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Disorders of esophagogastric junction (EGJ) outflow, including achalasia and EGJ outflow obstruction, are motility disorders characterized by inadequate relaxation of lower esophageal sphincter with or without impaired esophageal peristalsis. Current guidelines are technical and less practical in the Asia-Pacific region, and there are still massive challenges in timely diagnosis and managing these disorders effectively. Therefore, a Malaysian joint societies' task force has developed a consensus on disorders of EGJ outflow based on the latest evidence, while taking into consideration the practical relevance of local and regional context and resources. Twenty-one statements were established after a series of meetings and extensive review of literatures. The Delphi method was used in the consensus voting process. This consensus focuses on the definition, diagnostic investigations, the aims of treatment outcome, non-surgical or surgical treatment options, management of treatment failure or relapse, and the management of complications. This consensus advocates the use of high-resolution esophageal manometry for diagnosis of disorders of EGJ outflow. Myotomy, via either endoscopy or laparoscopy, is the preferred treatment option, while pneumatic dilatation can serve as a secondary option. Evaluation and management of complications including post-procedural reflux and cancer surveillance are recommended., (© 2023 The Authors. Journal of Gastroenterology and Hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Introduction: Per-Oral Endoscopic Myotomy (POEM) is an effective treatment for Esophageal Achalasia Cardia (EAC) but the endoscopic technique required is complex. As competency is crucial for patient safety, we believe that its' competency can be demonstrated when the complication rate equals that of an established procedure such as Laparoscopic Heller's Myotomy with Fundoplication (LHM + F)., Methods: A multicentre, ambi-directional, non-randomized comparison of intra-procedural complications during the learning curve of POEM was performed against a historical cohort of LHM + F. Demographic, clinicopathological, procedural data and complications were collected. A direct head-to-head comparison was performed, followed by a population pyramid of complication frequency. Case sequence was then divided into blocks of 5, and the complication rates during each block was compared to the historical cohort., Results: From January 2010 to April 2021, 60 patients underwent LHM + F and 63 underwent POEM. Mean age was lower for the POEM group (41.7 years vs 48.1 years, p = 0.03), but there was no difference in gender nor type of Achalasia. The POEM group recorded a shorter overall procedural time (125.9 min vs 144.1 min, p = 0.023) and longer myotomies (10.1 cm vs 6.2 cm, p = 0.023). The overall complication rate of POEM was 20.6%, whereas the historical cohort of LHM + F had a rate of 10.0%. On visual inspection of the population pyramid, complications were more frequent in the earlier procedures. On block sequencing, complication frequency could be seen tapering off dramatically after the 25th case, and subsequently equalled that of LHM + F., Conclusion: POEM is challenging even for experienced endoscopists. From our data, complication rates between POEM and LHM + F equalize after approximately 25 POEMs., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Peroral endoscopic myotomy (POEM) has rapidly gained popularity as an effective treatment modality for achalasia. However, POEM services in the South East Asian region are not widely available due to either a lack of expertise or interest. In this article, we describe how a POEM service can be developed through a combination of networking with regional experts, having prior experience of endoscopic submucosal dissection (ESD), attending animal model workshops, collaborating with upper gastrointestinal surgeons, and working together in a multidisciplinary team. A total of 68 POEM procedures have been performed since 2015, with a 94.1% technical and 93.4% clinical success rate, and a 21.5% minor complication rate. We believe that our model may be useful for other Endoscopy Units in the region, which are performing advanced therapeutic endoscopy, to develop a POEM service too., (© 2021 The Authors. JGH Open: An open access journal of gastroenterology and hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background: The association between dysregulated microRNAs (miRNAs) and acute myeloid leukemia (AML) is well known. However, our understanding of the regulatory role of miRNAs in the cytogenetically normal AML (CN-AML) subtype pathway is still poor. The current study integrated miRNA and mRNA profiles to explore novel miRNA-mRNA interactions that affect the regulatory patterns of de novo CN-AML. Methods: We utilized a multiplexed nanoString nCounter platform to profile both miRNAs and mRNAs using similar sets of patient samples ( n  = 24). Correlations were assessed, and an miRNA-mRNA network was constructed. The underlying biological functions of the mRNAs were predicted by gene enrichment. Finally, the interacting pairs were assessed using TargetScan and microT-CDS. We identified 637 significant negative correlations (false discovery rate <0.05). Results: Network analysis revealed a cluster of 12 miRNAs representing the majority of mRNA targets. Within the cluster, five miRNAs (miR-495-3p, miR-185-5p, let-7i-5p, miR-409-3p, and miR-127-3p) were posited to play a pivotal role in the regulation of CN-AML, as they are associated with the negative regulation of myeloid leukocyte differentiation, negative regulation of myeloid cell differentiation, and positive regulation of hematopoiesis. Conclusion: Three novel interactions in CN-AML were predicted as let-7i-5p: HOXA9 , miR-495-3p: PIK3R1 , and miR-495-3p: CDK6 may be responsible for regulating myeloid cell differentiation in CN-AML.

Background: Peritoneal recurrence of gastric cancer after curative surgical resection is common and portends a poor prognosis. Early studies suggest that extensive intraoperative peritoneal lavage (EIPL) might reduce the risk of peritoneal recurrence and improve survival. We aimed to evaluate the survival benefit of EIPL in patients with gastric cancer undergoing curative gastrectomy., Methods: In this open-label, phase 3, multicentre randomised trial, patients aged 21-80 years with cT3 or cT4 gastric cancer undergoing curative resection were enrolled at 22 centres from South Korea, China, Japan, Malaysia, Hong Kong, and Singapore. Patients were randomly assigned to receive surgery and EIPL (EIPL group) or surgery alone (standard surgery group) via a web-based programme in random permuted blocks in varying block sizes of four and six, assuming equal allocation between treatment groups. Randomisation was stratified according to study site and the sequence was generated using a computer program and concealed until the interventions were assigned. After surgery in the EIPL group, peritoneal lavage was done with 1 L of warm (42°C) normal 0·9% saline followed by complete aspiration; this procedure was repeated ten times. The primary endpoint was overall survival. All analyses were done assuming intention to treat. This trial is registered with ClinicalTrials.gov, NCT02140034., Findings: Between Sept 16, 2012, and Aug 3, 2018, 800 patients were randomly assigned to the EIPL group (n=398) or the standard surgery group (n=402). Two patients in the EIPL group and one in the standard surgery group withdrew from the trial immediately after randomisation and were excluded from the intention-to-treat analysis. At the third interim analysis on Aug 28, 2019, the predictive probability of overall survival being significantly higher in the EIPL group was less than 0·5%; therefore, the trial was terminated on the basis of futility. With a median follow-up of 2·4 years (IQR 1·5-3·0), the two groups were similar in terms of overall survival (hazard ratio 1·09 [95% CI 0·78-1·52; p=0·62). 3-year overall survival was 77·0% (95% CI 71·4-81·6) for the EIPL group and 76·7% (71·0-81·5) for the standard surgery group. 60 adverse events were reported in the EIPL group and 41 were reported in the standard surgery group. The most common adverse events included anastomotic leak (ten [3%] of 346 patients in the EIPL group vs six [2%] of 362 patients in the standard surgery group), bleeding (six [2%] vs six [2%]), intra-abdominal abscess (four [1%] vs five [1%]), superficial wound infection (seven [2%] vs one [<1%]), and abnormal liver function (six [2%] vs one [<1%]). Ten of the reported adverse events (eight in the EIPL group and two in the standard surgery group) resulted in death., Interpretation: EIPL and surgery did not have a survival benefit compared with surgery alone and is not recommended for patients undergoing curative gastrectomy for gastric cancer., Funding: National Medical Research Council, Singapore., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aim: Human leukocyte antigen (HLA) regions were highlighted as important genetic markers for various liver diseases by hepatology-related genome-wide association studies. Replication studies in non-alcoholic fatty liver disease (NAFLD) are limited and none has investigated the association of HLA alleles with non-alcoholic steatohepatitis (NASH) and other histological characteristics. In the current study, we examined the association of HLA-DQA1 and HLA-DQB1 alleles with NAFLD spectrum and its histological characteristics., Methods: Consecutive biopsy-proven NAFLD patients (n = 191) and healthy controls (n = 188) were enrolled and genotyped for HLA-DQA1 and HLA-DQB1 alleles using the sequence-specific oligonucleotide-polymerase chain reaction method., Results: No association was found between the HLA alleles and NAFLD or NASH in a case-control setting. Nevertheless, among NAFLD patients, the frequency of HLA-DQB1*06 allele was significantly the lowest in NASH with significant fibrosis (10.4%) and approximately similar for NASH without significant fibrosis (22.9%) and NAFL (22.5%) (P = 0.004). It is noteworthy that the association remains significant after correction for multiple comparisons (P c  = 0.04). Multivariate analysis revealed that HLA-DQB1*06 allele is also associated with fibrosis score (P = 0.001); the result remains significant after correction for multiple comparisons., Conclusion: These findings suggest that HLA-DQB1*06 is associated with lower fibrosis score in NAFLD patients., (© 2020 The Japan Society of Hepatology.)

Peanut allergy is the commonest cause of food-induced anaphylaxis in the world, and it can be fatal. There have been many recent improvements to achieve safe methods of peanut desensitisation, one of which is to use a combination of anti-immunoglobulin E and oral immunotherapy. We have treated 27 patients with anti-immunoglobulin E and oral immunotherapy, and report on the outcomes and incidence of adverse reactions encountered during treatment. The dose of peanut protein tolerated increased from a median baseline of 5 to 2000 mg after desensitisation, which is substantially more than would be encountered through accidental ingestion. The incidence of adverse reactions during the escalation phase of oral immunotherapy was 1.8%, and that during the maintenance phase was 0.6%. Most adverse reactions were mild; three episodes were severe enough to warrant withdrawal from oral immunotherapy, but none required epinephrine injection. Preliminary data suggest that unresponsiveness is lost when daily ingestion of peanuts is stopped after the maintenance period., Competing Interests: All authors have disclosed no conflicts of interest.

Bariatric surgery is currently the most durable weight loss solution for patients with morbid obesity. The extent of weight loss achieved, however, is subject to variation due to various factors, including patients' behaviour. In this study, we aimed to identify pre- and post-surgical predictors of weight loss following bariatric surgery. This prospective study included 57 participants who went through bariatric surgery (laparoscopic Roux-en-Y gastric bypass: n = 30; laparoscopic sleeve gastrectomy: n = 23; one anastomosis gastric bypass-mini gastric bypass: n = 4) in two tertiary referral hospitals. Consenting participants were assessed prior to surgery (T₀), and three months (T₁) and six months (T₂) after surgery. The assessment included interview and anthropometric measurements. The interview was done with the aid of instruments, including the Hospital Anxiety and Depression Scale (HADS) for anxiety and depression screening and the Dutch Eating Behaviour Questionnaire (DEBQ) for eating behaviour assessment. Baseline comorbidity status was obtained from medical records. A Generalised Estimating Equation (GEE) was developed to determine predictors of weight loss. Participants in the study were mostly women ( n = 37, 65%) with a mean age of 39.4 (SD = 10.01) years. The mean excess BMI loss (EBMIL) and total weight loss (TWL) at the sixth month was 63.31% and 23.83%, respectively. Anxiety, depression, and external eating scores reduced over time. Advancing age, high BMI, and higher scores for emotional and external eating emerged as significant negative predictors for TWL%. It can be concluded that the patients experienced substantial weight loss after surgery. Continuous monitoring of psychological well-being and eating behaviour are essential for optimal weight loss.

Objective: With an age-standardized incidence rate of 2 per 100 000, esophageal cancer is not common among Malaysians, but they are nevertheless important due to its poor prognosis. The study is to clarify whether the human papillomavirus (HPV) is associated with esophageal cancer in Malaysians as there has been no report to date on this in Malaysians and other South East Asians., Methods: Altogether 67 esophageal squamous cell carcinomas histologically diagnosed between 1 January 2004 and 31 December 2014 at the Department of Pathology, University of Malaya Medical Center, Malaysia were considered for HPV analysis using two commercially available methods, polymerase chain reaction with flow-through hybridization (21 HPV GenoArray Diagnostic Kit) and multiplex real-time polymerase chain reaction (Anyplex II HPV28 Detection). The DNA amplifiability of the formalin-fixed, paraffin-embedded tumor was checked by amplification of a 268 bp segment of the human β-globin gene (GH20/PC04) prior to HPV detection., Results: HPV detection was finally carried out in 51 patients. HPV16 was detected in the moderately differentiated, stage IV lower esophageal tumor of a 32-year-old Malaysian-born Chinese woman by both methods. Except for a predilection for Indians, the clinical characteristics of esophageal squamous cell carcinomas in this Malaysian cohort were generally similar to those of other populations., Conclusion: It appears that HPV is rare and an unlikely oncovirus in esophageal squamous cell carcinomas of Malaysians., (© 2018 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

To further expand the substrate range of the cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans, a library of diverse mutants was created and assayed toward a group of structurally diverse substrates. Among them, mutants T198A and M226A exhibited enhanced activity relative to wt CHAO for most (S)-enantiomers of primary amines and some secondary amines. While mutants T198I, L199I, L199F, M226I and M226T were more active than wt CHAO toward the primary amines, mutants T198F, L199T, Y321A, Y321T, Y321I and Y321F enhanced the enzyme activity toward the secondary amines. In particular, mutant Y321I displayed an enhanced catalytic efficiency toward 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13). Whereas a double mutant, Y321I/M226T, acted on (S)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine [(S)-8]. Since (R)-8 is an irreversible inhibitor of monoamine oxidase and (S)-13 is an intermediate of dextromethorphan, a cough suppressant drug, deracemizations of 8 and 13 were carried out with crude enzyme extracts of the respective mutants. This resulted in 51% and 78% isolated yields of (R)-8 and (S)-13, respectively, each with high enantiomeric excess (93% and 99% ee). The results demonstrated the application potential of the evolved CHAO mutants in drug synthesis requiring chiral secondary amines.

The utilization of bioethanol as the alcohol source for biodiesel production is more environmentally advantageous over methanol owing to its lower toxicity, lower flammability and its sustainable supply from renewable agricultural resources. However, as the presence of water in crude bioethanol is the critical factor limiting the biodiesel production process, the energy-intensive and costly purification of bioethanol is necessary for biodiesel application. Manganese glycerolate (MnGly) is reported the first time here as a robust heterogeneous catalyst that exhibited over 90% conversion by using aqueous ethanol containing 80 wt.% of water in the production of fatty acid ethyl ester (FAEE). The employment of 95 wt.% ethanol with respect to water could achieve 99.7% feedstock conversion in 6 hours under the optimal reaction conditions: reaction temperature (150 °C), feedstock-to-ethanol molar ratio (1:20) and catalyst loading (6 wt.%). Commercially available low grade crude bioethanol with the presence of impurities like sugars were applied which demonstrated remarkable catalytic activity in 24 hours. The high water tolerance of MnGly towards biodiesel production could eventually simplify the purification of bioethanol that consumes less energy and production cost.

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.

We discuss fundamental differences in electronic structure as reflected in one- and two-photon absorption spectra of semiconductor quantum dots and organic molecules by performing systematic experimental and theoretical studies of the size-dependent spectra of colloidal quantum dots. Quantum-chemical and effective-mass calculations are used to model the one- and two-photon absorption spectra and compare them with the experimental results. Currently, quantum-chemical calculations are limited to only small-sized quantum dots (nanoclusters) but allow one to study various environmental effects on the optical spectra such as solvation and various surface functionalizations. The effective-mass calculations, on the other hand, are applicable to the larger-sized quantum dots and can, in general, explain the observed trends but are insensitive to solvent and ligand effects. Careful comparison of the experimental and theoretical results allows for quantifying the range of applicability of theoretical methods used in this work. Our study shows that the small clusters can be in principle described in a manner similar to that used for organic molecules. In addition, there are several important factors (quality of passivation, nature of the ligands, and intraband/interband transitions) affecting optical properties of the nanoclusters. The larger-size quantum dots, on the other hand, behave similarly to bulk semiconductors, and can be well described in terms of the effective-mass models.

The Baeyer-Villiger monooxygenases (BVMOs) are microbial enzymes that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. The available BVMO crystal structures all lack a substrate or product bound in a position that would determine the substrate specificity and stereospecificity of the enzyme. Here, we report two crystal structures of cyclohexanone monooxygenase (CHMO) with its product, ε-caprolactone, bound: the CHMO(Tight) and CHMO(Loose) structures. The CHMO(Tight) structure represents the enzyme state in which substrate acceptance and stereospecificity is determined, providing a foundation for engineering BVMOs with altered substrate spectra and/or stereospecificity. The CHMO(Loose) structure is the first structure where the product is solvent accessible. This structure represents the enzyme state upon binding and release of the substrate and product. In addition, the role of the invariant Arg329 in chaperoning the substrate/product during the catalytic cycle is highlighted. Overall, these data provide a structural framework for the engineering of BVMOs with altered substrate spectra and/or stereospecificity.

The only available genome sequence for Rhizopus oryzae strain 99-880 was annotated to not encode any β-1,4-endoxylanase encoding genes of the glycoside hydrolase (GH) family 10 or 11. Here, we report the identification and cloning of two such members in R. oryzae strain NRRL 29086. Strain 29086 was one of several selected fungi grown on wheat or triticale bran and screened for xylanase activity among other hydrolytic actions. Its high activity (138 U/ml) in the culture supernatant led to the identification of two activity-stained proteins, designated Xyn-1 and Xyn-2 of respective molecular masses 32,000 and 22,000. These proteins were purified to electrophoretic homogeneity and characterized. The specific activities of Xyn-1 and Xyn-2 towards birchwood xylan were 605 and 7,710 U/mg, respectively. Kinetic data showed that the lower molecular weight Xyn-2 had a higher affinity (K m=3.2 ± 0.2 g/l) towards birchwood xylan than Xyn-1 by about 4-fold. The melting temperature (T m) of the two proteins, estimated to be in the range of 49.5-53.7 °C indicated that they are rather thermostable proteins. N-terminal and internal peptide sequences were obtained by chemical digestion of the purified xylanases to facilitate cloning, expression in Escherichia coli, and sequencing of the respective gene. The cloned Rhizopus xylanases were used to demonstrate release of xylose from flax shives-derived hemicellulose as model feedstock. Overall, this study expands the catalytic toolbox of GH10 and 11 family proteins that have applications in various industrial and bioproducts settings.

Peanut allergy is one of the commonest food hypersensitivities causing fatal or near-fatal reactions. There is, currently, no preventive treatment and the incidence of severe allergic reactions during peanut desensitisation has limited its clinical use. Anti-immunoglobulin E therapy has been shown to be effective in preventing peanut-induced reactions but it does not result in long-term tolerance. Two important advances have recently been reported. One involves gradual oral introduction of peanut protein to desensitise, whereas the other approach uses a combination of anti-immunoglobulin E and oral peanut immunotherapy. Both approaches could offer a way to desensitise with a far greater margin of safety than has, hitherto, been reported. This article provides an overview of the literature on peanut immunotherapy and describes the experience in a small group of children in Hong Kong who were treated successfully using anti-immunoglobulin E combined with oral peanut desensitisation.

Specially-treated glass substrates coated with a thin film of water soluble mercaptopropionic acid (MPA) capped CdTe nanocrystals (NCs) were prepared and found to undergo photoluminescence changes by as much as 40% when micro-droplets of organic molecules were placed in the nanometer-range proximity of the NCs. This imaging technique involving close proximity between a nano-crystal and an organic molecule is found to provide a 2 × -3 × enhanced contrast ratio over the conventional method of fluorescence imaging. Photoluminescence of NCs is recoverable upon removal of the organic molecules, therefore validating these NCs as potential all-optical organic molecular nanosensors. Upon optimization and with proper instrumentation, these nano-crystals could eventually serve as point-detectors for purposes of super-resolution optical microscopy. No solvents are required for the proposed sensing mechanism since all solutions were dried under argon flow. Fluorophores and fluorescent proteins were investigated, including fluorescein, Rhodamine 6G, and green fluorescent protein (GFP). Furthermore, NC photoluminescence changes were systematically quantified as a function of the solution pH and of the organic molecule concentration. Long duration (> 40 minutes) continuous excitation studies were conducted in order to evaluate the reliability of the proposed sensing scheme.

A cyclohexylamine oxidase (CHAO) of bacterial origin was previously shown to be a potentially useful catalyst in the deracemization of racemic primary amines. To further explore the properties and application of this enzyme, five single-amino acid substitution mutants (L199A, M226A, Y321A, Y321F, and L353M) were created based on superimposition of the tertiary structure of CHAO and the monoamine oxidase (MAO) B homolog. The substrate specificity of the purified wild-type and five mutant enzymes were examined towards 38 structurally diverse amines. All the enzymes exhibited better activity for primary amines than secondary and tertiary amines and in general exhibited high stereoselectivity. Among the mutant enzymes, M226A displayed an enhanced activity (5-400%) towards most substrates, and L353M showed 7-445% higher activity towards primary aliphatic amines with cycloalkane or aromatic moieties. Kinetic parameters revealed that both Y321 mutants showed higher catalytic efficiency towards cyclooctanamine, whereas the wild-type CHAO (wt CHAO) was most efficient towards cyclohexylamine. The wt CHAO or variant L353M in combination with a borane-ammonia complex as reducing agent was applied to the deracemization of 1-aminotetraline to give the (R)-enantiomer, a precursor of an antidepressant drug Norsertraline, in good yield (73-76%), demonstrating their application potential in chiral amine synthesis.

Mining fungal genomes for glucoamylase and α-amylase encoding sequences led to the selection of 23 candidates, two of which (designated TSgam-2 and NFamy-2) were advanced to testing for cooked or raw starch hydrolysis. TSgam-2 is a 66-kDa glucoamylase recombinantly produced in Pichia pastoris and originally derived for Talaromyces stipitatus. When harvested in a 20-L bioreactor at high cell density (OD600 > 200), the secreted TSgam-2 enzyme activity from P. pastoris strain GS115 reached 800 U/mL. In a 6-L working volume of a 10-L fermentation, the TSgam-2 protein yield was estimated to be ∼8 g with a specific activity of 360 U/mg. In contrast, the highest activity of NFamy-2, a 70-kDa α-amylase originally derived from Neosartorya fischeri, and expressed in P. pastoris KM71 only reached 8 U/mL. Both proteins were purified and characterized in terms of pH and temperature optima, kinetic parameters, and thermostability. TSgam-2 was more thermostable than NFamy-2 with a respective half-life (t1/2) of >300 min at 55 °C and >200 min at 40 °C. The kinetic parameters for raw starch adsorption of TSgam-2 and NFamy-2 were also determined. A combination of NFamy-2 and TSgam-2 hydrolyzed cooked potato and triticale starch into glucose with yields, 71-87 %, that are competitive with commercially available α-amylases. In the hydrolysis of raw starch, the best hydrolysis condition was seen with a sequential addition of 40 U of a thermostable Bacillus globigii amylase (BgAmy)/g starch at 80 °C for 16 h, and 40 U TSgam-2/g starch at 45 °C for 24 h. The glucose released was 8.7 g/10 g of triticale starch and 7.9 g/10 g of potato starch, representing 95 and 86 % of starch degradation rate, respectively.

We demonstrate that core/graded-shell CdSe/CdSe1-xSx/CdS giant semiconductor nanocrystals (g-NCs) have robust photoluminescence (PL) temperature response. At a size of 10.2 nm in diameter, these g-NCs undergo a PL drop of only 30% at 355 K relative to their PL intensity at 85 K. In comparison, the core/step-shell CdSe/CdS g-NCs at the same diameter exhibit 80% PL drop at 355 K. Spectral shifting and broadening were found to be, respectively, 5-10 times and 2-4 times smaller than those observed in standard CdSe core and CdSe/CdS core/shell NCs. These core/graded-shell g-NCs are largely blinking suppressed and have insignificant photoluminescence decay even after excitation at very high irradiance (44 kW cm(-2)) for over an hour. These types of g-NC have potential applications as the active medium for thermally robust laser devices (in the visible range) or as temperature-insensitive bioprobes for biomedical imaging.

There are few entries of carbon-carbon bond hydrolases (EC 3.7.1.-) in the ExPASy database. In microbes, these enzymes play an essential role in the metabolism of alicyclic or aromatic compounds as part of the global carbon cycle. CpdC is a ω-pentadecalactone hydrolase derived from the degradation pathway of cyclopentadecanol or cyclopentadecanone by Pseudomonas sp. strain HI-70. CpdC was purified to homogeneity and characterized. It is active as a dimer of 56,000 Da with a subunit molecular mass of 33,349. Although CpdC has the highest activity and reaction rate (kcat) toward ω-pentadecalactone, its catalytic efficiency favors lauryl lactone as a substrate. The melting temperature (Tm) of CpdC was estimated to be 50.9 ± 0.1°C. The half-life of CpdC at 35°C is several days. By virtue of its high level of expression in Escherichia coli, the intact CpdC-encoding gene and progressive 3'-end deletions were employed in the construction of a series of fusion plasmid system. Although we found them in inclusion bodies, proof-of-concept of overproduction of three microbial cutinases of which the genes were otherwise expressed poorly or not at all in E. coli was demonstrated. On the other hand, two antigenic proteins, azurin and MPT63, were readily produced in soluble form.

Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10(-9) M) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 10(2)-10(4) times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO.

Context and Objective: Scientific evidence has shown Coriolus versicolor (L. ex Fr.) Quel (also known as Yunzhi) has the role of immunomodulator in therapeutic effect. The aim of this in vitro study was to investigate the antioxidative effect of Yunzhi and to explore the mechanisms behind its DNA protection., Materials and Methods: Commercial Yunzhi extract was dissolved in water and diluted in five concentrations (10(1)-10(5) μg/L) with appropriate buffers. Lymphocytes harvested from three healthy subjects were incubated with Yunzhi extract for 30 min. Cells were then subjected to 5 min oxidant challenge by 45 μM hydrogen peroxide. The standard alkaline comet (SAC) assay and lysed cell comet (LCC) assay were performed in parallel. DNA damage of each treatment was scored under a fluorescence microscope and compared with the cells without Yunzhi pretreatment., Results: U-shaped dose-response was seen in both versions of the comet assay. Yunzhi at 10(4) μg/L demonstrated a genoprotective effect against oxidative damage in the SAC assay (25% decrease in comet score). In the LCC assay, a trend of protection in lymphocytes was observed but it did not reach statistical significance., Conclusion: A direct antioxidant effect of Yunzhi against oxidant challenge on the DNA of lymphocytes was evidenced. The active component in Yunzhi was likely to be membrane permeable.

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (Km = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (Km = 3.6 μM; kcat = 283 s(-1)) in preference to flavin adenine dinucleotide (FAD) (Km = 19 μM; kcat = 128 s(-1)). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25-1 for 2,5-DKCMO-1 and camE25-2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.

CdTe semiconductor nanocrystals (NCs) with 3-mercaptopropionic acid as the ligand exhibit a reversible response towards inter-switching oxygen and argon environments. The photoluminescence response is investigated at multiple oxygen concentrations, NC coverage and excitation intensities, in which all conditions exhibit full recovery upon exposure to flowing argon. The CdTe NC's large surface-to-volume ratio results in high sensitivity towards oxygen molecules with significant photoluminescence quenching at a concentration of 40 ppm. This suggests a novel approach to the creation of simple, inexpensive and ultrasensitive oxygen nanosensors.

Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme.

2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant Δ65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Δ193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5'-dithio-bis-(2-nitrobenzoic acid) and ZnCl(2), which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H(2)O(2). SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions.

OBJECTIVES. To clarify the use of ultrasonography by determining the frequency of developmental dysplasia of the hip among breech-presented Chinese neonates in Hong Kong. DESIGN. Prospective case series. SETTING. Regional hospital, Hong Kong. PATIENTS. All breech-presented Chinese neonates born during January 2008 to June 2009 were included (except premature neonates). They were examined clinically from birth till the age of 1 year. Ultrasound of the hips was performed at the age of 2 weeks, and X-ray of the pelvis at the age of 1 year. RESULTS. A total of 209 breech-presented neonates were born during the study period; 110 neonates completed all necessary investigations and follow-up. Among the latter, there were three neonates with developmental dysplasia of the hip warranting treatment, which amounted to a frequency of 2.7%. CONCLUSION. Developmental dysplasia of the hip among breech-presented Chinese babies is only slightly less common than in corresponding populations in other regions in the world. Since early diagnosis is important, ultrasonography screening in high-risk cases such as those with breech presentation may be useful.

In the Sorangium cellulosum strain So ce56 genome, two putative esterase-encoding genes (loci sce1896 and sce8927) were cloned, expressed in Escherichia coli, and the resulting enzymes (designated ScFAE1 and ScFAE2) were used to assess the possible release of ferulic acid (FA) from triticale and wheat brans, and an aqueous fraction of steam-exploded wheat straw. The two polypeptides, sharing only 30% sequence identity, exhibit a typical catalytic Ser-Asp-His triad, a characteristic of α/β-hydrolase fold proteins. Both ScFAE1 (35 kDa) and ScFAE2 (34 kDa) were purified to apparent homogeneity and comparison of their kinetic parameters indicated an apparent higher affinity of ScFAE2 than ScFAE1 towards the various feruloyl substrates. This property was reflected by the observation that ScFAE2 was capable of yielding up to 85% of FA from destarched triticale bran. In the steam-exploded wheat sample, more than 85% yield of FA or p-coumaric acid was also effected by ScFAE2 without the decomposition of valuable chemical such as furfural. The two cloned FAEs represent the first of myxobacterial origin to be characterized and they are classified as new members of the type D family of FAEs.

The Baeyer-Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP(+) and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP(+) and FAD, to 2.4 Å resolution. This structure shows a drastic rotation of the NADP(+) cofactor in comparison to previously reported NADP(+)-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP(+) permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme's chemospecificity, to a location above the flavin-peroxide adduct where catalysis occurs.

This study describes the release of antioxidant ferulic acid from wheat and triticale brans by mixtures of extracellular enzymes produced in culture by a strain FC007 of Alternaria alternata, a dark mold originally isolated from Canadian wood log. The genus of the mold was confirmed as Alternaria by 18S ribosomal DNA characterization. Enzyme activities for feruloyl esterase (FAE) and polysaccharide hydrolyzing enzymes were measured, and conditions for release of ferulic acid and reducing sugars from the mentioned brans were evaluated. The highest level of FAE activity (89 ± 7 mU ml(-1) fermentation culture) was obtained on the fifth day of fermentation on wheat bran as growth substrate. Depending on biomass and processing condition, up to 91.2 or 72.3% of the ferulic acid was released from wheat bran and triticale bran, respectively, indicating the proficiency of A. alternata extracellular enzymes in plant cell wall deconstruction. The apparent high extraction of ferulic acid from wheat and triticale brans represents a potential advantage of using a whole fungal cell enzyme complement over yields reported previously through an artificial assembly of cloned FAE with a particular xylanase in a cocktail format.

Nasopharyngeal carcinoma (NPC) represents a common cancer in endemic areas with high invasive and metastatic potential. It is now known that the HGF-MET signaling pathway plays an important role in mediating the invasive growth of many different types of cancer, including head and neck squamous cell carcinoma. HGF has been shown to stimulate NPC cell growth and invasion in cell line model. The current study aims at demonstrating the effect of MET inhibition by small molecule tyrosine kinase inhibitor PHA665752 on the growth and invasive potential of NPC cell lines. NPC cell lines were used for immunohistochemistry for the MET protein, as well as western blot analysis on MET together with its downstream cascade signaling proteins after treatment with PHA665752. The effect on cell growth, migration and invasion after PHA665752 treatment was also studied. MET inhibition by PHA665752 resulted in highly significant inhibition on NPC cell growth, migration and invasion in vitro. Down-regulation of phospho-MET, phospho-Akt, phospho-MAPK, phospho-STAT3, cyclin D1, β-catenin and PCNA was detected in NPC cells after PHA665752 treatment. MET inhibition with tyrosine kinase inhibitor resulted in suppression of NPC cell growth and invasive potential via down-regulation of a variety of signaling onco-proteins. MET is an important therapeutic target for NPC that warrants further studies and clinical trials.

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140-152, 1983). Here we cloned and overexpressed the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP(+) at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP(+). A comparison of several crystal forms of OTEMO bound to FAD and NADP(+) revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (k(cat)/K(m)) favors 2-n-hexyl cyclopentanone (4.3 × 10(5) M(-1) s(-1)) as a substrate, although its affinity (K(m) = 32 μM) was lower than that of the CoA-activated substrate (K(m) = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

Head and neck squamous cell carcinoma (HNSCC) represents a devastating type of malignancy characterized by its high incidence of regional and distant metastases at the time of diagnosis. Vital physiological functions in the upper aerodigestive tract are often impaired as a result of the disease and treatment for the disease, giving rise to severe morbidity in patients suffering from this type of cancer. It is crucial to delineate the aberrant growth signaling pathways in HNSCC cells and develop specific target therapies for the disease to improve the treatment outcome. Although the epidermal growth factor receptor pathway has been extensively studied in HNSCC and anti-epidermal growth factor receptor therapy has already shown promise in treating HNSCC in phase III clinical trials, the signaling pathway that accounts for the highly invasive phenotype of HNSCC needs to be defined and also therapeutically targeted. The hepatocyte growth factor-MET signaling pathway has been studied extensively over the past two decades and it is now clear that it plays an important role in mediating invasive growth of many types of cancer. Here, we review comprehensively the evidence on hepatocyte growth factor-MET cascade being a key in the signaling pathway in mediating invasive growth of HNSCC and the potential of this signaling pathway to be a therapeutic target for the treatment of HNSCC.

The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavouring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a β-barrel structure and two α-helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the β-barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site.

The existence of flat adenomas in the colon is well recognized. Whether they represent a distinct disease with a pathogenetic pathway different from that of the classical adenoma-carcinoma sequence in colorectal tumorigenesis and have higher malignant potential remains a matter of debate. To review the epidemiology, clinical features, detection and management of flat and depressed (non-polypoid) colonic neoplasm, we performed a thorough literature review on studies focusing on the prevalence, histological features, genetics, detection and treatment of flat and depressed (non-polypoid) colonic neoplasm. A high percentage of severe dysplasia in flat colonic adenomas has not been consistently demonstrated. Their malignant potential appears to be size-dependent. Flat adenomas are found to have a lower incidence of major genetic abnormalities involved in the classical adenoma-carcinoma sequence and that has raised suspicions that they may have a different pathogenesis. The depressed type of colorectal carcinoma is uncommon but shows more aggressive behavior. More advanced colonoscopic techniques, such as chromoendoscopy, may enhance the detection of small and inconspicuous colonic neoplastic lesions that lack a protruding configuration. It is essential for endoscopists to appreciate the existence and clinical significance of flat and depressed colonic lesions as an important variant of colonic neoplasms so that the goal of reducing colorectal carcinoma incidence by polypectomy can be better achieved.

A putative alpha/beta hydrolase fold-encoding gene (locus tag TTE1809) from the genome of Thermoanaerobacter tengcongensis was cloned and expressed in Escherichia coli as a possible source of thermostable feruloyl esterase (FAE) for the production of antioxidant phenolic acids from biomass. Designated as TtFAE, the 33-kDa protein was purified to apparent homogeneity. The lipase-like sequence characteristics of TtFAE and its substrate specificity towards methyl ferulate, methyl sinapate, and methyl p-coumarate classify it as a new member of the type A FAEs. At 75 degrees C, the enzyme retained at least 95% of its original activity for over 80 min; at 80 degrees C, its half-life was found to be 50 min, rendering TtFAE a highly thermostable protein. Under different hydrolytic conditions, ferulic acid (FA) was shown to be released from feruloylated oligosaccharides prepared from triticale bran. An estimated recovery of 68 mg FA/100 g triticale bran was demonstrated by a 30% release of the total FA from triticale bran within a 5-h incubation period. Both the oxygen radical absorbing capacity values of the feruloylated oligosaccharides and free FA were also determined. Overall, this work introduces a new bacterial member to the growing family of plant cell wall degrading FAEs that at present is largely of fungal origin, and it benchmarks the bioproduction of FA from triticale bran.

We studied the decolorization of malachite green (MG) by the fungus Cunninghamella elegans. The mitochondrial activity for MG reduction was increased with a simultaneous increase of a 9-kDa protein, called CeCyt. The presence of cytochrome c in CeCyt protein was determined by optical absorbance spectroscopy with an extinction coefficient (E(550-535)) of 19.7+/-6.3 mM(-1) cm(-1) and reduction potential of + 261 mV. When purified CeCyt was added into the mitochondria, the specific activity of CeCyt reached 440 +/- 122 micromol min(-1) mg(-1) protein. The inhibition of MG reduction by stigmatellin, but not by antimycin A, indicated a possible linkage of CeCyt activity to the Qo site of the bc1 complex. The RT-PCR results showed tight regulation of the cecyt gene expression by reactive oxygen species. We suggest that CeCyt acts as a protein reductant for MG under oxidative stress in a stationary or secondary growth stage of this fungus., (2009 Elsevier Inc. All rights reserved.)