Identification and characterization of two O-methyltransferases involved in biosynthesis of methylated 2-(2-phenethyl)chromones in agarwood.

The 2-(2-phenethyl)chromones (PECs) are the signature constituents responsible for the fragrance and pharmacological properties of agarwood. O-Methyltransferases (OMTs) are necessary for the biosynthesis of methylated PECs, but there is little known about OMTs in Aquilaria sinensis. In this study, we identified 29 OMT genes from the A. sinensis genome. Expression analysis showed they were differentially expressed in different tissues and responded to drill wounding. Comprehensive analysis of the gene expression and methylated PEC content revealed that AsOMT2, AsOMT8, AsOMT11, AsOMT16, and AsOMT28 could potentially be involved in methylated PECs biosynthesis. In vitro enzyme assays and functional analysis in Nicotiana benthamiana demonstrated that AsOMT11 and AsOMT16 could methylate 6-hydroxy-2-(2-phenylethyl)chromone to form 6-methoxy-2-(2-phenylethyl)chromone. A transient overexpression experiment in the variety 'Qi-Nan' revealed that AsOMT11 and AsOMT16 could significantly promote the accumulation of three major methylated PECs. Our results provide candidate genes for the mass production of methylated PECs using synthetic biology.

Evaluating the evidence for a liver shrinkage diet for obese patients prior to laparoscopic cholecystectomy: A systematic review and meta-analysis.

BACKGROUND: The role of a very low-calorie diet (VLCD) before cholecystectomy in obese patients is unclear. This study evaluated whether VLCD could be used as a risk mitigation strategy for this high-risk patient cohort. PATIENTS AND METHODS: A systematic review and meta-analysis was performed (PROSPERO ID CRD42022374610). The primary outcome was to determine the impact of pre-operative VLCD on the operative findings and ease of dissection during laparoscopic cholecystectomy (LC). RESULTS: Two studies were included with a total of 84 patients. VLCD was associated with a significantly easier Calot's dissection (MD: -0.58 (95% confidence interval [CI] [ -1.03, -0.13], P = 0.01) and was associated with a significantly higher rate of pre-operative weight loss (MD; 2.92 (95% CI [2.23, 3.62], P = 0.00001). CONCLUSIONS: The published evidence regarding VLCD before cholecystectomy in obese patients is limited. After acknowledging the limitations of the data, VLCD is associated with a significantly higher rate of weight loss preoperatively and directly impacts the ease of intraoperative dissection of Calot's triangle. Routine use of VLCD should be considered for all obese patients undergoing elective LC.

Tablet-based tests of everyday visual function in a diabetic macular oedema (DME) clinic waiting area: A feasibility study.

PURPOSE: (1) To assess the feasibility of conducting tablet-based vision tests in hospital clinic waiting areas; (2) To test the hypothesis that increasing severity of diabetic macular oedema (DME) is associated with the performance of tablet-based surrogates of everyday tasks and self-reported visual function. METHODS: Sixty-one people with mild (n = 28), moderate (n = 24) or severe (n = 9) DME performed two tablet-based tests of 'real-world' visual function (visual search and face recognition) while waiting for appointments in a hospital outpatient clinic. Participants also completed a tablet-based version of a seven-item, visual-functioning (VF-7) patient-reported outcome measure. Test performance was compared to previously published 99% normative limits for normally sighted individuals. RESULTS: Thirty-four participants (56%; 95% confidence interval [CI] 43%-68%) exceeded normative limits for visual search, while eight (13%; 95% CI 65%-24%) exceeded normative limits for face discrimination. Search duration was significantly longer for people with severe DME than those with mild and moderate DME (p = 0.01). Face discrimination performance was not significantly associated with DME severity. VF-7 scores were statistically similar across DME severity groups. Median time to complete all elements (eligibility screening, both tablet-based tasks and the VF-7) was 22 (quartiles 19, 25) min. Further, 98% and 87% of participants, respectively, reported the search task and face discrimination task to be enjoyable, while 25% and 97%, respectively, reported finding the two tasks to be difficult. CONCLUSIONS: Portable tablet-based tests are quick, acceptable to patients and feasible to be performed in a clinic waiting area with minimal supervision. They have the potential to be piloted in patients' homes for self-monitoring.

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Molecular characterisation of Entamoeba histolytica UDP-glucose 4-epimerase, an enzyme able to provide building blocks for cyst wall formation.

In the human host, the protozoan parasite Entamoeba histolytica is adapted to a non-invasive lifestyle in the colon as well as to an invasive lifestyle in the mesenterial blood vessels and the liver. This means to cope with bacteria and human cells as well as various metabolic challenges. Galactose and N-acetylgalactosamine (GalNAc) are sugars of great importance for the amoebae, they attach to the host mucus and enterocytes via their well-studied Gal/GalNAc specific lectin, they carry galactose residues in their surface glycans, and they cleave GalNAc from host mucins. The enzyme UDP-glucose 4-epimerase (GalE) works as a bridge between the galactose and glucose worlds, it can help to generate glucose for glycolysis from phagocytosis products containing galactose as well as providing UDP-galactose necessary for the biosynthesis of galactose-containing surface components. E. histolytica contains a single galE gene. We recombinantly expressed the enzyme in Escherichia coli and used a spectrophotometric assay to determine its temperature and pH dependency (37°C, pH 8.5), its kinetics for UDP-glucose (Km = 31.82 µM, Vmax = 4.31 U/mg) and substrate spectrum. As observed via RP-HPLC, the enzyme acts on UDP-Glc/Gal as well as UDP-GlcNAc/GalNAc. Previously, Trypanosoma brucei GalE and the bloodstream form of the parasite were shown to be susceptible to the three compounds ebselen, a selenoorganic drug with antioxidant properties, diethylstilbestrol, a mimic of oestrogen with anti-inflammatory properties, and ethacrynic acid, a loop diuretic used to treat oedema. In this study, the three compounds had cytotoxic activity against E. histolytica, but only ebselen inhibited the recombinant GalE with an IC50 of 1.79 µM (UDP-Gal) and 1.2 µM (UDP-GalNAc), suggesting that the two other compounds are active against other targets in the parasite. The importance of the ability of GalE to interconvert UDP-GalNAc and UDP-GlcNAc may be that the trophozoites can generate precursors for their own cyst wall from the sugar subunits cleaved from host mucins. This finding advances our understanding of the biochemical interactions of E. histolytica in its colonic environment.

Laparoscopy in high-risk emergency general surgery reduces intensive care stay, length of stay and mortality.

PURPOSE: Emergency general surgery patients undergoing laparoscopic surgery are at reduced risk of mortality and may require reduced length of critical care stay. This study investigated the effect of laparoscopy on high-risk patients' post-operative care requirements. METHODS: Data were retrieved for all patients entered into the NELA database between 2013 and 2018. Only high-risk surgical patients (P-POSSUM predicted mortality risk of ≥ 5%) were included. Patients undergoing laparoscopic and open emergency general surgical procedures were compared using a propensity score weighting approach. Outcome measures included total length of critical care (level 3) stay, overall length of stay and inpatient mortality. RESULTS: A total of 66,517 high-risk patients received emergency major abdominal surgery. A laparoscopic procedure was attempted in 6998 (10.5%); of these, the procedure was competed laparoscopically in 3492 (49.9%) and converted to open in 3506 (50.1%). Following inverse probability treatment weighting adjustment for patient disease and treatment characteristics, high-risk patients undergoing laparoscopic surgery had a shorter median ICU stay (1 day vs 2 days p < 0.001), overall hospital length of stay (11 days vs 14 days p < 0.001) and a lower inpatient mortality (16.0% vs 18.8%, p < 0.001). They were also less likely to have a prolonged ICU stay with an OR of 0.78 (95% CI 0.74-0.83, p < 0.001). CONCLUSION: The results of this study suggest that in patients at high risk of post-operative mortality, laparoscopic emergency bowel surgery leads to a reduced length of critical care stay, overall length of stay and inpatient mortality compared to traditional laparotomy.

Updating the Chronic Freshwater Ecotoxicity Database and Biotic Ligand Model for Nickel for Regulatory Applications in Europe.

Bioavailability has been taken into account in the regulation of nickel in freshwater ecosystems in Europe for over a decade; during that time a significant amount of new information has become available covering both the sensitivity of aquatic organisms to nickel toxicity and bioavailability normalization. The ecotoxicity database for chronic nickel toxicity to freshwater organisms has been updated and now includes 358 individual data points covering a total of 53 different species, all of which are suitable for bioavailability normalization to different water chemistry conditions. The bioavailability normalization procedure has also been updated to include updates to the bioavailability models that enable more sensitive water chemistry conditions to be covered by the model predictions. The updated database and bioavailability normalization procedure are applicable to more than 95% of regulated European surface water conditions and have been used to calculate site-specific criteria for a variety of different water chemistry scenarios, to provide an indication of how the sensitivity to nickel varies between different water types. The hazardous concentration for 5% of a species (HC5) values for this diverse selection of water types range from 1.6 to 36 µg L-1 , clearly demonstrating the importance of accounting for nickel bioavailability in freshwaters. This updated database and bioavailability normalization procedure provide a robust basis for the derivation of regulatory thresholds for chronic nickel toxicity in freshwaters such as predicted no-effect concentrations and Environmental Quality Standards and are protective of the results of several mesocosm studies. Environ Toxicol Chem 2023;42:566-580. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Assessing the Extent of Environmental Risks From Nickel in European Freshwaters: A Critical Reflection of the European Commission's Current Approach.

Nickel (Ni) has a been a Priority Substance under the European Water Framework Directive since 2008. As such it is deemed to present an European Union-wide risk to surface waters. Since 2013, the Ni Environmental Quality Standard (EQS) has been bioavailability-based, and new European Guidance supports accounting for bioavailability in assessing Ni compliance with the EQS. The European Commission has developed an approach to determine whether Priority Substances present a sufficient European Union-wide risk to justify an ongoing statutory monitoring programme, effectively to deselect a substance. This is a key step to ensure that finite monitoring resources are targeted at delivering environmental benefit, when there is an ever-growing burden of determinands to measure for all regulators. When the European Commission performed this exercise for Ni without accounting for bioavailability, they concluded that Ni should not be deselected, and Ni is an European Union-wide risk. Performing this same exercise with the same methodology, using regulatory monitoring data for over 300 000 samples, from more than 19 000 sites across Europe, and accounting for bioavailability, as detailed in the Directive, >99% of sites comply with the Ni EQS. Nickel shows very low risks for all of the criteria identified by the European Commission that need to be met for deselection. Accounting for bioavailability is key in the assessment of Ni risks in surface waters to deliver ecologically relevant outcomes. Environ Toxicol Chem 2022;41:1604-1612. © 2022 NiPERA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The Research Progress of Taxol in Taxus.

BACKGROUND: Taxus is a valuable woody species with important medicinal value. The bark of Taxus can produce taxol, a natural antineoplastic drug that is widely used in the treatment of breast, ovarian and lung cancers. However, the low content of taxol in the bark of Taxus can not meet the growing clinical demands, so the current research aims at finding ways to increase taxol production. OBJECTIVE: In this review, the research progress of taxol including the factors affecting the taxol content, biosynthesis pathway of taxol, production of taxol in vitro and the application of multi-omics approaches in Taxus as well as future research prospects will be discussed. RESULTS: The taxol content is not only dependent on the species, age and tissues but is also affected by light, moisture levels, temperature, soil fertility and microbes. Most of the enzymes in the taxol biosynthesis pathway have been identified and characterized. Total chemical synthesis, semi-synthesis, plant cell culture and biosynthesis in endophytic fungi have been explored to product taxol. Multi-omics have been used to study Taxus and taxol. CONCLUSION: Further efforts in the identification of unknown enzymes in the taxol biosynthesis pathway, establishment of the genetic transformation system in Taxus and the regulatory mechanism of taxol biosynthesis and Taxus cell growth will play a significant role in improving the yield of taxol in Taxus cells and plants.

A Semiautomated, Phenotypic, In Vitro Scratch Assay for Assessing Retinal Pigment Epithelial Cell Wound Healing.

Purpose: Age-related macular degeneration leads to retinal pigment epithelium (RPE) cell death and loss of central vision. In vivo studies have shown that the RPE layer has an innate, but limited, ability to repopulate atrophic areas. We aimed to establish a semiautomated, in vitro, wound healing assay workflow for targeted screening of compounds able to influence RPE wound healing. Methods: The ARPE-19 phenotype was evaluated using bright-field microscopy, immunocytochemistry, and quantitative real-time polymerase chain reaction. ARPE-19 monolayers were simultaneously scratched in a 96-well format and treated with Hoechst-33342 and an array of compounds. Initial wound dimensions and wound healing were subsequently evaluated using the EVOS FL Auto 2.0 imaging platform combined with automated image analyses. Results: Long-term cultured ARPE-19 cells displayed a more in vivo RPE-like phenotype compared with recently seeded or short-term cultured cells. No statistical difference of initial scratch width was observed between short-term and long-term cultured cells, but more wells were excluded from analyses in total in the latter case due to scratch width, scratch smoothness, and imaging errors. Furthermore, the previous time spent in continuous culture had an effect on the observation of an altered wound healing response to different treatment conditions. Conclusions: We have established a semiautomated, 96-well format, in vitro wound healing assay with a reproducible workflow. This would enable screening of a significant number of compounds and greatly advances the potential of identifying novel therapeutics that may enhance the innate ability of RPE cells to repopulate atrophic areas.

Comparison of the Chronic Toxicity of Nickel to Temperate and Tropical Freshwater Species.

Reliable ecotoxicity data are required to derive thresholds protective for aquatic life that are scientifically defensible and practically implementable as environmental risk assessment and management tools. Much of the data that have been used to derive thresholds for environmental quality have been collected for temperate species. There are concerns that due to a lack of data for the different species present in tropical ecosystems, they may not be adequately protected by thresholds derived from temperate species. In the present study, chronic ecotoxicity data for nickel and freshwater species from different climatic regions have been collated. Comparisons were performed between tropical and temperate datasets on the basis of the threshold values and overall distributions of the ecotoxicity data, as well as between groups of species and closely related species from different climatic regions. The analysis indicated that the sensitivities of tropical and temperate species cover similar ranges. An approach based on the inclusion of as diverse a range of taxa as possible is recommended to ensure the protection of sensitive species in both temperate and tropical ecosystems. Environ Toxicol Chem 2019;38:1211-1220. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Protein-Specific Analysis of Invertebrate Glycoproteins.

N-Glycans are posttranslational modifications of proteins attached to the amide side chains of asparagine residues, with possible heterogeneity due to different structures being possible at the same glycosylation site. In contrast to the mammalian systems, invertebrate N-glycosylation presents a challenge in analysis as there exist unfamiliar epitopes and a high degree of structural and isomeric variation between different species. A simple analytical approach to analyze N-glycans on specific glycoproteins is presented, which involves a combination of tryptic peptide mass spectrometry and "off-line" RP-HPLC MALDI-TOF MS/MS complemented by blotting to recognize specific epitopes. An additional N-glycan enrichment and labeling step can facilitate the analysis of single structures and even provide isomeric separation of N-glycans from specific proteins.

World Health Organization-World Federation of Societies of Anaesthesiologists (WHO-WFSA) International Standards for a Safe Practice of Anesthesia.

The International Standards for a Safe Practice of Anesthesia were developed on behalf of the World Federation of Societies of Anaesthesiologists (WFSA), a nonprofit organization representing anesthesiologists in 150 countries, and the World Health Organization (WHO). The recommendations have been approved by WHO and the membership of WFSA. These Standards are applicable to all anesthesia providers throughout the world. They are intended to provide guidance and assistance to anesthesia providers, their professional organizations, hospital and facility administrators, and governments for maintaining and improving the quality and safety of anesthesia care. The Standards cover professional aspects; facilities and equipment; medications and intravenous fluids; monitoring; and the conduct of anesthesia. HIGHLY RECOMMENDED standards, the functional equivalent of mandatory standards, include (amongst other things): the continuous presence of a trained and vigilant anesthesia provider; continuous monitoring of tissue oxygenation and perfusion by clinical observation and a pulse oximeter; intermittent monitoring of blood pressure; confirmation of correct placement of an endotracheal tube (if used) by auscultation and carbon dioxide detection; the use of the WHO Safe Surgery Checklist; and a system for transfer of care at the end of an anesthetic. The International Standards represent minimum standards and the goal should always be to practice to the highest possible standards, preferably exceeding the standards outlined in this document.

N-Benzyl Substitution of Polyhydroxypyrrolidines: The Way to Selective Inhibitors of Golgi α-Mannosidase†II.

Inhibition of the biosynthesis of complex N-glycans in the Golgi apparatus influences progress of tumor growth and metastasis. Golgi α-mannosidase II (GMII) has become a therapeutic target for drugs with anticancer activities. One critical task for successful application of GMII drugs in medical treatments is to decrease their unwanted co-inhibition of lysosomal α-mannosidase (LMan), a weakness of all known potent GMII inhibitors. A series of novel N-substituted polyhydroxypyrrolidines was synthesized and tested with modeled GH38 α-mannosidases from Drosophila melanogaster (GMIIb and LManII). The most potent structures inhibited GMIIb (Ki =50-76 µm, as determined by enzyme assays) with a significant selectivity index of IC50 (LManII)/IC50 (GMIIb) >100. These compounds also showed inhibitory activities in in vitro assays with cancer cell lines (leukemia, IC50 =92-200 µm) and low cytotoxic activities in normal fibroblast cell lines (IC50 >200 µm). In addition, they did not show any significant inhibitory activity toward GH47 Aspergillus saitoiα1,2-mannosidase. An appropriate stereo configuration of hydroxymethyl and benzyl functional groups on the pyrrolidine ring of the inhibitor may lead to an inhibitor with the required selectivity for the active site of a target α-mannosidase.

Percutaneous hepatic perfusion with melphalan in uveal melanoma: A safe and effective treatment modality in an orphan disease.

BACKGROUND: Metastatic uveal melanoma (UM) carries a poor prognosis; liver is the most frequent and often solitary site of recurrence. Available systemic treatments have not improved outcomes. Melphalan percutaneous hepatic perfusion (M-PHP) allows selective intrahepatic delivery of high dose cytotoxic chemotherapy. METHODS: Retrospective analysis of outcomes data of UM patients receiving M-PHP at two institutions was performed. Tumor response and toxicity were evaluated using RECIST 1.1 and Common Terminology Criteria for Adverse Events (CTCAE) v4.03, respectively. RESULTS: A total of 51 patients received 134 M-PHP procedures (median of 2 M-PHPs). 25 (49%) achieved a partial (N = 22, 43.1%) or complete hepatic response (N = 3, 5.9%). In 17 (33.3%) additional patients, the disease stabilized for at least 3 months, for a hepatic disease control rate of 82.4%. After median follow-up of 367 days, median overall progression free (PFS) and hepatic progression free survival (hPFS) was 8.1 and 9.1 months, respectively and median overall survival was 15.3 months. There were no treatment related fatalities. Non-hematologic grade 3-4 events were seen in 19 (37.5%) patients and were mainly coagulopathic (N = 8) and cardiovascular (N = 9). CONCLUSIONS: M-PHP results in durable intrahepatic disease control and can form the basis for an integrated multimodality treatment approach in appropriately selected UM patients.

Impact of Surgical Lighting on Intraoperative Safety in Low-Resource Settings: A Cross-Sectional Survey of Surgical Providers.

BACKGROUND: Safe surgery requires high-quality, reliable lighting of the surgical field. Little is reported on the quality or potential safety impact of surgical lighting in low-resource settings, where power failures are common and equipment and resources are limited. METHODS: Members of the Lifebox Foundation created a novel, non-mandatory, 18-item survey tool using an iterative process. This was distributed to surgical providers practicing in low-resource settings through surgical societies and mailing lists. RESULTS: We received 100 complete responses, representing a range of surgical centres from 39 countries. Poor-quality surgical field lighting was reported by 40% of respondents, with 32% reporting delayed or cancelled operations due to poor lighting and 48% reporting electrical power failures at least once per week. Eighty per cent reported the quality of their surgical lighting presents a patient safety risk with 18% having direct experience of poor-quality lighting leading to negative patient outcomes. When power outages occur, 58% of surgeons rely on a backup generator and 29% operate by mobile phone light. Only 9% of respondents regularly use a surgical headlight, with the most common barriers reported as unaffordability and poor in-country suppliers. CONCLUSIONS: In our survey of surgeons working in low-resource settings, a majority report poor surgical lighting as a major risk to patient safety and nearly one-third report delayed or cancelled operations due to poor lighting. Developing and distributing robust, affordable, high-quality surgical headlights could provide an ideal solution to this significant surgical safety issue.