Explainable artificial intelligence prediction-based model in laparoscopic liver surgery for segments 7 and 8: an international multicenter study.

BACKGROUND: Artificial intelligence (AI) is becoming more useful as a decision-making and outcomes predictor tool. We have developed AI models to predict surgical complexity and the postoperative course in laparoscopic liver surgery for segments 7 and 8. METHODS: We included patients with lesions located in segments 7 and 8 operated by minimally invasive liver surgery from an international multi-institutional database. We have employed AI models to predict surgical complexity and postoperative outcomes. Furthermore, we have applied SHapley Additive exPlanations (SHAP) to make the AI models interpretable. Finally, we analyzed the surgeries not converted to open versus those converted to open. RESULTS: Overall, 585 patients and 22 variables were included. Multi-layer Perceptron (MLP) showed the highest performance for predicting surgery complexity and Random Forest (RF) for predicting postoperative outcomes. SHAP detected that MLP and RF gave the highest relevance to the variables "resection type" and "largest tumor size" for predicting surgery complexity and postoperative outcomes. In addition, we explored between surgeries converted to open and non-converted, finding statistically significant differences in the variables "tumor location," "blood loss," "complications," and "operation time." CONCLUSION: We have observed how the application of SHAP allows us to understand the predictions of AI models in surgical complexity and the postoperative outcomes of laparoscopic liver surgery in segments 7 and 8.

Major Susceptibility Gene Epistasis over Minor Gene Resistance to Spot Form Net Blotch in a Commercial Barley Cultivar.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a significant global disease of barley (Hordeum vulgare). Baudin, a barley cultivar that was until recently extensively grown in Western Australia, was reported as having minor seedling resistance. However, Baudin was highly susceptible to a local isolate, M3, suggesting that this isolate had gained virulence against a major susceptibility gene. M3 causes atypical lesions with pale centers early in the infection, with initial screens of a segregating population indicating that this was determined by a single locus in the Baudin genome. The susceptibility was semidominant in F1 progeny and the susceptibility gene, designated Spm1 (Susceptibility to P. teres f. maculata 1), mapped to a 190-kb section of the resistance gene-rich Mla region of chromosome 1H. Phenotyping with Ptm SP1, a non-M3 pathotype, identified a seedling resistance locus on 2H. Minor gene resistance is generally regarded as potentially durable, but our findings suggest the resistance to spot form net blotch in Baudin is nullified by strong susceptibility conferred by a separate locus on 1H. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

In-cell kinetic stability is an essential trait in metallo-ß-lactamase evolution.

Protein stability is an essential property for biological function. In contrast to the vast knowledge on protein stability in vitro, little is known about the factors governing in-cell stability. Here we show that the metallo-ß-lactamase (MBL) New Delhi MBL-1 (NDM-1) is a kinetically unstable protein on metal restriction that has evolved by acquiring different biochemical traits that optimize its in-cell stability. The nonmetalated (apo) NDM-1 is degraded by the periplasmic protease Prc that recognizes its partially unstructured C-terminal domain. Zn(II) binding renders the protein refractory to degradation by quenching the flexibility of this region. Membrane anchoring makes apo-NDM-1 less accessible to Prc and protects it from DegP, a cellular protease degrading misfolded, nonmetalated NDM-1 precursors. NDM variants accumulate substitutions at the C terminus that quench its flexibility, enhancing their kinetic stability and bypassing proteolysis. These observations link MBL-mediated resistance with the essential periplasmic metabolism, highlighting the importance of the cellular protein homeostasis.

Widespread genetic heterogeneity and genotypic grouping associated with fungicide resistance among barley spot form net blotch isolates in Australia.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a major foliar disease of barley worldwide. Knowledge of the pathogen's genetic diversity and population structure is critical for a better understanding of inherent evolutionary capacity and for the development of sustainable disease management strategies. Genome-wide, single nucleotide polymorphism data of 254 Australian isolates revealed genotypic diversity and an absence of population structure, either between states, or between fields and cultivars in different agro-ecological zones. This indicates there is little geographical isolation or cultivar directional selection and that the pathogen is highly mobile across the continent. However, two cryptic genotypic groups were found only in Western Australia, predominantly associated with genes involved in fungicide resistance. The findings in this study are discussed in the context of current cultivar resistance and the pathogen's adaptive potential.

Histidine 19 Residue Is Essential for Cell Internalization of Antifungal Peptide SmAPα1-21 Derived from the α-Core of the Silybum marianum Defensin DefSm2-D in Fusarium graminearum.

The synthetic peptide SmAPα1-21 (KLCEKPSKTWFGNCGNPRHCG) derived from DefSm2-D defensin α-core is active at micromolar concentrations against the phytopathogenic fungus Fusarium graminearum and has a multistep mechanism of action that includes alteration of the fungal cell wall and membrane permeabilization. Here, we continued the study of this peptide's mode of action and explored the correlation between the biological activity and its primary structure. Transmission electron microscopy was used to study the ultrastructural effects of SmAPα1-21 in conidial cells. New peptides were designed by modifying the parent peptide SmAPα1-21 (SmAPH19R and SmAPH19A, where His19 was replaced by Arg or Ala, respectively) and synthesized by the Fmoc solid phase method. Antifungal activity was determined against F. graminearum. Membrane permeability and subcellular localization in conidia were studied by confocal laser scanning microscopy (CLSM). Reactive oxygen species (ROS) production was assessed by fluorescence spectroscopy and CLSM. SmAPα1-21 induced peroxisome biogenesis and oxidative stress through ROS production in F. graminearum and was internalized into the conidial cells' cytoplasm. SmAPH19R and SmAPH19A were active against F. graminearum with minimal inhibitory concentrations (MICs) of 38 and 100 µM for SmAPH19R and SmAPH19A, respectively. The replacement of His19 by Ala produced a decrease in the net charge with a significant increase in the MIC, thus evidencing the importance of the positive charge in position 19 of the antifungal peptide. Like SmAPα1-21, SmAP2H19A and SmAP2H19R produced the permeabilization of the conidia membrane and induced oxidative stress through ROS production. However, SmAPH19R and SmAPH19A were localized in the conidia cell wall. The replacement of His19 by Ala turned all the processes slower. The extracellular localization of peptides SmAPH19R and SmAPH19A highlights the role of the His19 residue in the internalization.

Case Report: Mini-endoscopic combined intrarenal surgery in an en-bloc kidney transplant.

BACKGROUND: Overall incidence of stones in kidney transplant recipients is 1%. En-bloc kidney transplant is a rare anatomical condition in which kidney stones treatment can be extremely difficult to treat. As far as we know, no cases of staghorn calculi in en-bloc kidney transplant have been published so far. CASE PRESENTATION: A 27-year-old woman presented to the Emergency Department because of asthenia, adynamia and weight loss associated with lower urinary tract symptoms and subfebrile temperature. Ten years before, she had undergone an en-bloc kidney transplant because of end-stage renal disease secondary to perinatal asphyxia syndrome. One kidney was implanted capo-volta in the right iliac fossa and the other one in the right flank. NCCT scan showed incomplete staghorn calculi in the iliac fossa transplanted kidney. Besides, severe dilation of the native and the right flank transplanted kidney, due to two ureteral stones of 6 and 7 mm impacted in the uretero-ureteral anastomosis, was found. After hospital admission and under ceftriaxone prophylaxis, an attempt to perform primary RIRS following our COVID protocol was carried out. Nevertheless, we ended up placing a JJ stent because once the guidewire passed through the ureteral stones, purulent material came out from the ureteral orifice. She stayed 9 days in-hospital for management of postobstructive polyuria and was discharged with oral antibiotics. Three weeks afterward, we removed the stent and performed flexible ureteroscopy and holmium laser lithotripsy of the ureteral stones. In the same procedure, we performed Mini-ECIRS (21 French) previous ultrasound-guided upper pole puncture. Postoperative NCCT scan showed neither residual fragments nor operative complications. CONCLUSION: This is the first clinical case reporting Mini-ECIRS in a patient with an en-bloc kidney transplant. This endourological approach seems to be a feasible, safe and effective approach to treat stones in this anatomically challenging condition.

Evaluation of Avian Reovirus S1133 Vaccine Strain in Neonatal Broiler Chickens in Gastrointestinal Integrity and Performance in a Large-Scale Commercial Field Trial.

Avian reovirus (ARV) is the principal cause of several diseases. The vaccination of breeders allows for the control of viral arthritis and delivery of maternal-derived antibodies to the progeny. The vaccination of broiler chickens with ARV strain S1133 is used to prevent viral arthritis. However, the post-vaccination enteric effects have not been well-characterized. The purpose of this study was to evaluate the effect of vaccination with the S1133 strain on the weight gain and feed conversion of broiler chickens and to characterize the gastric, enteric, and pancreatic lesions that the strain could induce. A total of 672,000 chickens were divided into two groups: a group vaccinated with ARV strain S1133 (S1133ARV) and a control group (not vaccinated). Upon histological analysis, the vaccine group showed less proventricular glandular tissue and atrophy of the pancreas and duodenal villi, as well as having a lower average daily profit. The conclusion based on the results of this investigation is that neonatal vaccination with S1133ARV causes atrophy of the pancreatic acini, proventricular glands, and intestinal villi, leading to an increased diameter of the glandular lumen and atrophy of the enteric villous, as well as weight loss, in broiler chickens.

Flexible fibre optic vs digital ureteroscopy and enhanced vs unenhanced imaging for diagnosis and treatment of upper tract urothelial carcinoma (UTUC): results from the Clinical Research Office of the Endourology Society (CROES)-UTUC registry.

OBJECTIVES: To compare the oncological outcomes of patients with upper tract urothelial carcinoma (UTUC) undergoing kidney-sparing surgery (KSS) with fibre-optic (FO) vs digital (D) ureteroscopy (URS). To evaluate the oncological impact of image-enhancement technologies such as narrow-band imaging (NBI) and Image1-S in patients with UTUC. PATIENTS AND METHODS: The Clinical Research Office of the Endourology Society (CROES)-UTUC registry is an international, multicentre, cohort study prospectively collecting data on patients with UTUC. Patients undergoing flexible FO- or D-URS for diagnostic or diagnostic and treatment purposes were included. Differences between groups in terms of overall survival (OS) and disease-free survival (DFS) were evaluated. RESULTS: The CROES registry included 2380 patients from 101 centres and 37 countries, of whom 401 patients underwent URS (FO-URS 186 and D-URS 215). FO-URS were performed more frequently for diagnostic purposes, while D-URS was peformed when a combined diagnostic and treatment strategy was planned. Intra- and postoperative complications did not differ between the groups. The 5-year OS and DFS rates were 91.5% and 66.4%, respectively. The mean OS was 42 months for patients receiving FO-URS and 39 months for those undergoing D-URS (P = 0.9); the mean DFS was 28 months in the FO-URS group and 21 months in the D-URS group (P < 0.001). In patients who received URS with treatment purposes, there were no differences in OS (P = 0.9) and DFS (P = 0.7). NBI and Image1-S technologies did not improve OS or DFS over D-URS. CONCLUSIONS: D-URS did not provide any oncological advantage over FO-URS. Similarly, no differences in terms of OS and DFS were found when image-enhancement technologies were compared to D-URS. These findings underline the importance of surgeon skills and experience, and reinforce the need for the centralisation of UTUC care.

Intra- and inter-rater reliability of electrical impedance myography using adhesive electrodes in healthy volunteers.

In spite of the growing use of the electrical impedance myography (EIM) measures for clinical assessment and follow-up of diseased muscle tissue, reliability studies are scarce. We evaluate the reliability of the (EIM) technique using four adhesive electrodes over the muscle of interest. Intra- and inter-rater reliability was studied within the same session and between sessions. Thirty-one healthy and volunteer subjects aged between 20 and 26 years were recruited. Phase angle, reactance and resistance were assessed for each EIM measurement. Intraclass correlation coefficient (ICC) was used to determine the relative reliability. Absolute reliability was expressed as the standard error of measurement and the minimum detectable change. Relative reliability within the same session and between sessions for the EIM technique was excellent (ICCs > 0.9) concerning both intra- and inter-rater reliability, except for the component reactance. The absolute reliability was very high for the three EIM components. EIM measures using four adhesive electrodes over the area of interest is a reliable technique to assess muscle tissue status. This study confirms that these measurement results barely vary depending on the examiner and the moment. The present study also confirms phase angle as the least affected EIM component by examiner and evaluation moment.

Endourology and benign prostatic hyperplasia in COVID-19 pandemic

ABSTRACT The new disease COVID-19 pandemic has completely modified our lifestyle, changing our personal habits and daily activities and strongly our professional activity. Following World Health Organization (WHO) and health care authorities around the World recommendations, all elective surgeries from benign diagnose procedures must be postponed and imperatively continue working on emergent and oncological urgent pathologies. Surgical elective treatment of benign prostatic hyperplasia (BPH) is not considered as a priority. During BPH endoscopic surgeries, urine and blood are mixed with the irrigation liquid implying a risk of viral presence. Furthermore, a steam and smoke bubble is being accumulated inside the bladder implying the risk of splashing and aerosols. The risks of other viral infections have been identified during endourological procedures and they are related to splashing events. Several studies observed 33-100% of splashing on goggles. All BPH endoscopic procedures must be postponed. In case of complete urinary obstruction, this event can be adequately treated by urethral or suprapubic catheter under local anesthesia. As soon as local COVID-19 prevalence decreases, endourological procedures could be restarted. As protocols are being validating around the World to redeem elective surgeries, a symptomatic obstructed patient could be operated knowing his COVID-19 status with a molecular PCR, a cleaned epidemiological interview with a normal preoperative protocol. If patient is COVID-19+, surgery must be delayed until complete recovery, because mortality could increase as Lei from Wuhan describes. Informed consent must include risks of complications related to COVID-19 disease. Surgery must be performed by an experienced surgeon in order to avoid increase of operating time and risks of complications. Surgical approach of BPH must be considered depending on availability of disposable material, infrastructure, and the epidemiological COVID-19 status of your area. The main aim is patients and healthcare staff safety.

Endourology and Benign Prostatic Hyperplasia in COVID-19 Pandemic.

The new disease COVID-19 pandemic has completely modified our lifestyle, changing our personal habits and daily activities and strongly our professional activity. Following World Health Organization (WHO) and health care authorities around the World recommendations, all elective surgeries from benign diagnose procedures must be postponed and imperatively continue working on emergent and oncological urgent pathologies. Surgical elective treatment of benign prostatic hyperplasia (BPH) is not considered as a priority. During BPH endoscopic surgeries, urine and blood are mixed with the irrigation liquid implying a risk of viral presence. Furthermore, a steam and smoke bubble is being accumulated inside the bladder implying the risk of splashing and aerosols. The risks of other viral infections have been identified during endourological procedures and they are related to splashing events. Several studies observed 33-100% of splashing on goggles. All BPH endoscopic procedures must be postponed. In case of complete urinary obstruction, this event can be adequately treated by urethral or suprapubic catheter under local anesthesia. As soon as local COVID-19 prevalence decreases, endourological procedures could be restarted. As protocols are being validating around the World to redeem elective surgeries, a symptomatic obstructed patient could be operated knowing his COVID-19 status with a molecular PCR, a cleaned epidemiological interview with a normal preoperative protocol. If patient is COVID-19+, surgery must be delayed until complete recovery, because mortality could increase as Lei from Wuhan describes. Informed consent must include risks of complications related to COVID-19 disease. Surgery must be performed by an experienced surgeon in order to avoid increase of operating time and risks of complications. Surgical approach of BPH must be considered depending on availability of disposable material, infrastructure, and the epidemiological COVID-19 status of your area. The main aim is patients and healthcare staff safety.

Expansion and Conservation of Biosynthetic Gene Clusters in Pathogenic Pyrenophora spp.

Pyrenophora is a fungal genus responsible for a number of major cereal diseases. Although fungi produce many specialised or secondary metabolites for defence and interacting with the surrounding environment, the repertoire of specialised metabolites (SM) within Pyrenophora pathogenic species remains mostly uncharted. In this study, an in-depth comparative analysis of the P. teres f. teres, P teres f. maculata and P. tritici-repentis potential to produce SMs, based on in silico predicted biosynthetic gene clusters (BGCs), was conducted using genome assemblies from PacBio DNA reads. Conservation of BGCs between the Pyrenophora species included type I polyketide synthases, terpene synthases and the first reporting of a type III polyketide synthase in P teres f. maculata. P. teres isolates exhibited substantial expansion of non-ribosomal peptide synthases relative to P. tritici-repentis, hallmarked by the presence of tailoring cis-acting nitrogen methyltransferase domains. P. teres isolates also possessed unique non-ribosomal peptide synthase (NRPS)-indole and indole BGCs, while a P. tritici-repentis phytotoxin BGC for triticone production was absent in P. teres. These differences highlight diversification between the pathogens that reflects their different evolutionary histories, host adaption and lifestyles.

Volatile Molecules Secreted by the Wheat Pathogen Parastagonospora nodorum Are Involved in Development and Phytotoxicity.

Septoria nodorum blotch is a major disease of wheat caused by the fungus Parastagonospora nodorum. Recent studies have demonstrated that secondary metabolites, including polyketides and non-ribosomal peptides, produced by the pathogen play important roles in disease and development. However, there is currently no knowledge on the composition or biological activity of the volatile organic compounds (VOCs) secreted by P. nodorum. To address this, we undertook a series of growth and phytotoxicity assays and demonstrated that P. nodorum VOCs inhibited bacterial growth, were phytotoxic and suppressed self-growth. Mass spectrometry analysis revealed that 3-methyl-1-butanol, 2-methyl-1-butanol, 2-methyl-1-propanol, and 2-phenylethanol were dominant in the VOC mixture and phenotypic assays using these short chain alcohols confirmed that they were phytotoxic. Further analysis of the VOCs also identified the presence of multiple sesquiterpenes of which four were identified via mass spectrometry and nuclear magnetic resonance as ß-elemene, α-cyperone, eudesma-4,11-diene and acora-4,9-diene. Subsequent reverse genetics studies were able to link these molecules to corresponding sesquiterpene synthases in the P. nodorum genome. However, despite extensive testing, these molecules were not involved in either of the growth inhibition or phytotoxicity phenotypes previously observed. Plant assays using mutants of the pathogen lacking the synthetic genes revealed that the identified sesquiterpenes were not required for disease formation on wheat leaves. Collectively, these data have significantly extended our knowledge of the VOCs in fungi and provided the basis for further dissecting the roles of sesquiterpenes in plant disease.

Stability and Predictors of Poor 6-min Walking Test Performance over 2 Years in Patients with COPD.

Poor performance in the 6-min walk test (6MWT < 350 m) is an important prognostic indicator of mortality and risk of exacerbations in patients with chronic obstructive pulmonary disease (COPD). Little is known about the stability of this state over time and what factors might predict a poor 6MWT performance. To determine the stability of 6MWT performance over a 2-year period in COPD patients participating in annual medical follow-up visits, and to assess the ability of several clinical, pulmonary, and non-pulmonary factors to predict poor 6MWT performance, we prospectively included 137 patients with stable COPD (mean age, 66.9 ± 8.3 years). The 6MWT was scored at baseline and 2-year follow-up. To evaluate clinical, pulmonary, and non-pulmonary variables as potential predictors of poor 6MWT performance, we used multiple logistic regression models adjusted for age, sex, weight, height, and 6MWT performance at baseline. Poor 6MWT performance was stable over 2 years for 67.4% of patients. Predictors of poor 6MWT performance included a five-repetition sit-to-stand test score ≤2 (OR, 3.01; 95% CI, 1.22-7.42), the percentage of mobility activities with limitations (OR, 1.03; 95% CI, 1.00-1.07), and poor 6MWT performance at baseline (OR, 4.64; 95% CI, 1.88-11.43). Poor 6MWT performance status was stable for the majority of COPD patients. Lower scores on the five-repetition sit-to-stand test and a higher number of mobility activities with limitations were relevant predictors of poor 6MWT performance over 2 years. Prognostic models based on these non-pulmonary factors can provide non-inferior discriminative ability in comparison with prognostic models based on only pulmonary factors.

High incidence of advanced colorectal neoplasia during endoscopic surveillance in serrated polyposis syndrome.

BACKGROUND: Serrated polyposis syndrome (SPS) has been associated with an increased risk of colorectal cancer (CRC). Accordingly, intensive surveillance with annual colonoscopy is advised. The aim of this multicenter study was to describe the risk of advanced lesions in SPS patients undergoing surveillance, and to identify risk factors that could guide the prevention strategy. METHODS: From March 2013 to April 2015, 296 patients who fulfilled criteria I and/or III for SPS were retrospectively recruited at 18 centers. We selected patients in whom successful clearing colonoscopy had been performed and who underwent subsequent endoscopic surveillance. Advanced neoplasia was defined as CRC, advanced adenoma, or advanced serrated lesion that were ≥ 10 mm and/or with dysplasia. Cumulative incidence of advanced neoplasia was calculated and independent predictors of advanced neoplasia development were identified. RESULTS: In 152 SPS patients a total of 315 surveillance colonoscopies were performed (median 2, range 1 - 7). The 3-year cumulative incidence of CRC and advanced neoplasia were 3.1 % (95 % confidence interval [CI] 0 - 6.9) and 42.0 % (95 %CI 32.4 - 51.7), respectively. Fulfilling both I + III criteria and the presence of advanced serrated lesions at baseline colonoscopy were independent predictors of advanced neoplasia development (odds ratio [OR] 1.85, 95 %CI 1.03 - 3.33, P  = 0.04 and OR 2.62, 95 %CI 1.18 - 5.81, P  = 0.02, respectively). During follow-up, nine patients (5.9 %) were referred for surgery for invasive CRC (n = 4, 2.6 %) or because of polyp burden (n = 5, 3.3 %). After total colectomy, 17.9 % patients developed advanced neoplasia in the retained rectum. CONCLUSIONS: Patients with SPS have a substantial risk of developing advanced neoplasia under endoscopic surveillance, whereas CRC incidence is low. Personalized endoscopic surveillance based on polyp burden and advanced serrated histology could help to optimize prevention in patients with SPS.

Transposable Element Genomic Fissuring in Pyrenophora teres Is Associated With Genome Expansion and Dynamics of Host-Pathogen Genetic Interactions.

Pyrenophora teres, P. teres f. teres (PTT) and P. teres f. maculata (PTM) cause significant diseases in barley, but little is known about the large-scale genomic differences that may distinguish the two forms. Comprehensive genome assemblies were constructed from long DNA reads, optical and genetic maps. As repeat masking in fungal genomes influences the final gene annotations, an accurate and reproducible pipeline was developed to ensure comparability between isolates. The genomes of the two forms are highly collinear, each composed of 12 chromosomes. Genome evolution in P. teres is characterized by genome fissuring through the insertion and expansion of transposable elements (TEs), a process that isolates blocks of genic sequence. The phenomenon is particularly pronounced in PTT, which has a larger, more repetitive genome than PTM and more recent transposon activity measured by the frequency and size of genome fissures. PTT has a longer cultivated host association and, notably, a greater range of host-pathogen genetic interactions compared to other Pyrenophora spp., a property which associates better with genome size than pathogen lifestyle. The two forms possess similar complements of TE families with Tc1/Mariner and LINE-like Tad-1 elements more abundant in PTT. Tad-1 was only detectable as vestigial fragments in PTM and, within the forms, differences in genome sizes and the presence and absence of several TE families indicated recent lineage invasions. Gene differences between P. teres forms are mainly associated with gene-sparse regions near or within TE-rich regions, with many genes possessing characteristics of fungal effectors. Instances of gene interruption by transposons resulting in pseudogenization were detected in PTT. In addition, both forms have a large complement of secondary metabolite gene clusters indicating significant capacity to produce an array of different molecules. This study provides genomic resources for functional genetics to help dissect factors underlying the host-pathogen interactions.

A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-ß-lactamases.

Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-ß-lactamases (MßLs) are the main mechanism of resistance against carbapenems. Clinically approved inhibitors of MBLs are currently unavailable as design has been limited by the incomplete knowledge of their mechanism. Here, we report a biochemical and biophysical study of carbapenem hydrolysis by the B1 enzymes NDM-1 and BcII in the bi-Zn(II) form, the mono-Zn(II) B2 Sfh-I and the mono-Zn(II) B3 GOB-18. These MßLs hydrolyse carbapenems via a similar mechanism, with accumulation of the same anionic intermediates. We characterize the Michaelis complex formed by mono-Zn(II) enzymes, and we identify all intermediate species, enabling us to propose a chemical mechanism for mono and binuclear MßLs. This common mechanism open avenues for rationally designed inhibitors of all MßLs, notwithstanding the profound differences between these enzymes' active site structure, ß-lactam specificity and metal content.Carbapenem-resistant bacteria pose a major health threat by expressing metallo-ß-lactamases (MßLs), enzymes able to hydrolyse these life-saving drugs. Here the authors use biophysical and computational methods and show that different MßLs share the same reaction mechanism, suggesting new strategies for drug design.

Enantioselective synthesis of new oxazolidinylthiazolidines as enzyme inhibitors.

The synthesis of new oxazolidinylthiazolidines bicycles, oxygen analogues of bisthiazolidines, also known as metallo-ß-lactamase inhibitors is described. The reaction of ß-aminoalcohols and 2,5-dihydroxy-1,4-dithiane led to oxazolidinylthiazolidines and/or dithia-azabicycles as the main products. The distribution pattern depends mainly on the aminoalcohol substituents. In a one-pot reaction, four new bonds are formed in good yields and with high atom efficiency. When the oxazolidinylthiazolidines are formed, two stereogenic centres are generated with high enantiospecificity. The reaction mechanism is discussed based on crystallographic data and interconversion studies. Two oxazolidinylthiazolidines were evaluated as inhibitors of the potent lactamase NDM-1 and compound 4f displayed competitive inhibition with Ki = 1.6 ± 0.6 µM.

Heterologous expression of cytotoxic sesquiterpenoids from the medicinal mushroom Lignosus rhinocerotis in yeast.

BACKGROUND: Genome mining facilitated by heterologous systems is an emerging approach to access the chemical diversity encoded in basidiomycete genomes. In this study, three sesquiterpene synthase genes, GME3634, GME3638, and GME9210, which were highly expressed in the sclerotium of the medicinal mushroom Lignosus rhinocerotis, were cloned and heterologously expressed in a yeast system. RESULTS: Metabolite profile analysis of the yeast culture extracts by GC-MS showed the production of several sesquiterpene alcohols (C15H26O), including cadinols and germacrene D-4-ol as major products. Other detected sesquiterpenes include selina-6-en-4-ol, ß-elemene, ß-cubebene, and cedrene. Two purified major compounds namely (+)-torreyol and α-cadinol synthesised by GME3638 and GME3634 respectively, are stereoisomers and their chemical structures were confirmed by 1H and 13C NMR. Phylogenetic analysis revealed that GME3638 and GME3634 are a pair of orthologues, and are grouped together with terpene synthases that synthesise cadinenes and related sesquiterpenes. (+)-Torreyol and α-cadinol were tested against a panel of human cancer cell lines and the latter was found to exhibit selective potent cytotoxicity in breast adenocarcinoma cells (MCF7) with IC50 value of 3.5 ± 0.58 µg/ml while α-cadinol is less active (IC50 = 18.0 ± 3.27 µg/ml). CONCLUSIONS: This demonstrates that yeast-based genome mining, guided by transcriptomics, is a promising approach for uncovering bioactive compounds from medicinal mushrooms.