, p = 0.002, OR = 0.94), higher serum cystatin C levels (1.2 vs. 3.2 mg/L, p < 0.001, OR = 0.46), and the presence of septic shock (28.1% vs. 41.5%, p < 0.001, OR = 0.63) were associated with reduced likelihood of RRT weaning. Conversely, a positive furosemide stress test (FST) (60.2% vs. 40.7%, p < 0.001, OR = 2.75) and higher total urine volume 3 d before RRT withdrawal (755 vs. 125 mL/d, p < 0.001, OR = 2.12) were associated with an increased likelihood of successful weaning from RRT. Next, we demonstrated that machine learning models, especially Random Forest and XGBoost, achieving an AUROC of 0.95. The XGBoost model exhibited superior accuracy, yielding an AUROC of 0.849. CONCLUSION: High-risk factors for unsuccessful RRT weaning in severe AKI patients include prolonged RRT duration. Machine learning prediction models, when compared to models based on multivariate logistic regression using these indicators, offer distinct advantages in predictive accuracy.

[2-DG improves lung ischemia/reperfusion injury by inhibiting NLRP3-mediated pyroptosis in rats].

The aim of this study was to investigate whether the protective effect of 2-deoxyglucose (2-DG) on lung ischemia/reperfusion (I/R) injury is mediated by inhibiting nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)-mediated pyroptosis in rats. Male Sprague-Dawley rats were randomly divided into control group, 2-DG group, lung I/R injury group (I/R group) and 2-DG+I/R group. 2-DG (0.7 g/kg) was intraperitoneally injected 1 h prior to lung ischemia. The tissue structure was measured under light microscope. Lung injury parameters were detected. The contents of malondialdehyde (MDA), myeloperoxidase (MPO) and lactate were determined by commercially available kits. ELISA was used to detect the levels of IL-1ß and IL-18. Western blot, qRT-PCR and immunofluorescence staining were used to measure the expression changes of glycolysis and pyroptosis related indicators. The results showed that there was no significant difference in the parameters between the control group and the 2-DG group. However, the lung injury parameters, oxidative stress response, lactic acid content, IL-1ß, and IL-18 levels were significantly increased in the I/R group. The protein expression levels of glycolysis and pyroptosis related indicators including hexokinase 2 (HK2), pyruvate kinase 2 (PKM2), NLRP3, Gasdermin superfamily member GSDMD-N, cleaved-Caspase1, cleaved-IL-1ß and cleaved-IL-18, and the gene expression levels of HK2, PKM2 and NLRP3 were markedly up-regulated in the I/R group compared with those in the control group. The expression of HK2 and NLRP3 was also increased detected by immunofluorescence staining. Compared with the I/R group, the 2-DG+I/R group exhibited significantly improved alveolar structure and inflammatory infiltration, reduced lung injury parameters, and decreased expression of glycolysis and pyroptosis related indicators. These results suggest that 2-DG protects against lung I/R injury possibly by inhibiting NLRP3-mediated pyroptosis in rats.

Immune response and protective efficacy of mannosylated polyethylenimine (PEI) as an antigen delivery vector, administered with a Streptococcus agalactiae DNA vaccine in Nile tilapia (Oreochromis niloticus).

This study examined the effectiveness of a DNA vaccine for S. agalactiae that was delivered by mannose-based polyethyleneimine (Man-PEI). The results showed that Man-PEI/pcDNA-Sip stimulated a higher serum antibody titer compared to control or other vaccine groups (p < 0.05). Additionally, it induced higher expression of immune-related genes, and increased activities of superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Furthermore, the Man-PEI/pcDNA-Sip group showed an improved relative percent survival (RPS) of 85.71%. These results demonstrate the potential value of Man-PEI as a vaccine delivery vehicle, and suggest that it can be effective in boosting the immune protective rate induced by pcDNA-Sip vaccines.

Serum Ferritin in Geriatric Individuals in the Shanghai Region: Distribution, Correlations, and Reference Intervals.

BACKGROUND: Serum ferritin levels have a clinical application in diagnosing diseases. However, the clinical standard levels and distribution characteristics of serum ferritin based on reference intervals (RIs) in the geriatric Han Chinese population in the East China region have not previously been well reported. This work aimed to investigate the correlation between serum ferritin levels and 14 metabolic markers, analyse the distribution of serum ferritin, and establish serum ferritin RIs for geriatric (> 60 years) individuals in Shanghai. METHODS: Four hundred and sixty-nine healthy Chinese Han subjects (age, 61 - 95 years; median, 71 years) were recruited from the Health Examination Center of Shanghai Fourth People's Hospital in 2021. Serum ferritin was measured on a Roche Cobas 8000 e602, and 14 biochemical parameters were measured on a Siemens Atellica CH-930 to analyse distributions and correlations and to establish serum ferritin RIs for the elderly population in Shanghai. RESULTS: Serum ferritin levels were significantly different between genders (p = 0.06). The established RIs for serum ferritin were 24.44 - 627.09 ng/mL and 48.18 - 554.88 ng/mL in males and females, respectively. Correlation analyses revealed that ferritin levels were correlated with 7 parameters, including body mass index (BMI, p = 0.02), gamma-glutamyl transferase (GGT, p < 0.01), alanine aminotransferase (ALT, p < 0.01), triglycerides (TGs, p < 0.01), high-density lipoprotein (HDL, p < 0.01), total protein (TP, p < 0.01) and prealbumin (PAB, p < 0.01). When the participants were further divided by BMI, aspartate aminotransferase (AST) was an additional variable that was positively correlated only in the overweight/obese group (p = 0.04), while globulin (GLO) was an additional variable that was positively correlated only in the other group (p < 0.01). CONCLUSIONS: Nutrition and metabolism may play a great role in the regulation of serum ferritin levels in geriatric individuals in vivo. The RIs established for serum ferritin may provide precise references for further studies on ferritin-related disease in geriatric individuals.

In Situ Electrochemically Activated Vanadium Oxide Cathode for Advanced Aqueous Zn-Ion Batteries.

The search for large-capacity and high-energy-density cathode materials for aqueous Zn-ion batteries is still challenging. Here, an in situ electrochemical activation strategy to boost the electrochemical activity of a carbon-confined vanadium trioxide (V2O3@C) microsphere cathode is demonstrated. Tunnel-structured V2O3 undergoes a complete phase transition to a layered, amorphous, and oxygen-deficient Zn0.4V2O5-m·nH2O on the first charge, thus allowing subsequent (de)intercalation of zinc cations on the basis of the latter structure, which can be regulated by the amount of H2O in the electrolyte. The electrode thus delivers excellent stability with a significantly high capacity of 602 mAh g-1 over 150 cycles upon being subjected to a low-current-rate cycling, as well as a high-energy density of 439.6 Wh kg-1 and extended life up to 10000 cycles with a 90.3% capacity retention. This strategy will be exceptionally desirable to achieve ultrafast Zn-ion storage with high capacity and energy density.

Increasing astrogenesis in the developing hippocampus induces autistic-like behavior in mice via enhancing inhibitory synaptic transmission.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized primarily by impaired social communication and rigid, repetitive, and stereotyped behaviors. Many studies implicate abnormal synapse development and the resultant abnormalities in synaptic excitatory-inhibitory (E/I) balance may underlie many features of the disease, suggesting aberrant neuronal connections and networks are prone to occur in the developing autistic brain. Astrocytes are crucial for synaptic formation and function, and defects in astrocytic activation and function during a critical developmental period may also contribute to the pathogenesis of ASD. Here, we report that increasing hippocampal astrogenesis during development induces autistic-like behavior in mice and a concurrent decreased E/I ratio in the hippocampus that results from enhanced GABAergic transmission in CA1 pyramidal neurons. Suppressing the aberrantly elevated GABAergic synaptic transmission in hippocampal CA1 area rescues autistic-like behavior and restores the E/I balance. Thus, we provide direct evidence for a developmental role of astrocytes in driving the behavioral phenotypes of ASD, and our results support that targeting the altered GABAergic neurotransmission may represent a promising therapeutic strategy for ASD.

Rationally Designed Three-Layered TiO2 @amorphous MoS3 @Carbon Hierarchical Microspheres for Efficient Potassium Storage.

Amorphous MoS3 has been an attractive electrode material for sodium-ion batteries and lithium-sulfur batteries. However, the potassium storage capability of amorphous MoS3 remains unreported. Herein, the construction of hybrid hierarchical microspheres composed of amorphous MoS3 nanosheets dual-confined with TiO2 core, and nitrogen-doped carbon shell layer (denoted as TiO2 @A-MoS3 @NC) via a self-templating method, combined with a low-temperature sulfurization process as a new anode material for potassium-ion batteries (PIBs), is reported. Benefitting from the unique structural merits including unique 1D chain structure, disordered arrangement of atoms and a large number of defects of amorphous MoS3 , more active heterointerfacial sites, effectively mitigated volume change, good electrical contact, and easy K+ ion migration, the TiO2 @A-MoS3 @NC microspheres exhibit excellent potassium-storage performance with high specific capacity, superior rate capability, and cycling stability.

Coupling cell growth and biochemical pathway induction in Saccharomyces cerevisiae for production of (+)-valencene and its chemical conversion to (+)-nootkatone.

(+)-Nootkatone is a valuable, functional sesquiterpene that is widely used in food, cosmetics, pharmaceutical, agriculture, and other fields. However, only traces of it accumulate in plants, which is insufficient to meet the market demand. Therefore, commercial (+)-nootkatone is currently synthesized from (+)-valencene. Here, we engineered Saccharomyces cerevisiae to achieve high production of (+)-valencene. Employing gene screening, protein engineering and biosynthetic pathway optimization, we achieved 12.4 g/L (+)-valencene production with the mutant strain. This titer was further increased to 16.6 g/L, the highest titer reported to date, by coupling critical factors for cell growth and biochemical pathway induction. Subsequently, (+)-nootkatone was chemically synthesized from bio-fermented (+)-valencene with a yield of 80%. This study achieved efficient microbial synthesis of (+)-valencene, which may be utilized in industrial production and stabilize the supply of (+)-nootkatone.

Systematic identification of Ocimum sanctum sesquiterpenoid synthases and (-)-eremophilene overproduction in engineered yeast.

Plant-derived natural active products have attracted increasing attention for use in flavors and perfumes. These compounds also have applications in insect pest control because of their environment-friendly properties. Holy basil (Ocimum sanctum), a famous herb used in Ayurveda in India, is a natural source of medical healing agents and insecticidal repellents. Despite the available genomic sequences and genome-wide bioinformatic analysis of terpene synthase genes, the functionality of the sesquiterpene genes involved in the unique fragrance and insecticidal activities of Holy basil are largely unknown. In this study, we systematically screened the sesquiterpenoid biosynthesis genes in this plant using a precursor-providing yeast system. The enzymes that synthesize ß-caryophyllene and its close isomer α-humulene were successfully identified. The enzymatic product of OsaTPS07 was characterized by in vivo mining, in vitro reaction, and NMR detection. This product was revealed as (-)-eremophilene. We created a mutant yeast strain that can achieve a high-yield titer by adjusting the gene copy number and FPP precursor enhancement. An optimized two-stage fed-batch fermentation method achieved high biosynthetic capacity, with a titer of 34.6 g/L cyclic sesquiterpene bioproduction in a 15-L bioreactor. Further insect-repelling assays demonstrated that (-)-eremophilene repelled the insect pest, fall leafworm, suggesting the potential of (-)-eremophilene as an alternative to synthetic chemicals for agricultural pest control. This study highlights the potential of our microbial platform for the bulk mining of plant-derived ingredients and provides an impressive cornerstone for their industrial utilization.

Concurrent definitive chemoradiation incorporating intensity-modulated radiotherapy followed by adjuvant chemotherapy in high risk locally advanced cervical squamous cancer: a phase II study.

BACKGROUND: Although the prognosis of locally advanced cervical cancer has improved dramatically, survival for those with stage IIIB-IVA disease or lymph nodes metastasis remains poor. It is believed that the incorporation of intensity-modulated radiotherapy into the treatment of cervical cancer might yield an improved loco-regional control, whereas more cycles of more potent chemotherapy after the completion of concurrent chemotherapy was associated with a diminished distant metastasis. We therefore initiated a non-randomized prospective phaseII study to evaluate the feasibility of incorporating both these two treatment modality into the treatment of high risk locally advanced cervical cancer. OBJECTIVES: To determine whether the incorporation of intensity-modulated radiotherapy and the addition of adjuvant paclitaxel plus cisplatin regimen into the treatment policy for patients with high risk locally advanced cervical cancer might improve their oncologic outcomes. STUDY DESIGN: Patients were enrolled if they had biopsy proven stage IIIA-IVA squamous cervical cancer or stage IIB disease with metastatic regional nodes. Intensity-modulated radiotherapy was delivered with dynamic multi-leaf collimators using 6MV photon beams. Prescription for PTV ranged from 45.0 ~ 50.0 Gy at 1.8 Gy ~ 2.0 Gy/fraction in 25 fractions. Enlarged nodes were contoured separately and PTV-nodes were boosted simultaneously to a total dose of 50.0-65 Gy at 2.0- 2.6 Gy/fraction in 25 fractions. A total dose of 28 ~ 35 Gy high-dose- rate brachytherapy was prescribed to point A in 4 ~ 5 weekly fractions using an iridium- 192 source. Concurrent weekly intravenous cisplatin at 30 mg/m2 was initiated on the first day of radiotherapy for over 1-h during external-beam radiotherapy. Adjuvant chemotherapy was scheduled within 4 weeks after the completion of concurrent chemo-radiotherapy and repeated 3 weeks later. Paclitaxel 150 mg/m2 was given as a 3-h infusion on day1, followed by cisplatin 35 mg/m2 with 1-h infusion on day1-2 (70 mg/m2 in total). RESULTS: Fifty patients achieved complete response 4 weeks after the completion of the treatment protocol, whereas 2 patients had persistent disease. After a median follow-up period of 66 months, loco-regional (including 2 persistent disease), distant, and synchronous treatment failure occurred in 4,5, and 1, respectively. The 5-year disease-free survival, loco-regional recurrence-free survival, distant-metastasis recurrence-free survival was 80.5%, 90.3%, and 88.0%, respectively. Four of the patients died of the disease, and the 5-year overall survival was 92.1%. Most of the toxicities reported during concurrent chemo-radiotherapy were mild and transient. The occurrence of hematological toxicities elevated mildly during adjuvant chemotherapy, as 32% (16/50) and 4% (2/50) patients experienced grade 3-4 leukopenia and thrombocytopenia, respectively. Grade 3-4 late toxicities were reported in 3 patients. CONCLUSIONS: The incorporation of intensity-modulated radiotherapy and adjuvant paclitaxel plus cisplatin chemotherapy were highly effective and well-tolerated in the treatment of high-risk locally advanced cervical cancer. The former yields an improved loco-regional control, whereas distant metastases could be effectively eradicated with mild toxicities when adjuvant regimen was prescribed.

Fabrication of Adsorption-Type Hierarchical Functional Films by Using a Facile Swollen Based Breath Figure Method.

The morphology transition from primary to hierarchical adsorption-type microporous domains of amphiphilic block copolymer (BCP) honeycomb-structured films is demonstrated by a facile swollen based breath figure (BF) method. The characteristic parameters of poly(4-vinylpyridine)-block-polystyrene (P4VP-b-PS) hierarchical micro- and submicroporous films can be controlled by changing the length of segments or subsequent swelling conditions. A plausible mechanism is demonstrated in this research. A typical amphiphilic BCP with very low volume content of hydrophilic blocks (fP4VP ≤ 0.050) can efficiently stabilize water droplets and inherently assist in the formation of morphology transition. This BCP film can be used for Cr(VI) removal from wastewater, which additionally has enormous potential application in the field of novel optical devices, soft lithography, size-selective separation, etc.

Enrichment of Tumor-Infiltrating B Cells in Group 4 Medulloblastoma in Children.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is classified into core molecular subgroups (wingless activated (WNT), sonic hedgehog activated (SHH), Group 3 (G3), and Group 4 (G4)). In this study, we analyzed the tumor-infiltrating immune cells and cytokine profiles of 70 MB patients in Taiwan using transcriptome data. In parallel, immune cell composition in tumors from the SickKids cohort dataset was also analyzed to confirm the findings. The clinical cohort data showed the WNT and G4 MB patients had lower recurrence rates and better 5-year relapse-free survival (RFP) compared with the SHH and G3 MB patients, among the four subgroups of MB. We found tumor-infiltrating B cells (TIL-Bs) enriched in the G4 subgroups in the Taiwanese MB patients and the SickKids cohort dataset. In the G4 subgroups, the patients with a high level of TIL-Bs had better 5-year overall survival. Mast cells presented in G4 MB tumors were positively correlated with TIL-Bs. Higher levels of CXCL13, IL-36γ, and CCL27 were found compared to other subgroups or normal brains. These three cytokines, B cells and mast cells contributed to the unique immune microenvironment in G4 MB tumors. Therefore, B-cell enrichment is a G4-subgroup-specific immune signature and the presence of B cells may be an indicator of a better prognosis in G4 MB patients.