M6A-modified lncRNA FAM83H-AS1 promotes colorectal cancer progression through PTBP1.

LncRNA plays a crucial role in cancer progression and targeting, but it has been difficult to identify the critical lncRNAs involved in colorectal cancer (CRC) progression. We identified FAM83H-AS1 as a tumor-promoting associated lncRNA using 21 pairs of stage IV CRC tissues and adjacent normal tissues. In vitro and in vivo experiments revealed that knockdown of FAM83H-AS1 in CRC cells inhibited tumor proliferation and metastasis, and vice versa. m6A modification is critical for FAM83H-AS1 RNA stability through the writer METTL3 and the readers IGF2BP2/IGFBP3. PTBP1-an RNA binding protein-is responsible for the FAM83H-AS1 function in CRC. T4 (1770-2440nt) and T5 (2440-2743nt) on exon 4 of FAM83H-AS1 provide a platform for PTBP1 RRM2 interactions. Our results demonstrated that m6A modification dysregulated the FAM83H-AS1 oncogenic role by phosphorylated PTBP1 on its RNA splicing effect. In patient-derived xenograft models, ASO-FAM83H-AS1 significantly suppressed the growth of gastrointestinal (GI) tumors, not only CRC but also GC and ESCC. The combination of ASO-FAM83H-AS1 and oxaliplatin/cisplatin significantly suppressed tumor growth compared with treatment with either agent alone. Notably, there was pathological complete response in all these three GI cancers. Our findings suggest that FAM83H-AS1 targeted therapy would benefit patients primarily receiving platinum-based therapy in GI cancers.

HIPK3 maintains sensitivity to platinum drugs and prevents disease progression in gastric cancer.

In the realm of cancer therapeutics and resistance, kinases play a crucial role, particularly in gastric cancer (GC). Our study focused on platinum-based chemotherapy resistance in GC, revealing a significant reduction in homeodomain-interacting protein kinase 3 (HIPK3) expression in platinum-resistant tumors through meticulous analysis of transcriptome datasets. In vitro and in vivo experiments demonstrated that HIPK3 knockdown enhanced tumor proliferation and metastasis, while upregulation had the opposite effect. We identified the myocyte enhancer factor 2C (MEF2C) as a transcriptional regulator of HIPK3 and uncovered HIPK3's role in downregulating the morphogenesis regulator microtubule-associated protein (MAP7) through ubiquitination. Phosphoproteome profiling revealed HIPK3's inhibitory effects on mTOR and Wnt pathways crucial in cell proliferation and movement. A combined treatment strategy involving oxaliplatin, rapamycin, and IWR1-1-endo effectively overcame platinum resistance induced by reduced HIPK3 expression. Monitoring HIPK3 levels could serve as a GC malignancy and platinum resistance indicator, with our proposed treatment strategy offering novel avenues for reversing resistance in gastric cancer.

Long noncoding RNA Regulating ImMune Escape regulates mixed lineage leukaemia protein-1-H3K4me3-mediated immune escape in oesophageal squamous cell carcinoma.

BACKGROUND: Predictive biomarkers for oesophageal squamous cell carcinoma (ESCC) immunotherapy are lacking, and immunotherapy resistance remains to be addressed. The role of long noncoding RNA (lncRNA) in ESCC immune escape and immunotherapy resistance remains to be elucidated. METHODS: The tumour-associated macrophage-upregulated lncRNAs and the exosomal lncRNAs highly expressed in ESCC immunotherapy nonresponders were identified by lncRNA sequencing and polymerase chain reaction assays. CRISPR-Cas9 was used to explore the functional roles of the lncRNA. RNA pull-down, MS2-tagged RNA affinity purification (MS2-TRAP) and RNA-binding protein immunoprecipitation (RIP) were performed to identify lncRNA-associated proteins and related mechanisms. In vivo, the humanized PBMC (hu-PBMC) mouse model was established to assess the therapeutic responses of specific lncRNA inhibitors and their combination with programmed cell death protein 1 (PD-1) monoclonal antibody (mAb). Single-cell sequencing, flow cytometry, and multiplex fluorescent immunohistochemistry were used to analyze immune cells infiltrating the tumour microenvironment. RESULTS: We identified a lncRNA that is involved in tumour immune evasion and immunotherapy resistance. High LINC02096 (RIME) expression in plasma exosomes correlates with a reduced response to PD-1 mAb treatment and poor prognosis. Mechanistically, RIME binds to mixed lineage leukaemia protein-1 (MLL1) and prevents ankyrin repeat and SOCS box containing 2 (ASB2)-mediated MLL1 ubiquitination, improving the stability of MLL1. RIME-MLL1 increases H3K4me3 levels in the promoter regions of programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1), constitutively increasing the expression of PD-L1/IDO-1 in tumour cells and inhibiting CD8+ T cells infiltration and activation. RIME depletion in huPBMC-NOG mice significantly represses tumour development and improves the effectiveness of PD-1 mAb treatment by activating T-cell-mediated antitumour immunity. CONCLUSIONS: This study reveals that the RIME-MLL1-H3K4me3 axis plays a critical role in tumour immunosuppression. Moreover, RIME appears to be a potential prognostic biomarker for immunotherapy and developing drugs that target RIME may be a new therapeutic strategy that overcomes immunotherapy resistance and benefits patients with ESCC.

The Macrophage-Associated LncRNA MALR Facilitates ILF3 Liquid-Liquid Phase Separation to Promote HIF1α Signaling in Esophageal Cancer.

SIGNIFICANCE: Secretion of TNFα by tumor-associated macrophages stimulates cancer cells to upregulate lncRNA MALR, which induces ILF3 liquid-liquid phase separation and activation of HIF1α signaling to promote cancer progression.

Aortic dissection with epileptic seizure: A case report.

BACKGROUND: Aortic dissection (AoD) is a life-threatening disease. Its diversified clinical manifestations, especially the atypical ones, make it difficult to diagnose. The epileptic seizure is a neurological problem caused by various kinds of diseases, but AoD with epileptic seizure as the first symptom is rare. CASE SUMMARY: A 53-year-old male patient suffered from loss of consciousness for 1 h and tonic-clonic convulsion for 2 min. The patient performed persistent hypomania and chest discomfort for 30 min after admission. He had a history of hypertension without regular antihypertensive drugs, and the results of his bilateral blood pressure varied greatly. Then the electroencephalogram showed the existence of epileptic waves. The thoracic aorta computed tomography angiography showed the appearance of AoD, and it originated at the lower part of the ascending aorta. Finally, the diagnosis was AoD (DeBakey, type I), acute aortic syndrome, hypertension (Grade 3), and secondary epileptic seizure. He was given symptomatic treatment to relieve symptoms and prevent complications. Thereafter, the medical therapy was effective but he refused our surgical advice. CONCLUSION: The AoD symptoms are varied. When diagnosing the epileptic seizure etiologically, AoD is important to consider by clinical and imaging examinations.

Arginine methylation of MTHFD1 by PRMT5 enhances anoikis resistance and cancer metastasis.

Metastasis accounts for the major cause of cancer-related mortality. How disseminated tumor cells survive under suspension conditions and avoid anoikis is largely unknown. Here, using a metabolic enzyme-centered CRISPR-Cas9 genetic screen, we identified methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1) as a novel suppressor of anoikis. MTHFD1 depletion obviously restrained the capacity of cellular antioxidant defense and inhibited tumor distant metastasis. Mechanistically, MTHFD1 was found to bind the protein arginine methyltransferase 5 (PRMT5) and then undergo symmetric dimethylation on R173 by PRMT5. Under suspension conditions, the interaction between MTHFD1 and PRMT5 was strengthened, which increased the symmetric dimethylation of MTHFD1. The elevated methylation of MTHFD1 largely augmented its metabolic activity to generate NADPH, therefore leading to anoikis resistance and distant organ metastasis. Therapeutically, genetic depletion or pharmacological inhibition of PRMT5 declined tumor distant metastasis. And R173 symmetric dimethylation status was associated with metastasis and prognosis of ESCC patients. In conclusion, our study uncovered a novel regulatory role and therapeutic implications of PRMT5/MTHFD1 axis in facilitating anoikis resistance and cancer metastasis.

Glucose metabolism inhibitor PFK-015 combined with immune checkpoint inhibitor is an effective treatment regimen in cancer.

Metabolic inhibition via PFKFB3 inhibition has demonstrated considerable tumor inhibitory effects in various studies; however, PFKFB3 inhibition did not show satisfactory tumor inhibition when used in clinical trials. PFKFB3 is a crucial metabolic enzyme that is highly upregulated in cancer cells and directly affects tumor glycolysis. Here, we showed that PFKFB3 inhibition suppresses tumors in vitro and in vivo in immune-deficient xenografts. However, this inhibition induces the upregulation of PD-L1 levels, which inactivated cocultured T-cells in vitro, compromises anti-tumor immunity in vivo, and reduced anti-tumor efficacy in an immune-competent mouse model. Functionally, PD-1 mAb treatment enhances the efficacy of PFKFB3 inhibition in immunocompetent and hu-PBMC NOG mouse models. Mechanistically, PFKFB3 inhibition increases phosphorylation of PFKFB3 at residue Ser461, which increases interaction with HIF-1α, and their colocalization into the nucleus, where HIF-1α transcriptionally upregulate PD-L1 expression and causes subsequent tumor immune evasion. Higher phos-PFKFB3 correlated with higher PD-L1 expression, lower CD8 and GRZMB levels, and shorter survival time in ESCC patients.

LncRNA TMPO-AS1 promotes esophageal squamous cell carcinoma progression by forming biomolecular condensates with FUS and p300 to regulate TMPO transcription.

Esophageal squamous cell carcinoma (ESCC) is one of the most life- and health-threatening malignant diseases worldwide, especially in China. Long noncoding RNAs (lncRNAs) have emerged as important regulators of tumorigenesis and tumor progression. However, the roles and mechanisms of lncRNAs in ESCC require further exploration. Here, in combination with a small interfering RNA (siRNA) library targeting specific lncRNAs, we performed MTS and Transwell assays to screen functional lncRNAs that were overexpressed in ESCC. TMPO-AS1 expression was significantly upregulated in ESCC tumor samples, with higher TMPO-AS1 expression positively correlated with shorter overall survival times. In vitro and in vivo functional experiments revealed that TMPO-AS1 promotes the proliferation and metastasis of ESCC cells. Mechanistically, TMPO-AS1 bound to fused in sarcoma (FUS) and recruited p300 to the TMPO promoter, forming biomolecular condensates in situ to activate TMPO transcription in cis by increasing the acetylation of histone H3 lysine 27 (H3K27ac). Targeting TMPO-AS1 led to impaired ESCC tumor growth in a patient-derived xenograft (PDX) model. We found that TMPO-AS1 is required for cell proliferation and metastasis in ESCC by promoting the expression of TMPO, and both TMPO-AS1 and TMPO might be potential biomarkers and therapeutic targets in ESCC.

Bilateral Hypertrophic Olivary Degeneration after Pontine Hemorrhage: A Case Report.

BACKGROUND: Hemorrhage lesions may lead to bilateral hypertrophic olivary degeneration (HOD) through interruption of the dentato-rubral-olivary pathway. The pathological features of HOD are unusual neuronal trans-synaptic degenerative changes. CASE SUMMARY: A 56-year-old female was admitted to our hospital because her lower extremities and left upper ones were unable to move for 3 mo, and the swelling of her right lower extremities became worse 3 days ago. She had a hypertension history. Her characteristic clinical manifestations are palatal myoclonus and nystagmus. The patient's magnetic resonance imaging (MRI) results showed that she had bilateral HOD after an acute pontine hemorrhage. She was given symptomatic and supportive treatment. The gabapentin, the memantine and the trihexyphenidyl were taken twice a day each. The rehabilitation and psychotherapy were implemented. After 3 months of treatment, her eye symptoms improved. CONCLUSION: Bilateral HOD is a rare phenomenon after pontine hemorrhage. The key to diagnosis lies in the clinical manifestations and MRI results.

Population pharmacokinetics-pharmacodynamics of ceftazidime in neonates and young infants: Dosing optimization for neonatal sepsis.

Ceftazidime is a third-generation cephalosporin with high activity against many pathogens. But the ambiguity and diversity of the dosing regimens in neonates and young infants impair access to effective treatment. Thus, we conducted a population pharmacokinetic study of ceftazidime in this vulnerable population and recommended a model-based dosage regimen to optimize sepsis therapy. Totally 146 neonates and young infants (gestational age (GA): 36-43.4 weeks, postnatal age (PNA): 1-81 days, current weight (CW): 900-4500 g) were enrolled based on inclusion and exclusion criteria. Ceftazidime bloods samples (203) were obtained using the opportunistic sampling strategy and determined by the high-performance liquid chromatography. The population pharmacokinetic-pharmacodynamic analysis was conducted by nonlinear mixed effects model (NONMEM). A one-compartment model with first-order elimination best described the pharmacokinetic data. Covariate analysis showed the significance of GA, PNA, and CW on developmental pharmacokinetics. Monte Carlo simulation was performed based on above covariates and minimum inhibitory concentration (MIC). In the newborns with PNA ≤ 3 days (MIC=8 mg/L), the dose regimen was 25 mg/kg twice daily (BID). For the newborns with PNA > 3 days (MIC=16 mg/L), the optimal dose was 30 mg/kg three times daily (TID) for those with GA ≤ 37 weeks and 40 mg/kg TID for those with GA > 37 weeks. Overall, on the basis of the developmental population pharmacokinetic-pharmacodynamic analysis covering the whole range of neonates and young infants, the evidence-based ceftazidime dosage regimens were proposed to optimize neonatal early-onset and late-onset sepsis therapy.

MYC-Activated LncRNA MNX1-AS1 Promotes the Progression of Colorectal Cancer by Stabilizing YB1.

Long noncoding RNAs (lncRNA) are involved in tumorigenesis and drug resistance. However, the roles and underlying mechanisms of lncRNAs in colorectal cancer are still unknown. In this work, through transcriptomic profiling analysis of 21 paired tumor and normal samples, we identified a novel colorectal cancer-related lncRNA, MNX1-AS1. MNX1-AS1 expression was significantly upregulated in colorectal cancer and associated with poor prognosis. In vitro and in vivo gain- and loss-of-function experiments showed that MNX1-AS1 promotes the proliferation of colorectal cancer cells. MNX1-AS1 bound to and activated Y-box-binding protein 1 (YB1), a multifunctional RNA/DNA-binding protein, and prevented its ubiquitination and degradation. A marked overlap between genes that are differentially expressed in MNX1-AS1 knockdown cells and transcriptional targets of YB1 was observed. YB1 knockdown mimicked the loss of viability phenotype observed upon depletion of MNX1-AS1. In addition, MYC bound the promoter of the MNX1-AS1 locus and activated its transcription. In vivo experiments showed that ASO inhibited MNX1-AS1, which suppressed the proliferation of colorectal cancer cells in both cell-based and patient-derived xenograft models. Collectively, these findings suggest that the MYC-MNX1-AS1-YB1 axis might serve as a potential biomarker and therapeutic target in colorectal cancer. SIGNIFICANCE: This study highlights the discovery of a novel colorectal cancer biomarker and therapeutic target, MNX1-AS1, a long noncoding RNA that drives proliferation via a MYC/MNX1-AS1/YB1 signaling pathway. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2636/F1.large.jpg.

Novel Genetic and Epigenetic Biomarkers of Prognostic and Predictive Significance in Stage II/III Colorectal Cancer.

The therapeutic strategies of stage II/III colorectal cancer (CRC) patients after curative surgery remain controversial. In the clinical decision-making process, oncologists need to answer questions such as whether adjuvant chemotherapy is necessary or which therapeutic regimen should be given to each patient. At present, whether adjuvant chemotherapy should be applied is primarily based on histopathological features and clinical risk factors. However, only a fraction of patients can benefit from it. More rigorous stratifying biomarkers are urgently needed to help further distinguishing these populations of patients. Recent progress in next-generation sequencing and high-throughput technologies has greatly promoted biomarker discovery as well as our understanding of the underlying mechanisms in CRC. Novel genetic and epigenetic biomarkers that are associated with prognosis or therapeutic responses have emerged. In this review, we discuss the strategies of biomarker discovery and summarize the status and assess the utility of previously published biomarkers in CRC.

LncRNA LINRIS stabilizes IGF2BP2 and promotes the aerobic glycolysis in colorectal cancer.

BACKGROUND: Long noncoding RNAs (lncRNAs) play nonnegligible roles in the epigenetic regulation of cancer cells. This study aimed to identify a specific lncRNA that promotes the colorectal cancer (CRC) progression and could be a potential therapeutic target. METHODS: We screened highly expressed lncRNAs in human CRC samples compared with their matched adjacent normal tissues. The proteins that interact with LINRIS (Long Intergenic Noncoding RNA for IGF2BP2 Stability) were confirmed by RNA pull-down and RNA immunoprecipitation (RIP) assays. The proliferation and metabolic alteration of CRC cells with LINRIS inhibited were tested in vitro and in vivo. RESULTS: LINRIS was upregulated in CRC tissues from patients with poor overall survival (OS), and LINRIS inhibition led to the impaired CRC cell line growth. Moreover, knockdown of LINRIS resulted in a decreased level of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), a newly found N6-methyladenosine (m6A) 'reader'. LINRIS blocked K139 ubiquitination of IGF2BP2, maintaining its stability. This process prevented the degradation of IGF2BP2 through the autophagy-lysosome pathway (ALP). Therefore, knockdown of LINRIS attenuated the downstream effects of IGF2BP2, especially MYC-mediated glycolysis in CRC cells. In addition, the transcription of LINRIS could be inhibited by GATA3 in CRC cells. In vivo experiments showed that the inhibition of LINRIS suppressed the proliferation of tumors in orthotopic models and in patient-derived xenograft (PDX) models. CONCLUSION: LINRIS is an independent prognostic biomarker for CRC. The LINRIS-IGF2BP2-MYC axis promotes the progression of CRC and is a promising therapeutic target.

Population pharmacokinetics and dosing optimization of latamoxef in neonates and young infants.

OBJECTIVES: There has been recent renewed interest in historical antibiotics because of the increased antibiotic-resistant bacterial strains. Latamoxef, a semi-synthetic oxacephem antibiotic developed in 1980s, has recently been brought back into use for treatment of infections in newborns; however, it is still used off-label in neonatal clinical practice due to the lack of an evidence-based dosing regimen. This study was performed to evaluate the pharmacokinetics of latamoxef in neonates and young infants, and to provide an evidence-based dosing regimen for newborns based on developmental pharmacokinetics-pharmacodynamics (PK-PD). METHODS: Opportunistic blood samples from newborns treated with latamoxef were collected to determine the latamoxef concentration by high-performance liquid chromatography with UV detection. Population PK-PD analysis was conducted using NONMEM and R software. A total of 165 plasma samples from 128 newborns (postmenstrual age range 28.4-46.1 weeks) were available for analysis. RESULTS: A two-compartment model with first-order elimination showed the best fit with the data. Current body weight, birth weight, and postnatal age were identified as significant covariates influencing latamoxef clearance. Simulation indicated that the current dosing regimen (30 mg/kg q12h) is adequate with an MIC of 1 mg/L. For an MIC of 4 mg/L, 30 mg/kg q8h was required to achieve a target rate of 70% of patients having a free antimicrobial drug concentration exceeding the MIC during 70% of the dosing interval. CONCLUSIONS: Based on the developmental PK-PD analysis of latamoxef, a rational dosing regimen of 30 mg/kg q12h or q8h was required in newborns, depending on the pathogen.

Bioamination of alkane with ammonium by an artificially designed multienzyme cascade.

Biocatalytic C-H amination is one of the most challenging tasks. C-H amination reaction can hardly be driven efficiently by solely one enzyme so far. Thus, enzymatic synergy represents an alternative strategy. Herein, we report an "Artificially Bioamination Pathway" for C-H amination of cyclohexane as a model substrate. Three enzymes, a monooxygenase P450BM3 mutant, an alcohol dehydrogenase ScCR from Streptomyces coelicolor and an amine dehydrogenase EsLeuDH from Exiguobacterium sibiricum, constituted a clean cascade reaction system with easy product isolation. Two independent cofactor regeneration systems were optimized to avoid interference from the endogenous NADH oxidases in the host E. coli cells. Based on a stepwise pH adjustment and ammonium supplement strategy, and using an in vitro mixture of cell-free extracts of the three enzymes, cyclohexylamine was produced in a titer of 14.9 mM, with a product content of up to 92.5%. Furthermore, designer cells coexpressing the three required enzymes were constructed and their capability of alkane bio-amination was examined. This artificially designed bioamination paves an attractive approach for enzymatic synthesis of amines from accessible and cheap alkanes.

Distinct Effects of Ca2+ Sparks on Cerebral Artery and Airway Smooth Muscle Cell Tone in Mice and Humans.

The effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone, as well as the underlying mechanisms, are not clear. In this investigation, we elucidated the underlying mechanisms of the distinct effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone. In CASMCs, owing to the functional loss of Ca2+-activated Cl- (Clca) channels, Ca2+ sparks activated large-conductance Ca2+-activated K+ channels (BKs), resulting in a decreases in tone against a spontaneous depolarization-caused high tone in the resting state. In ASMCs, Ca2+ sparks induced relaxation through BKs and contraction via Clca channels. However, the integrated result was contraction because Ca2+ sparks activated BKs prior to Clca channels and Clca channels-induced depolarization was larger than BKs-caused hyperpolarization. However, the effects of Ca2+ sparks on both cell types were determined by L-type voltage-dependent Ca2+ channels (LVDCCs). In addition, compared with ASMCs, CASMCs had great and higher amplitude Ca2+ sparks, a higher density of BKs, and higher Ca2+ and voltage sensitivity of BKs. These differences enhanced the ability of Ca2+ sparks to decrease CASMC and to increase ASMC tone. The higher Ca2+ and voltage sensitivity of BKs in CASMCs than ASMCs were determined by the ß1 subunits. Moreover, Ca2+ sparks showed the similar effects on human CASMC and ASMC tone. In conclusions, Ca2+ sparks decrease CASMC tone and increase ASMC tone, mediated by BKs and Clca channels, respectively, and finally determined by LVDCCs.

Efficient Degradation of Malathion in the Presence of Detergents Using an Engineered Organophosphorus Hydrolase Highly Expressed by Pichia pastoris without Methanol Induction.

The biodegradation of pesticides by organophosphorus hydrolases (OPHs) requires an efficient enzyme production technology in industry. Herein, a Pichia pastoris strain was constructed for the extracellular expression of PoOPHM9, an engineered malathion-degrading enzyme. After optimization, the maximum titer and yield of fermentation reached 50.8 kU/L and 4.1 gprotein/L after 3 days, with the highest space-time yield (STY) reported so far, 640 U L-1 h-1. PoOPHM9 displayed its high activity and stability in the presence of 0.1% (w/w) plant-derived detergent. Only 0.04 mg/mL enzyme could completely remove 0.15 mM malathion in aqueous solution within 20 min. Furthermore, 12 µmol malathion on apples and cucumbers surfaces was completely removed by 0.05 mg/mL PoOPHM9 in tap water after 35 min washing. The efficient production of the highly active PoOPHM9 has cleared a major barrier to biodegradation of pesticide residues in food industry.

Improved efficiency of a novel methyl parathion hydrolase using consensus approach.

A methyl parathion hydrolase (MPH) gene, bjmpd, was cloned from a newly isolated MP-degrading bacterial strain, Burkholderia jiangsuensis MP-1T and heterologously expressed in Escherichia coli BL21 (DE3). Although the amino acid sequence of the bjmpd-encoded enzyme, named BjMPH, differed from that of MPH from Pseudomonas sp. WBC-3 (PsMPH) in only three residues, Ser132, Val247 and Ala267, a significantly higher specific activity towards MP was exhibited by BjMPH than PsMPH. Among them, Ala267 was identified as a key site affecting the catalytic efficiency, and it was rather conservative (Ala or Ser) in homologous proteins, suggesting that a simple substitution of the residue in conservative site with another conservative residue based on the consensus sequence approach might possibly enhance the catalytic efficiency of the MP-degrading enzyme. Inspired by such an observation, we identified a new mutant, BjMPHT64N, exhibiting 3.78-fold higher catalytic efficiency (kcat/KM) towards MP than its wild-type, reaching 4.20×106M-1s-1. The mutant BjMPHT64N also displayed enhanced reactivities (kcat/KM) towards other organophosphorus pesticides. Homology-modelling analysis indicates that enhanced polar contacts of the 64th residue in this mutant may contribute to stabilizing the structure of the enzyme and promote the interactions between enzyme and substrate. This study generated an efficient MP-degrading enzyme, and provides useful information for enhancing the catalytic efficiency of MPHs via conservative residue substitution based on the consensus approach.

Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by hierarchical iteration mutagenesis.

Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling. Finally, guided by in silico energy calculations (FoldX), thermostability of the variant was improved. The mutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening <9,000 mutants, the best variant PoOPHM9 showed 25-fold higher activity than wild-type PoOPHM2 , with a thermostability (T50 (15) ) of 67.6°C. Thus, PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification. Biotechnol. Bioeng. 2016;113: 2350-2357. © 2016 Wiley Periodicals, Inc.

Non-selective cation channels mediate chloroquine-induced relaxation in precontracted mouse airway smooth muscle.

Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca2+ channels (VDLCCs). In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca2+ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca2+-free conditions, and, following a restoration of Ca2+, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca2+ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.