Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB

Resistance–nodulation–division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics

Human DNA contamination of postmortem examination facilities: Impact of COVID-19 cleaning procedure

The DNA contamination of evidentiary trace samples, included those collected in the autopsy room, has significant detrimental consequences for forensic genetics investigation. After the COVID-19 pandemic, methods to prevent environmental contamination in the autopsy room have been developed and intensified. This study aimed to evaluate the level of human DNA contamination of a postmortem examination facility before and after the introduction of COVID-19-related disinfection and cleaning procedures. Ninety-one swabs were collected from the surfaces and the dissecting instruments, analyzed by real-time quantitative PCR (q-PCR), and typed for 21 autosomal STRs. Sixty-seven out of 91 samples resulted in quantifiable human DNA, ranging from 1 pg/μl to 12.4 ng/μl, including all the samples collected before the implementation of COVID-19 cleaning procedures (n = 38) and 29 out of 53 (54.7%) samples taken afterward. All samples containing human DNA were amplified, resulting in mixed (83.6%), single (13.4%), and incomplete (3%) profiles. A statistically significant decrease in DNA contamination was found for dissecting instruments after treatment with chlorhexidine and autoclave (p< 0.05). Environmental decontamination strategies adopted during the COVID-19 pandemic only partially solved the long-standing issue of DNA contamination of postmortem examination facilities. The pandemic represents an opportunity to further stress the need for standardized evidence-based protocols targeted to overcome the problem of DNA contamination in the autopsy room

Per- and polyfluoroalkyl substances (PFAS) presence in food: Comparison among fresh, frozen and ready-to-eat vegetables

There is a worldwide discussion to provide safety limits in food for per-and polyfluoroalkyl substances (PFAS), a group of persistent contaminants associated to human disease. Processed food is more at risk of containing increased amounts of PFAS as a consequence of intentionally or non-intentionally contamination during manipulation and packaging. Among food products, also vegetables can be submitted to industrial manipulation; therefore, a different PFAS content correlated to the level of vegetables processing is conceivable. This study assessed the amount and type of PFAS present in fresh, frozen and ready-to-eat vegetables. Differences have been observed between the three groups of samples in the average PFAS content; the difference between ready-to eat and frozen vegetables resulted statistically significative. Organic vegetables displayed a lower total amount of PFAS respect to the traditional counterpart. The impact of industrial manipulation remains to be cleared, but pesticides use during cultivation could be considered a source of PFAS contamination

Assessment of the structural and functional characteristics of human mesenchymal stem cells associated with a prolonged exposure of morphine

The discovery of the expression of opioid receptors in the skin and their role in orchestrating the process of tissue repair gave rise to questions regarding the potential effects of clinical morphine treatment in wound healing. Although short term treatment was reported to improve tissue regeneration, in vivo chronic administration was associated to an impairment of the physiological healing process and systemic fibrosis. Human mesenchymal stem cells (hMSCs) play a fundamental role in tissue regeneration. In this regard, acute morphine exposition was recently reported to impact negatively on the functional characteristics of hMSCs, but little is currently known about its long-term effects. To determine how a prolonged treatment could impair their functional characteristics, we exposed hMSCs to increasing morphine concentrations respectively for nine and eighteen days, evaluating in particular the fibrogenic potential exerted by the long-term exposition. Our results showed a time dependent cell viability decline, and conditions compatible with a cellular senescent state. Ultrastructural and protein expression analysis were indicative of increased autophagy, suggesting a relation to a detoxification activity. In addition, the enhanced transcription observed for the genes involved in the synthesis and regulation of type I collagen suggested the possibility that a prolonged morphine treatment might exert its fibrotic potential risk, even involving the hMSCs

Infection Induced Fetal Inflammatory Response Syndrome (FIRS): State-of- the-Art and Medico-Legal Implications—A Narrative Review

Fetal inflammatory response syndrome (FIRS) represents the fetal inflammatory reaction to intrauterine infection or injury, potentially leading to multiorgan impairment, neonatal mortality, and morbidity. Infections induce FIRS after chorioamnionitis (CA), defined as acute maternal inflammatory response to amniotic fluid infection, acute funisitis and chorionic vasculitis. FIRS involves many molecules, i.e., cytokines and/or chemokines, able to directly or indirectly damage fetal organs. Therefore, due to FIRS being a condition with a complex etiopathogenesis and multiple organ dysfunction, especially brain injury, medical liability is frequently claimed. In medical malpractice, reconstruction of the pathological pathways is paramount. However, in cases of FIRS, ideal medical conduct is hard to delineate, due to uncertainty in diagnosis, treatment, and prognosis of this highly complex condition. This narrative review revises the current knowledge of FIRS caused by infections, maternal and neonatal diagnosis and treatments, the main consequences of the disease and their prognoses, and discusses the medico-legal implications

Insights in opiates toxicity: impairment of human vascular mesenchymal stromal cells

: The most common pulmonary findings in opiate-related fatalities are congestion and oedema, as well as acute and/or chronic alveolar haemorrhage, the cause of which is thought to be a damage to the capillary endothelium related to ischemia. Human vascular mesenchymal stromal cells (vMSCs) play a fundamental role in tissue regeneration and repair after endothelial cell injury, and they express opioid receptors. The aim of this study was to assess the effect of in vitro morphine exposure on the physiological activity and maintenance of human vMSCs. vMSCs were obtained from abdominal aorta fragments collected during surgery repair and were exposed to incremental doses (0.1 mM, 0.4 mM, 0.8 mM and 1 mM) of morphine sulphate for 7 days. The effect was investigated through cell viability assessment, proliferation assay, reactive oxygen species (ROS) detection assay, senescence-associated β-galactosidase assay, senescent-related markers (p21WAF1/CIP1 and p16INK4) and the apoptosis-related marker caspase 3. Moreover, an ultrastructural analysis by transmission electron microscopy and in vitro vascular differentiation were evaluated. Results showed a decrease of the cellular metabolic activity, a pro-oxidant and pro-senescence effect, an increase in intracellular ROS and the activation of the apoptosis signalling, as well as ultrastructural modifications and impairment of vascular differentiation after morphine treatment of vMSC. Although confirmation studies are required on real fatal opiate intoxications, the approach based on morphological and immunofluorescence methodologies may have a high potential also as a useful tool or as a complementary method in forensic pathology. The application of these techniques in the future may lead to the identification of new markers and morphological parameters useful as complementary investigations for drug-related deaths

Structural and Functional Characterization of a New Double Variant Haemoglobin (HbG-Philadelphia/Duarte α268Asn→Lysβ262Ala→Pro)

We report the first case of cosegregation of two haemoglobins (Hbs): HbG-Philadelphia [α68(E17)Asn → Lys] and HbDuarte [β62(E6)Ala → Pro]. The proband is a young patient heterozygous also for β°-thalassaemia. We detected exclusively two haemoglobin variants: HbDuarte and HbG-Philadelphia/Duarte. Functional study of the new double variant HbG-Philadelphia/Duarte exhibited an increase in oxygen affinity, with a slight decrease of cooperativity and Bohr effect. This functional behaviour is attributed to β62Ala → Pro instead of α68Asn → Lys substitution. Indeed, HbG-Philadelphia isolated in our laboratory from blood cells donor carrier for this variant is not affected by any functional modification, whereas purified Hb Duarte showed functional properties very similar to the double variant. NMR and MD simulation studies confirmed that the presence of Pro instead of Ala at the β62 position produces displacement of the E helix and modifications of the tertiary structure. The substitution α68(E17)Asn → Lys does not cause significant structural and dynamical modifications of the protein. A possible structure-based rational of substitution effects is suggested

Combination therapy of high-dose rabeprazole plus metronomic capecitabine in advanced gastrointestinal cancer: a randomized phase II trial

Abstract: Background: In recent years, proton pump inhibitors (PPIs) have been investigated at high-dose tomodulate tumormicroenvironment acidification thus restoring chemotherapeutic sensitivity. This is the first trial to study activity and safety of repurposing high dose rabeprazole combined with metronomic capecitabine (mCAP). Methods: A phase II study in which patients with gastrointestinal cancer, refractory to standard treatments, who had a life expectancy >3 months, were blind randomized 1:1 to mCAP, 1500 mg/daily, continuously with or without rabeprazole 1.5 mg/kg bid, three days a week. The primary endpoint was 3-months progression-free survival (PFS). The secondary endpoints were clinical benefit (CB) and overall survival (OS). Safety and plasma concentrations of capecitabine and its metabolites (50-DFUR and 5-FU) were also evaluated. Results: Sixty-seven (median age 69 years; 63% male; 84% colorectal cancer, 76% ECOG-PS 1; 84% pretreated with two or more lines of chemotherapy) out of 90 patients screened for eligibility, were randomized to receive mCAP+rabeprazole (n = 32) vs. mCAP (n = 35). All patients were evaluable for response. No significant dierence between mCAP+rabeprazole vs. mCAP, in terms of 3-months PFS rate (HR = 1.43, 95%CI 0.53–3.85; p = 0.477), median PFS (HR = 1.22, 95%CI 0.75–2.00, p = 0.420), CB (RR = 0.85, 95%CI 0.29–2.44; p = 0.786) and median OS (HR = 0.89, 95%CI 0.54–1.48; p = 0.664) was observed. However, a 3-year OS rate of 10% and 12% was reported in the mCAP-rabeprazole and mCAP groups, respectively. Overall, no grade 3 or 4 toxicity occurred but grade 1 or 2 adverse event of any type were more frequently in the mCAP+rabeprazole group than in the mCAP (OR 2.83, 95%CI 1.03–7.79; p = 0.043). Finally, there was not statistically significant dierence in the plasma concentration of capecitabine and its metabolites between the two groups. Conclusions: Although the adjunct of high dose rabeprazole to mCAP was not shown to aect mCAP activity, as PPI are being investigated worldwide as drugs to be repositioned in cancer treatment and also considering the limited sample size as well as the favorable safety profile of the combination in the present study, further clinical investigations are desirable

Morphological and immunoistochemical evaluation of cadaveric gingival specimens to estimate the post-mortem Interval

The estimation of the post-mortem interval (PMI) is a critical step in forensic medicine and remains one of the most challenging variables to determine. In general, the numerous methods currently used in estimating post-mortem interval yield to large post-mortem windows, are influenced by several factors and sometime contradict each other.  With the aim to obtain a much more accurate determination of the post-mortem interval we combined morphological ultrastructural and immunoistochemical analyses to reach a more detailed knowledge on tissue organization and degradation after death. Samples of gingival tissues obtained from 20 cadavers at different post-mortem intervals were processed for transmission electron microscopy to evaluate ultrastructural modifications in the epithelium and connective tissue. Gingival cells and the extracellular matrix of gingival tissues have been observed by a transmission electron microscopy (TEM) in combination with the evaluation of potential post-mortem biochemical markers, with the final goal to find a tight correlation between the ultrastructural modifications, the biomarker expression and the time of death. All the samples were also immunostained with anti-hypoxia-induced factor 1-α (HIF1-α) antibody, a transcription factor expressed in response to hypoxia, in order to evaluate the expression of HIF1-α, and to establish a correlation between the protein presence and the time of death. Results showed nuclear chromatin changes and cytoplasmic vacuolization in both epithelial and connective tissues and a different pattern of expression of HIF1alpha protein that correlate to the time of death. In conclusion, our preliminary findings suggest that ultrastructural investigations in combination with immunohistochemistry techniques in gingival specimens may represent a new tool to accurately and systematically estimate post-mortem interval