Cryoelectron-microscopy structure of the enteropathogenic Escherichia coli type III secretion system EspA filament.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) utilize a macromolecular type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. This apparatus spans the inner and outer bacterial membranes and includes a helical needle protruding into the extracellular space. Thus far observed only in EPEC and EHEC and not found in other pathogenic Gram-negative bacteria that have a T3SS is an additional helical filament made by the EspA protein that forms a long extension to the needle, mediating both attachment to eukaryotic cells and transport of effector proteins through the intestinal mucus layer. Here, we present the structure of the EspA filament from EPEC at 3.4 Å resolution. The structure reveals that the EspA filament is a right-handed 1-start helical assembly with a conserved lumen architecture with respect to the needle to ensure the seamless transport of unfolded cargos en route to the target cell. This functional conservation is despite the fact that there is little apparent overall conservation at the level of sequence or structure with the needle. We also unveil the molecular details of the immunodominant EspA epitope that can now be exploited for the rational design of epitope display systems.

Design and Implementation of a Prototype Seismogeodetic System for Tectonic Monitoring.

This manuscript describes the design, development, and implementation of a prototype system based on seismogeodetic techniques, consisting of a low-cost MEMS seismometer/accelerometer, a biaxial inclinometer, a multi-frequency GNSS receiver, and a meteorological sensor, installed at the Doñana Biological Station (Huelva, Spain) that transmits multiparameter data in real and/or deferred time to the control center at the University of Cadiz. The main objective of this system is to know, detect, and monitor the tectonic activity in the Gulf of Cadiz region and adjacent areas in which important seismic events occur produced by the interaction of the Eurasian and African plates, in addition to the ability to integrate into a regional early warning system (EWS) to minimize the consequences of dangerous geological phenomena.

Clinical, Histopathologic and Genetic Features of Rhabdoid Meningiomas.

Rhabdoid meningiomas (RM) shows heterogeneous histological findings, and a wide variety of chromosomal copy number alterations (CNA) are associated with an unpredictable course of the disease. In this study, we analyzed a series of 305 RM samples from patients previously reported in the literature and 33 samples from 23 patients studied in our laboratory. Monosomy 22-involving the minimal but most common recurrent region loss of the 22q11.23 chromosomal region was the most observed chromosomal alteration, followed by losses of chromosomes 14, 1, 6, and 19, polysomies of chromosomes 17, 1q, and 20, and gains of 13q14.2, 10p13, and 21q21.2 chromosomal regions. Based on their CNA profile, RM could be classified into two genetic subgroups with distinct clinicopathologic features characterized by the presence of (1) chromosomal losses only and (2) combined losses and gains of several chromosomes. The latter displays a higher frequency of WHO grade 3 tumors and poorer clinical outcomes.

A standardized stepwise approach to minimally invasive ileocolic anastomosis: Tips and tricks for laparoscopic and robotic surgery.

AIM: Intracorporeal anastomosis has been associated with earlier recovery of postoperative bowel function, shorter length of stay and lower surgical site infection rates. The aim of this work is to describe a step-by-step standardized technique for intracorporeal ileocolic and ileosigmoid anastomosis suitable for laparoscopic and robotic colectomy. METHOD: Each step of the technique is illustrated using a composite collection of three operative patient videos. Two procedures were performed robotically and one was laparoscopic. Tips are provided to construct a two-layer anastomosis (both posteriorly and anteriorly). The procedures are presented in stepwise fashion, discussing the advantages and feasibility of the technique. RESULTS: The standardized technique described herein was used in three patients for this report, of whom two underwent right colectomy and one subtotal colectomy for cancer. The median operating time was 255 (206-333) min. There were no intraoperative complications. No major postoperative complications or 30-day readmissions occurred. The median length of stay was 4 (3-5) days. CONCLUSION: The described technique of a two-layer anastomosis can be used with any available minimally invasive approach. It is safe and feasible. Using a standardized approach, the technique can be easily taught and mastered, optimizing operating times and reducing adverse events.

Robotic abdominal resection of tailgut cysts - A technical note with step-by-step description.

AIM: Here, we describe a step-by-step standardized technique for tailgut cyst resection using a single-docking robotic approach. METHOD: Each step of the technique is illustrated using a composite collection of four operative patient videos to demonstrate the advantages and feasibility of this technique. The robot platform utilised is Da Vinci Xi. RESULTS: Five female patients have undergone this operation in our unit. The size of tumours ranged from 12 to 45 mm. Median operating time was 100 min (range 90-150). Mean blood loss in all the patients was less than 50 ml. There were no major intraoperative complications. One patient had a postoperative presacral collection which required radiological drainage. Length of stay in all patients was one day. CONCLUSIONS: This technique using a single-docking robotic approach appears safe and feasible. The robotic approach results in improved dexterity and more accurate dissection, better retraction and excellent vision which improves the ease of operating in the pelvis. Therefore, this approach can be replicated for use in a wide variety of patients with tailgut cysts.

Life with a stoma across five European countries-a cross-sectional study on long-term rectal cancer survivors.

PURPOSE: Stoma-related problems are known to be important to patients and potentially affect everyday life. The prevalence of stoma-related problems in rectal cancer survivors remains undetermined. This study aimed to examine aspects of life with a long-term stoma, stoma management, and stoma-related problems and explore the impact of stoma-related problems on daily life. METHODS: In total, 2262 patients from 5 European countries completed a multidimensional survey. Stoma-related problems were assessed using the Colostomy Impact score. Multivariable regression analysis, after adjusting for potential confounding factors, provided odds ratio (OR) and 95% confidence intervals (CI) for stoma-related problems' association with restrictions in daily life. RESULTS: The 2262 rectal cancer survivors completed the questionnaire at a median of 5.4 years (interquartile range 3.8-7.6) after stoma formation. In the total sample, leakage (58%) and troublesome odour (55%) were most prevalent followed by skin problems (27%) and pain (21%). Stoma-related problems were more prevalent in patients with parastomal bulging. A total of 431 (19%) reported feeling restricted in daily activities in life with a stoma. Leakage, odour, skin problems, stool consistency, and frequent appliance changes were significantly associated with restrictions in daily life. The highest risk of experiencing restrictions was seen for patients having odour (OR 2.74 [95% CI: 1.99-3.78]) more than once a week and skin problems (OR 1.77 [95% CI: 1.38-2.27]). CONCLUSION: In this large cohort with rectal cancer, stoma-related problems were highly prevalent and impacted daily life. Supportive care strategies should entail outreach to patients with a long-term stoma.

Preoperative oral antibiotics with versus without mechanical bowel preparation to reduce surgical site infections following colonic resection: Protocol for an international randomized controlled trial (ORALEV2).

AIM: Surgical site infections (SSIs) are common after colonic surgery. SSIs can cause relevant morbidity and increase costs of care. Preoperative oral antibiotics can reduce the incidence of SSIs after resection of the colon, but the role of mechanical bowel preparation (MBP) is debated. This study aims to assess the impact of a combined regimen of oral antibiotics and MBP on SSIs after colonic surgery. METHODS: An international, multicentre, pragmatic, adaptive, parallel-group, randomized controlled trial will be conducted across Europe. Adult patients scheduled to undergo elective colonic resection will be assessed for inclusion. Patients will be randomized into one of two treatment arms: (1) preoperative oral antibiotics without MBP (control); (2) preoperative oral antibiotics with MBP (experimental). All patients will receive intravenous antibiotics at anaesthetic induction. The primary aim will be 30-day SSI, assessed by a blinded nurse. Additional end-points include safety, morbidity and mortality, satisfaction with the preparation, time to return of bowel function, time to complete recovery and time to discharge, long-term results. Analyses will be performed with a modified intention-to-treat approach. Interim analyses are planned. DISCUSSION: This will be the first randomized clinical trial to assess the efficacy and safety of preoperative oral antibiotics plus MBP versus preoperative oral antibiotics only, before colonic surgery. The knowledge obtained could help to establish the ideal preparation for patients scheduled to undergo resection of the colon. Full protocol NCT04161599.

Translation and international validation of the Colostomy Impact score.

AIM: Optimal oncological resection in cancers of the lower rectum often requires a permanent colostomy. However, in some patients a colostomy may have a negative impact on health-related quality of life (HRQoL). The Colostomy Impact (CI) score is a simple questionnaire that identifies patients with stoma dysfunction that impairs HRQoL by dividing patients into 'minor' and 'major' CI groups. This aim of this study is to evaluate construct and discriminative validity, sensitivity, specificity and reliability of the CI score internationally, making it applicable for screening and identification of patients with stoma-related impaired HRQoL. METHOD: The CI score was translated in agreement with WHO recommendations. Cross-sectional cohorts of rectal cancer survivors with a colostomy in Australia, China, Denmark, the Netherlands, Portugal, Spain and Sweden were asked to complete the CI score, the European Organization for Research and Treatment of Cancer (EORTC) quality of life 30-item core questionnaire, the stoma-specific items of the EORTC quality of life 29-item colorectal-specific questionnaire and five anchor questions assessing the impact of colostomy on HRQoL. RESULTS: A total of 2470 patients participated (response rate 51%-93%). CI scores were significantly higher in patients reporting reduced HRQoL due to their colostomy than in patients reporting no reduction. Differences in EORTC scale scores between patients with minor and major CI were significant and clinically relevant. Sensitivity was high regarding dissatisfaction with a colostomy. Regarding evaluation of discriminative validity, the CI score relevantly identified groups with differences in HRQoL. The CI score proved reliable, with equal CI scores between test and retest and an intraclass correlation coefficient in the moderate to excellent range. CONCLUSION: The CI score is internationally valid and reliable. We encourage its use in clinical practice to identify patients with stoma dysfunction who require further attention.

Incidence, features, outcome and impact on health system of de-novo abdominal surgical diseases in patients admitted with COVID-19.

OBJECTIVE: to assess the epidemiology and features of de novo surgical diseases in patients admitted with COVID-19, and their impact on patients and healthcare system. SUMMARY BACKGROUND DATA: Gastrointestinal involvement has been described in COVID-19; however, no clear figures of incidence, epidemiology and economic impact exist for de-novo surgical diseases in hospitalized patients. METHODS: This is a prospective study including all patients admitted with confirmed SARS-CoV-2 rT-PCR, between 1 March and 15 May 2020 at two Tertiary Hospitals. Patients with known surgical disease at admission were excluded. Sub-analyses were performed with a consecutive group of COVID-19 patients admitted during the study period, who did not require surgical consultation. RESULTS: Ten out of 3089 COVID-19 positive patients (0.32%) required surgical consultation. Among those admitted in intensive care unit (ICU) incidence was 1.9%. Mortality was 40% in patients requiring immediate surgery and 20% in those suitable for conservative management. The overall median length of stay (LOS) of patients admitted to ICU was longer in those requiring surgical consultation compared with those who did not (51.5 vs 25 days, p = 0.0042). Patients requiring surgical consultation and treatment for de-novo surgical disease had longer median ICU-LOS (31.5 vs 12 days, p = 0.0004). A median of two post-surgical complications were registered for each patient undergoing surgery. Complication-associated costs were as high as 38,962 USD per patient. CONCLUSIONS: Incidence of de-novo surgical diseases is low in COVID-19, but it is associated with significant morbidity and mortality. Future studies should elucidate the mechanism underlying the condition and identify strategies to prevent the need for surgery.

Structural basis of human kinesin-8 function and inhibition.

Kinesin motors play diverse roles in mitosis and are targets for antimitotic drugs. The clinical significance of these motors emphasizes the importance of understanding the molecular basis of their function. Equally important, investigations into the modes of inhibition of these motors provide crucial information about their molecular mechanisms. Kif18A regulates spindle microtubules through its dual functionality, with microtubule-based stepping and regulation of microtubule dynamics. We investigated the mechanism of Kif18A and its inhibition by the small molecule BTB-1. The Kif18A motor domain drives ATP-dependent plus-end microtubule gliding, and undergoes conformational changes consistent with canonical mechanisms of plus-end-directed motility. The Kif18A motor domain also depolymerizes microtubule plus and minus ends. BTB-1 inhibits both of these microtubule-based Kif18A activities. A reconstruction of BTB-1-bound, microtubule-bound Kif18A, in combination with computational modeling, identified an allosteric BTB-1-binding site near loop5, where it blocks the ATP-dependent conformational changes that we characterized. Strikingly, BTB-1 binding is close to that of well-characterized Kif11 inhibitors that block tight microtubule binding, whereas BTB-1 traps Kif18A on the microtubule. Our work highlights a general mechanism of kinesin inhibition in which small-molecule binding near loop5 prevents a range of conformational changes, blocking motor function.

Proton Conductive Zr-Phosphonate UPG-1-Aminoacid Insertion as Proton Carrier Stabilizer.

Proton exchange membrane fuel cells (PEMFCs) are an attractive green technology for energy generation. The poor stability and performances under working conditions of the current electrolytes are their major drawbacks. Metal-Organic Frameworks (MOFs) have recently emerged as an alternative to overcome these issues. Here, we propose a robust Zr-phosphonate MOF (UPG-1) bearing labile protons able to act a priori as an efficient electrolyte in PEMFCs. Further, in an attempt to further enhance the stability and conductivity of UPG-1, a proton carrier (the amino acid Lysine, Lys) was successfully encapsulated within its porosity. The behaviors of both solids as an electrolyte were investigated by a complete experimental (impedance spectroscopy, water sorption) and computational approach (MonteCarlo, water sorption). Compared with the pristine UPG-1, the newly prepared Lys@UPG-1 composite showed similar proton conductivity but a higher stability, which allows a better cyclability. This improved cyclability is mainly related to the different hydrophobic-hydrophilic balance of the Lys@UPG-1 and UPG-1 and the steric protection of the reactive sites of the MOF by the Lys.

"In vitro" capacitation and further progesterone-induced acrosome exocytosis are linked to specific changes in the expression and location of threonine phosphorylation of boar spermatozoa.

The aim of this study was to determine if the achievement of the "in vitro" capacitation (IVC) status and subsequent progesterone-induced "in vitro" acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono- and bi-dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono-dimensional Western blot in non-capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi-dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non-capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm.

Output ordering and prioritisation system (OOPS): ranking biosynthetic gene clusters to enhance bioactive metabolite discovery.

The rapid increase of publicly available microbial genome sequences has highlighted the presence of hundreds of thousands of biosynthetic gene clusters (BGCs) encoding valuable secondary metabolites. The experimental characterization of new BGCs is extremely laborious and struggles to keep pace with the in silico identification of potential BGCs. Therefore, the prioritisation of promising candidates among computationally predicted BGCs represents a pressing need. Here, we propose an output ordering and prioritisation system (OOPS) which helps sorting identified BGCs by a wide variety of custom-weighted biological and biochemical criteria in a flexible and user-friendly interface. OOPS facilitates a judicious prioritisation of BGCs using G+C content, coding sequence length, gene number, cluster self-similarity and codon bias parameters, as well as enabling the user to rank BGCs based upon BGC type, novelty, and taxonomic distribution. Effective prioritisation of BGCs will help to reduce experimental attrition rates and improve the breadth of bioactive metabolites characterized.

Efficacy of a New Recrystallized Enrofloxacin Hydrochloride-Dihydrate against Leptospirosis in a Hamster Model.

A trial on Syrian hamsters (Mesocricetus auratus) infected with Leptospira interrogans serovar Canicola was established to compare treatment efficacies of daily intramuscular (i.m.) injections of either 10 mg/kg of 5% enrofloxacin (Baytril [BE]; Bayer Animal Health, Mexico) or the same dose of enrofloxacin hydrochloride-dihydrate (enro-C). Hamsters were experimentally infected via the oral submucosa with 400 microorganisms/animal, in a sequential time schedule aligned to the initial treatment day, and were treated in groups as follows: a group treated with 5% enrofloxacin daily for 7 days after 24 h of infection (group BE24); a group treated as described for group BE24 but with enro-C (enro-C24); a group also treated with 5% enrofloxacin but starting at 72 h after infection (BE74); a group treated as described for group BE74 but with injection of enro-C (enro-C74). An untreated-uninfected control group (group CG-) and an infected-untreated control group (group CG+) were assembled (n = 18 in all groups). Weights and temperatures of the hamsters were monitored daily for 28 days. After hamsters were euthanatized or following death, necropsy, histopathology, macroscopic agglutination tests (MAT), bacterial culture, and PCR were performed. The mortality rates were 38.8% in group BE24 and 100% in group BE74 No mortality was observed in group enro-C24, and 11.1% mortality was recorded in group enro-C74 The mortality rates in groups CG+ and CG- were 100% and zero, respectively. Combined necropsy and histopathologic findings revealed signs of septicemia and organ damage in groups BE24, BE72, and CG+ Groups enro-C24 and CG- showed no lesions. Moderated lesions were registered in 3 hamsters in group enro-C72 MAT results were positive in 83.3% of BE24 hamsters (83.3%) and 100% of BE72 and CG+ hamsters; MAT results were positive in 16.7% in group Enro-C24 and 38.9% in group enro-C72 Only 4/18 were PCR positive in group enro-C72 and only 1 in group enro-C24 (P < 0.05). It can be concluded that enro-C may be a viable option to treat leptospirosis in hamsters and that this may be the case in other species.

Effects of reduced glutathione on acrosin activity in frozen-thawed boar spermatozoa.

In pigs, acrosin activity in extended semen is correlated with reproductive performance and has recently been identified as a freezability marker. Reduced glutathione (GSH) is known to decrease sperm cryodamage and increase the reproductive performance of frozen-thawed boar spermatozoa. However, the effects of GSH on the acrosin activity of good and poor freezability ejaculates (GFE and PFE, respectively) is yet to be examined. The present study investigated how supplementing cryopreservation media with GSH affected acrosin activity in GFE and PFE, as well as the relationship between acrosin activity and reproductive performance in frozen-thawed boar spermatozoa. In addition, we examined whether the increase in fertility rates and litter sizes observed after the addition of 2mM GSH to cryopreservation extenders was related to acrosin activity. Supplementing freezing media with 2mM GSH partially counteracted the cryopreservation-related decrease in acrosin activity in GFE but not PFE. Acrosin activity was found to be significantly correlated with in vivo reproductive performance of frozen-thawed boar semen. In conclusion, the effects of adding GSH to freezing extenders on the acrosin activity of frozen-thawed boar spermatozoa rely on the intrinsic freezability of the ejaculate. Furthermore, the maintenance of proper acrosin activity could contribute to the increase in reproductive performance mediated by GSH.

Tuning of the Electronic Levels of Oligothiophene-Naphthalimide Assemblies by Chemical Modification.

Inversion of the connections of amidine linkers combined with controlled oligothiophene chain catenation in oligothiophene-naphthalimide assemblies provides an efficient method to tune the HOMO and LUMO values in this type of assemblies. This modification also suppresses the intramolecular charge transfer (ICT) band normally found in this type of derivatives, also delocalizing the frontier molecular orbitals over the whole conjugated skeleton. The resultant assemblies were used in the fabrication of field-effect transistors, which showed well-balanced ambipolar transport.

Benzotrithiophene versus Benzo/Naphthodithiophene Building Blocks: The Effect of Star-Shaped versus Linear Conjugation on Their Electronic Structures.

The synthesis, characterization, and optical properties of a novel star-shaped oligothiophene with a central rigid trithienobenzene (BTT) core and diketopyrrolopyrrole (DPP) units are reported and compared with homologous linear systems based on the benzodithiophene (BDT) and the naphthodithiophene (NDT) units end capped with DPPs. This comparison is aimed at elucidating the effect of the star-shaped configuration versus linear conformation on the optical and electrical properties. Electronic and vibrational spectroscopies, together with transient absorption spectroscopy, scanning electronic microscopy, and DFT calculations are used to understand not only the molecular properties of these semiconductors, but also to analyze the supramolecular aggregation in these derivatives. We conclude that although the subject star-shaped derivative is not optimal in terms of π-conjugation, its extended BTT unit significantly favors intermolecular π-stacking interactions, which is interesting for their applications in devices. Field-effect transistors and solar cells were fabricated with these new molecular semiconductors and the performance difference discussed.

Assessment of the shaping ability of R-Motion 30 .04 and 25 .06 in mesial canals of mandibular molars based on automated computational measurement.

INTRODUCTION: The aim of this study was to evaluate changes in geometry following root canal preparation using R-Motion instruments with different apical sizes and tapers. METHODS: 54 mesial canals of mandibular molars with single curvature of angles ranging between 20° and 30° were stratified into 3 groups according to their internal anatomy (R-Motion 25/.06, 30/.04, Reciproc Blue) (n=18 per group). Micro-computed tomography was used to standardize the samples before instrumentation and, after instrumentation, to assess canal transportation, changes in canal volume and centering ability. Canals were irrigated with 17% EDTA and sodium hypochlorite, and the final rinse included subsonic agitation of these solutions. Measurements were analyzed automatically using the Dragonfly software (Come, Montreal, Canada) and were confirmed by a technician and an endodontist, based on a previously validated methodology. The results were analyzed using the Kruskal-Wallis's and Mann-Whitney´s tests. The level of statistical significance was set at 5%. RESULTS: Significant differences were found in the coronal third for canal transportation, with Reciproc Blue R25 having greater values compared with both R-Motion instruments (p<0.05) and greater changes in volume when compared with R-Motion 30/.04 (p<0.05). CONCLUSIONS: R-Motion of apical size and taper 25/.06 and 30/.04 were associated with similar changes in geometry following root canal preparation in curved mesial canals of mandibular molars, whereas Reciproc Blue was associated with greater canal transportation in the coronal root third.