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.

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.

Global Spread, Genetic Differentiation, and Selection of Barley Spot Form Net Blotch Isolates.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a significant necrotrophic disease of barley that spread worldwide in the twentieth century. Genetic relationships were analyzed to determine the diversity, survival, and dispersal of a diverse collection of 346 isolates from Australia, Southern Africa, North America, Asia Minor, and Europe. The results, based on genome-wide DArTseq data, indicated that isolates from Turkey were the most differentiated with regional sub-structuring, together with individuals closely related to geographically distant genotypes. Elsewhere, population subdivision related to country of origin was evident, although low levels of admixturing was found that may represent rare genotypes or migration from unsampled populations. Canadian isolates were the next most diverged, and Australian and South African the most closely related. With the exception of Turkish isolates, multiple independent Cyp51A mutation events (which confer insensitivity to demethylation inhibitor fungicides) between countries and within regions was evident, with strong selection for a transposable element insertion at the 3' end of the promoter and counterselection elsewhere. Individuals from Western Australia shared genomic regions and Cyp51A haplotypes with South African isolates, suggesting a recent common origin. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

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.

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.

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.

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.

Cross-class metallo-ß-lactamase inhibition by bisthiazolidines reveals multiple binding modes.

Metallo-ß-lactamases (MBLs) hydrolyze almost all ß-lactam antibiotics and are unaffected by clinically available ß-lactamase inhibitors (ßLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of ßLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both l- and d-BTZ enantiomers are micromolar competitive ßLIs of all MBL classes in vitro, with Kis of 6-15 µM or 36-84 µM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 µM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the l-BTZ enantiomers exhibit 100-fold lower Kis (0.26-0.36 µM) than d-BTZs (26-29 µM). Importantly, cell-based time-kill assays show BTZs restore ß-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate ß-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the l-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. d-BTZ complexes most closely resemble ß-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.

Optimization of Conformational Dynamics in an Epistatic Evolutionary Trajectory.

The understanding of protein evolution depends on the ability to relate the impact of mutations on molecular traits to organismal fitness. Biological activity and robustness have been regarded as important features in shaping protein evolutionary landscapes. Conformational dynamics, which is essential for protein function, has received little attention in the context of evolutionary analyses. Here we employ NMR spectroscopy, the chief experimental tool to describe protein dynamics at atomic level in solution at room temperature, to study the intrinsic dynamic features of a metallo- Β: -lactamase enzyme and three variants identified during a directed evolution experiment that led to an expanded substrate profile. We show that conformational dynamics in the catalytically relevant microsecond to millisecond timescale is optimized along the favored evolutionary trajectory. In addition, we observe that the effects of mutations on dynamics are epistatic. Mutation Gly262Ser introduces slow dynamics on several residues that surround the active site when introduced in the wild-type enzyme. Mutation Asn70Ser removes the slow dynamics observed for few residues of the wild-type enzyme, but increases the number of residues that undergo slow dynamics when introduced in the Gly262Ser mutant. These effects on dynamics correlate with the epistatic interaction between these two mutations on the bacterial phenotype. These findings indicate that conformational dynamics is an evolvable trait, and that proteins endowed with more dynamic active sites also display a larger potential for promoting evolution.

Functional genomics-guided discovery of a light-activated phytotoxin in the wheat pathogen Parastagonospora nodorum via pathway activation.

Parastagonospora nodorum is an important pathogen of wheat. The contribution of secondary metabolites to this pathosystem is poorly understood. A biosynthetic gene cluster (SNOG_08608-08616) has been shown to be upregulated during the late stage of P. nodorum wheat leaf infection. The gene cluster shares several homologues with the Cercospora nicotianae CTB gene cluster encoding the biosynthesis of cercosporin. Activation of the gene cluster by overexpression (OE) of the transcription factor gene (SNOG_08609) in P. nodorum resulted in the production of elsinochrome C, a perelyenequinone phytotoxin structurally similar to cercosporin. Heterologous expression of the polyketide synthase gene elcA from the gene cluster in Aspergillus nidulans resulted in the production of the polyketide precursor nortoralactone common to the cercosporin pathway. Elsinochrome C could be detected on wheat leaves infected with P. nodorum, but not in the elcA disruption mutant. The compound was shown to exhibit necrotic activity on wheat leaves in a light-dependent manner. Wheat seedling infection assays showed that ΔelcA exhibited reduced virulence compared with wild type, while infection by an OE strain overproducing elsinochrome C resulted in larger lesions on leaves. These data provided evidence that elsinochrome C contributes to the virulence of P. nodorum against wheat.

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.

Colorectal cancer risk factors in patients with serrated polyposis syndrome: a large multicentre study.

OBJECTIVE: Serrated polyposis syndrome (SPS) is associated with an increased colorectal cancer (CRC) risk, although the magnitude of the risk remains uncertain. Whereas intensive endoscopic surveillance for CRC prevention is advised, predictors that identify patients who have high CRC risk remain unknown. We performed a multicentre nationwide study aimed at describing the CRC risk in patients with SPS and identifying clinicopathological predictors independently associated with CRC. DESIGN: From March 2013 through September 2014, patients with SPS were retrospectively recruited at 18 Spanish centres. Data were collected from medical, endoscopy and histopathology reports. Multivariate logistic regression was performed to identify CRC risk factors. RESULTS: In 296 patients with SPS with a median follow-up time of 45 months (IQR 26-79.7), a median of 26 (IQR 18.2-40.7) serrated polyps and 3 (IQR 1-6) adenomas per patient were detected. Forty-seven patients (15.8%) developed CRC at a mean age of 53.9±12.8, and 4 out of 47 (8.5%) tumours were detected during surveillance (cumulative CRC incidence 1.9%). Patients with >2 sessile serrated adenomas/polyps (SSA/Ps) proximal to splenic flexure and ≥1 proximal SSA/P with high-grade dysplasia were independent CRC risk factors (incremental OR=2, 95% CI 1.22 to 3.24, p=0.006). Patients with no risk factors showed a 55% decrease in CRC risk (OR=0.45, 95% CI 0.24 to 0.86, p=0.01). CONCLUSIONS: Patients with SPS have an increased risk of CRC, although lower than previously published. Close colonoscopy surveillance in experienced centres show a low risk of developing CRC (1.9% in 5 years). Specific polyp features (SSA/P histology, proximal location and presence of high-grade dysplasia) should be used to guide clinical management.

SnPKS19 Encodes the Polyketide Synthase for Alternariol Mycotoxin Biosynthesis in the Wheat Pathogen Parastagonospora nodorum.

Alternariol (AOH) is an important mycotoxin from the Alternaria fungi. AOH was detected for the first time in the wheat pathogen Parastagonospora nodorum in a recent study. Here, we exploited reverse genetics to demonstrate that SNOG_15829 (SnPKS19), a close homolog of Penicillium aethiopicum norlichexanthone (NLX) synthase gene gsfA, is required for AOH production. We further validate that SnPKS19 is solely responsible for AOH production by heterologous expression in Aspergillus nidulans. The expression profile of SnPKS19 based on previous P. nodorum microarray data correlated with the presence of AOH in vitro and its absence in planta. Subsequent characterization of the ΔSnPKS19 mutants showed that SnPKS19 and AOH are not involved in virulence and oxidative stress tolerance. Identification and characterization of the P. nodorum SnPKS19 cast light on a possible alternative AOH synthase gene in Alternaria alternata and allowed us to survey the distribution of AOH synthase genes in other fungal genomes. We further demonstrate that phylogenetic analysis could be used to differentiate between AOH synthases and the closely related NLX synthases. This study provides the basis for studying the genetic regulation of AOH production and for development of molecular diagnostic methods for detecting AOH-producing fungi in the future.

Protective Effect of Plantago major Extract against t-BOOH-Induced Mitochondrial Oxidative Damage and Cytotoxicity.

Plantago major L. produces several chemical substances with anti-inflammatory and analgesic activities and its use in the treatment of oral and throat inflammation in popular medicine is well described. In this study, the antioxidant potential of the Plantago major hydroethanolic extract was screened and its protective action was evaluated against t-BOOH-induced oxidative stress. The extract was obtained by fractionated percolation using 50% ethanolic solution and, after drying, suspended in dimethyl sulfoxide. The chromatographic profile of crude extract was obtained with the identification of some phytochemical markers and the total phenols and flavonoids were quantified. The scavenger activity against DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals was determined and the antioxidant activity in biological systems was evaluated in isolated rat liver mitochondria and HepG2 cells. The extract exhibited a significant free radical scavenger activity at 0.1 mg/mL, and decreased the ROS (reactive oxygen species) generation in succinate-energized mitochondria. Such an effect was associated with the preservation of the intrinsic antioxidant defenses (reduced glutathione and NAD(P)H) against the oxidation by t-BOOH, and also to the protection of membranes from lipid oxidation. The cytoprotective effect of PmHE against t-BOOH induced cell death was also shown. These findings contribute to the understanding of the health benefits attributed to P. major.

Nanoscale markers of esophageal field carcinogenesis: potential implications for esophageal cancer screening.

BACKGROUND AND STUDY AIMS: Esophageal adenocarcinoma (EAC) has a dismal prognosis unless treated early or prevented at the precursor stage of Barrett's esophagus-associated dysplasia. However, some patients with cancer or dysplastic Barrett's esophagus (DBE) may not be captured by current screening and surveillance programs. Additional screening techniques are needed to determine who would benefit from endoscopic screening or surveillance. Partial wave spectroscopy (PWS) microscopy (also known as nanocytology) measures the disorder strength (Ld ), a statistic that characterizes the spatial distribution of the intracellular mass at the nanoscale level and thus provides insights into the cell nanoscale architecture beyond that which is revealed by conventional microscopy. The aim of the present study was to compare the disorder strength measured by PWS in normal squamous epithelium in the proximal esophagus to determine whether nanoscale architectural differences are detectable in the field area of EAC and Barrett's esophagus. METHODS: During endoscopy, proximal esophageal squamous cells were obtained by brushings and were fixed in alcohol and stained with standard hematoxylin and Cyto-Stain. The disorder strength of these sampled squamous cells was determined by PWS. RESULTS: A total of 75 patient samples were analyzed, 15 of which were pathologically confirmed as EAC, 13 were DBE, and 15 were non-dysplastic Barrett's esophagus; 32 of the patients, most of whom had reflux symptoms, acted as controls. The mean disorder strength per patient in cytologically normal squamous cells in the proximal esophagus of patients with EAC was 1.79-times higher than that of controls (P<0.01). Patients with DBE also had a disorder strength 1.63-times higher than controls (P<0.01). CONCLUSION: Intracellular nanoarchitectural changes were found in the proximal squamous epithelium in patients harboring distal EAC and DBE using PWS. Advances in this technology and the biological phenomenon of the field effect of carcinogenesis revealed in this study may lead to a useful tool in non-invasive screening practices in DBE and EAC.

Violence and social capital in post-conflict Guatemala.

OBJECTIVE: Violence in post-conflict Guatemala has serious public health consequences for the population. The objective of this study was to assess the relationship between violence and social capital. METHODS: Data from a cross-sectional victimization survey conducted in 2008 - 2010 in Guatemala were analyzed. Two-stage proportionate sampling was used in the survey. Households (n = 1 300) were randomly sampled within a random sample of communities (n = 118) in five administrative departments. The survey collected information on the six-month violence exposure of 6 335 individuals. Social capital was measured at the household level using the short version of the Adapted Social Capital Tool (SASCAT). The odds ratio for household violence exposure was estimated using multiple logistic regression. Community-level data from the latest national census were included as explanatory factors at the community level. Income, ethnicity, and social capital were included at the household level. Data were analyzed using SPSS 18.0. RESULTS: In total, 2.7% of individuals and 11.7% of households had been exposed to violence within the past six months. The multivariate analysis showed that 1) structural social capital (in this case, the level of participation in social networks and civil society) was a risk factor for violence and 2) cognitive social capital (measured as trust, norms, and sense of belonging) was a protective factor for violence. CONCLUSIONS: The opposite direction of the association between violence and structural and cognitive social capital challenges the use of social capital as a unified concept. If this finding is corroborated by other studies, structural and cognitive social capital will have to be treated as two distinctly different concepts.

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.