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Bacterial resistance to antibiotics has been long recognized as a priority to address for human health. Among all micro-organisms, the so-called multi-drug resistant (MDR) bacteria, which are resistant to most, if not all drugs in our current arsenal, are particularly worrisome. The World Health Organization has prioritized the ESKAPE ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa and Enterobacter species) pathogens, which include four Gram-negative bacterial species. In these bacteria, active extrusion of antimicrobial compounds out of the cell by means of ‘molecular guns’ known as efflux pumps is a main determinant of MDR phenotypes. The resistance-nodulation-cell division (RND) superfamily of efflux pumps connecting the inner and outer membrane in Gram-negative bacteria is crucial to the onset of MDR and virulence, as well as biofilm formation. Thus, understanding the molecular basis of the interaction of antibiotics and inhibitors with these pumps is key to the design of more effective therapeutics. With the aim to contribute to this challenge, and complement and inspire experimental research, in silico studies on RND efflux pumps have flourished in recent decades. Here, we review a selection of such investigations addressing the main determinants behind the polyspecificity of these pumps, the mechanisms of substrate recognition, transport and inhibition, as well as the relevance of their assembly for proper functioning, and the role of protein–lipid interactions. The journey will end with a perspective on the role of computer simulations in addressing the challenges posed by these beautifully complex machineries and in supporting the fight against the spread of MDR bacteria.

Graphical abstract, Efflux pumps of the Resistance-Nodulation-cell Division (RND) superfamily contribute to intrinsic and acquired resistance in Gram-negative pathogens by expelling chemically unrelated antibiotics with high efficiency. They are tripartite systems constituted by an inner-membrane-anchored transporter, an outer membrane factor protein, and a membrane fusion protein. Multimerization of the membrane fusion protein is an essential prerequisite for full functionality of these efflux pumps. In this work, we employed complementary computational techniques to investigate the stability of a dimeric unit of MexA (the membrane fusion protein of the MexAB-OprM RND efflux pump of Pseudomonas aeruginosa), and to provide a molecular rationale for the effect of the G72S substitution, which affects MexAB-OprM functionality by impairing the assembly of MexA. Our findings indicate that: i) dimers of this protein are stable in multiple µs-long molecular dynamics simulations; ii) the mutation drastically alters the conformational equilibrium of MexA, favouring a collapsed conformation that is unlikely to form dimers or higher order assemblies. Unveiling the mechanistic aspects underlying large conformational distortions induced by minor sequence changes is informative to efforts at interfering with the activity of this elusive bacterial weapon. In this respect, our work further confirms how molecular simulations can give important contribution and useful insights to characterize the mechanism of highly complex biological systems.

ZusammenfassungWir berichten über einen 64-jährigen Patienten mit einer Penismetastase zehn Jahre nach Erstdiagnose eines polymorphen Adenokarzinoms der kleinen Speicheldrüsen der Zungenbasis. Unserer Kenntnis gibt es bis dato nur zwei in der Literatur beschriebene Fälle.

Modern drug design relies on a detailed understanding of the molecular recognition process by which biological partners such as a protein and a drug interact and bind to each other. Accounting for the correct structural rearrangements accompanying this process by means of generally applicable and high-throughput in silico methods such as molecular docking represents a great challenge. In this review, we summarize the recent advances in the modeling of molecular recognition accounting for receptor flexibility. First, we outline the theoretical background behind molecular recognition events. Next, we introduce the reader to molecular docking, focusing on some of the current methodologies to treat receptor flexibility and highlighting some of the most successful approaches developed in the last years. Finally, we describe approaches based on molecular dynamics simulations and machine learning algorithms to simulate molecular recognition events. Along the review, we discuss the major strengths and pitfalls of current methodologies, mentioning possible future developments in the field.

Ventricular assist devices (VADs) are considered the standard of care for end-stage heart failure (HF) patients. Despite increasing confidence in the technology, evidence data, endorsement by scientific societies and guidelines, the number of implants reached a steady state and is not increasing at the expected pace. This is most likely related to complications that are still burdening the technology and consequently excluding the most needy, ill, and fragile population. In this manuscript we are reporting a single-center experience in a very fragile, elderly and end-stage HF population, with a superficial, partial support device: the CircuLite Synergy. The patients were included in the pre-CE mark clinical study and consequently the device underwent technical adjustment during the support, based on the complications recorded. At our institution were implanted 6 patients overall: 3 patients survived to discharge and 2 survived up to 5 years. Most of the complications recorded were due to patients' frailty and overall clinical conditions. The initial experience with the CircuLite Synergy device is proving that a more "superficial" device might be more tolerable in an elderly, frail population. Partial support has proven hemodynamically efficacious and efficient in relieving heart failure symptoms, improving medical therapy tolerability, and improving quality of life. Unfortunately, the technology was not made available due to financial uncertainty and poor management, but we hope that once the concept has been proved someone will collect the legacy.

Breast cancer (CM) is the most commonly diagnosed form of cancer in Italy and the most common malignancy in women (25%) with 53,000 new cases. This disease affects all age groups (1 in 40 women49 years, 1 in 20 between 50 and 69 years, 1 in 25 between 70 and 84 years). The percentage of patients diagnosed with cancer within 5 years of diagnosis is higher than for other cancers, and this conviction rate in these cancer patients has increased, leading to an ever-improving level of attention. The research aims to understand the main needs of women with breast cancer, compare them with those reported in the literature and in particular to identify which of these are still unsatisfied. Method The study is based on the use of the narrative interview conducted by the Case Manager, in the Breast Unit of Melegnano (Milan), selected with a sampling of convenience.The participants who joined the research project were 12 women with breast cancer, aged between 40 and 60, who are in the active treatment phase of the disease.As highlighted by literature, various unmet needs have emerged, afferent to well-defined areas: emotional health, continuity of care, information, adverse effects, educational strategies, economics, information and social support. The study showed that, the needs that are most unsatisfied relate to the information area and the psychological area.The interviews conducted underlined the importance of the role of the Case Care Manager as a facilitator of the disease path that patients are experiencing both in terms of the clinical and psychological aspects but also as a support in managing daily life. future research should investigate the role of the Case Vare manager in meeting these needs.

There are sex differences in vulnerability to Coronavirus disease 2019 (COVID-19). The coronavirus S protein mediates viral entry into target cells employing the host cellular serine protease TMPRSS2 for S-protein priming. The TMPRSS2 gene expression is responsive to androgen stimulation and it could partially explain sex differences. We hypothesized that men chronically exposed to 5-alpha reductase inhibitors (5ARIs) for benign prostate hyperplasia (BPH) have a lower risk of hospitalization for COVID-19.This is a population-based case-control study on consecutive patients positive for SARS-CoV-2 virus who required hospitalization for COVID-19 (cases), age-matched to beneficiaries of the Lombardy Regional Health Service (controls). Data were collected by two high-volume COVID-19 regional centers of Lombardy (Italy). The primary outcome was to compare the prevalence of patients chronically exposed to 5ARIs, who required hospitalization for COVID-19, with the one of controls.Overall, 943 males were enrolled; 45 (4.77%) were exposed to 5ARI. COVID-19 patients aged55 years under 5ARI treatment were significantly less than expected on the basis of the prevalence of 5ARI treatment among age-matched controls (5.57 vs. 8.14%; P=0.0083, 95% CI: 0.75-3.97%). This disproportion was higher for men aged65 (7.14 vs. 12.31%; P=0.0001, 95% CI: 2.83-6.97%). Eighteen 5ARIs-patients died; the mean age of men who died was higher than those who did not: 75.98±9.29 vs. 64.78±13.57 (P0.001). Cox-regression and multivariable models did not show correlation between 5ARIs exposure and protection against intensive care unit admission/death.Men exposed to 5ARIs might be less vulnerable to severe COVID-19, supporting its use in disease prophylaxis.

The drug/proton antiporter AcrB, part of the major efflux pump AcrABZ-TolC in Escherichia coli, is characterized by its impressive ability to transport chemically diverse compounds, conferring a multidrug resistance phenotype. However, the molecular features differentiating between good and poor substrates of the pump have yet to be identified. In this work, we combined molecular docking with molecular dynamics simulations to study the interactions between AcrB and two representative cephalosporins, cefepime and ceftazidime (a good and poor substrate of AcrB, respectively). Our analysis revealed different binding preferences of the two compounds toward the subsites of the large deep binding pocket of AcrB. Cefepime, although less hydrophobic than ceftazidime, showed a higher affinity than ceftazidime for the so-called hydrophobic trap, a region known for binding inhibitors and substrates. This supports the hypothesis that surface complementarity between the molecule and AcrB, more than the intrinsic hydrophobicity of the antibiotic, is a feature required for the interaction within this region. Oppositely, the preference of ceftazidime for binding outside the hydrophobic trap might not be optimal for triggering allosteric conformational changes needed to the transporter to accomplish its function. Altogether, our findings could provide valuable information for the design of new antibiotics less susceptible to the efflux mechanism.

Background and Aim of the Study: Barlow’s disease is characterized by excess myxomatous degenerative tissue, leaflets prolapse and/or billowing, chordal-elongation, and annular-dilation. We reviewed our experience in Minimally Invasive Mitral Valve Repair (MIMVR) techniques to treat this complex mitral pathology. Methods: Between 1999 and 2017 a group of 125 patients was identified as Barlow’s disease undergoing MIMVR. The diagnosis of Barlow disease was based upon preoperative transesophageal-echocardiography (TEE) and confirmed by the surgeon’s assessment during open-heart procedure. Operative technique and data were retrospectively collected along with intra-hospital and long-term follow-up. Results: Successful Mitral Repair was possible in 100% of cases (125 patents) within the first cross clamp. Most patients (118 – 94.4%) were treated by the Edge to Edge (Alfieri-Stich), while 2 (1.6%) where corrected by neochordae implant and 2 (1.6%) by quadrangular resection. 58 patients (47.1%) received complete semi-rigid ring, while 65 (52.9%) incomplete flexible partial ring excluding the LAM. Concomitant procedures were additional cleft occlusion in 10 cases (8%), tricuspid valve repair (ring implant or remodeling) in 29 cases (23.2%), left atrial ablation for atrial fibrillation in 4 cases (3.2%) and atrial septal repair in 4 (3.2%). Operative mortality was 0%. Average long-term follow-up was 85 ± 62 months, with a survival rate of 97.6%, freedom from redo mitral surgery of 98.4% and freedom from >2+ recurrent mitral regurgitation of 94.5%. No difference in outcome could be related to annuloplasty ring type. Conclusions: Mitral repair can be safely and successfully achieved though minimal-invasive approach, with optimal long-term results.

We report the performance of our newly introduced Ensemble Docking with Enhanced sampling of pocket Shape (EDES) protocol coupled to a template-based algorithm to generate near-native ligand conformations in the 2019 iteration of the Grand Challenge organized by the D3R consortium. Using either AutoDock4.2 or HADDOCK2.2 docking programs (each software in two variants of the protocol) our method generated native-like poses among the top 5 submitted for evaluation for most of the 20 targets with similar performances. The protein selected for GC4 was the human beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a transmembrane aspartic-acid protease. We identified at least one pose whose heavy-atoms RMSD was less than 2.5 {\AA} from the native conformation for 16 (80%) and 17 (85%) of the twenty targets using AutoDock and HADDOCK, respectively. Dissecting the possible sources of errors revealed that: i) our EDES protocol (with minor modifications) was able to sample sub-{\aa}ngstrom conformations for all 20 protein targets, reproducing the correct conformation of the binding site within ~1 {\AA} RMSD; ii) as already shown by some of us in GC3, even in the presence of near-native protein structures, a proper selection of ligand conformers is crucial for the success of ensemble-docking calculations. Importantly, our approach performed best among the protocols exploiting only structural information of the apo protein to generate conformations of the receptor for ensemble-docking calculations.

We report the case of a 64-year-old man, initially diagnosed with a polymorphous adenocarcinoma of the small salivary glands at the tongue base, with occurrence of a penile metastasis ten years after diagnosis. To our knowledge, only two such cases have been described in the literature to date.Wir berichten über einen 64-jährigen Patienten mit einer Penismetastase zehn Jahre nach Erstdiagnose eines polymorphen Adenokarzinoms der kleinen Speicheldrüsen der Zungenbasis. Unserer Kenntnis gibt es bis dato nur zwei in der Literatur beschriebene Fälle.

Summary Objectives To obtain a picture of the work done in Italian anatomical pathology centres in 2014, and evaluate differences between the various centres in terms of the workloads of medical and non-medical staff. Methods A self-administered questionnaire designed by a SIAPEC working group was e-mailed to 256 centres and subsequently collected by the Anatomical Pathology Service of Bolzano. QlikView software was used to prepare the final database and check the quality of the data, which were processed using version 18.0 of SPSS for Windows statistical software. Results The questionnaire was completed by 120 of the centres (46.9%), which were staffed by a mean number of 6.6 physicians (range 1-24), 1.6 biologists (range 0-7), 10.8 laboratory technicians (range 2-47) and 2.2 administrative personnel (range 0-9). During 2014, the centres carried out a mean of 15,000 histology examinations (range 3,215-50,680), almost 11,700 immunohistochemistry examinations (range 0-54,359), and a mean of 1,471 molecular biology examinations (range 0-31,322) relating to a mean of 704 patients (range 0-9,434), and a mean of 16,509 cytology examinations (range 0-150,000) relating to 13,383 patients (range 0-120,000). Each centre physician issued a mean of 2,444 histology examinations reports (range 613-11,000); the ratio between the number of immunohistochemistry examinations and the number of histology examinations was 0.8 (range 0-2.7); and each laboratory technician had a mean overall annual workload of 3,072 histology, molecular biology and cytology examinations (range 793-9,882/year). These values varied widely among the participating centres. The mean ratio between the number of histology examinations carried out and the number of physicians was 1,982.77:1 a year in the small centres (< 10,000 histology cases/year), 2,627:1 a year in the medium-sized centres (10-24,999 histology cases/year), and 2,881.34:1 in the large centres (> 25,000 histology cases/year). There were significant differences between the small and medium-sized centres (p = 0.004) and between the small and large centres (p = 0.001), but not between the medium-sized and large centres. The ratio between the total number of histology, molecular biology and cytology examinations and the number of laboratory technicians was 1,963.34 in the small centres (< 10,000 examinations/year), 2,717.11 in the medium-sized centres (10,000-24,999 examinations/year), and 3,531.56 in the large centres (≥ 25,000 examinations/year). There were significant differences between the small and large centres (p = 0.001) and between the medium-sized and large centres (p = 0.004), but not between the small and medium-sized centres. Conclusions The data collected by means of this survey provide an important, albeit partial, point of reference concerning the status of Italian anatomical pathology centres and their recent, everyday working situation.