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Graphene and hexagonal boron nitride (hBN) are two-dimensional (2D) materials with a similar atomic structure but drastically different although complementary electronic properties. The large-scale synthesis of h-BN/graphene heterostructures with high crystallographic quality is required to fully benefit of the graphene electronic properties. In this study, we examine numerically the interaction of graphene precursors on hBN and of hBN precursors on graphene to gain deep insight of the CVD and MBE growth mechanism of graphene/hBN heterostructures. Density functional theory (DFT) calculations reveal the adsorption and diffusion behaviors for B, N, and C atoms on these surfaces. In particular, the adsorption energy is found to be similar to the diffusion barriers, except for the nearly free diffusion of B atoms on both graphene and hBN. We have also investigated the transition from individual atoms to graphene or h-BN seeds by considering the stability of linear chains as well as branched and ring seeds. Furthermore, for larger clusters, the triangular h-BN domains are found to be equally thermodynamically stable on graphene regardless of their orientation. These findings provide preliminary hints for the ability of graphene to grow on hBN layers and hBN layer on graphene.

The therapeutic use of mesenchymal stem cells (MSCs) becomes more and more important due to their potential for cell replacement procedures as well as due to their immunomodulatory properties. However, protocols for MSCs differentiation can be lengthy and may result in incomplete or asynchronous differentiation. To ensure homogeneous populations for therapeutic purposes, it is crucial to develop protocols for separation of the different cell types after differentiation. In this article we show that, when MSCs start to differentiate towards adipogenic or osteogenic progenies, their dielectrophoretic behavior changes. The values of cell electric parameters which can be obtained by dielectrophoretic measurements (membrane permittivity, conductivity, and cytoplasm conductivity) change before the morphological features of differentiation become microscopically visible. We further demonstrate, by simulation, that these electric modifications make possible to separate cells in their early stages of differentiation by using the dielectrophoretic separation technique. A label free method which allows obtaining cultures of homogenously differentiated cells is thus offered., (© 2024. The Author(s).)

Escherichia coli bacterium is a rod-shaped organism composed of a complex double membrane structure. Knowledge of electric field driven ion transport through both membranes and the evolution of their induced permeabilization has important applications in biomedical engineering, delivery of genes and antibacterial agents. However, few studies have been conducted on Gram-negative bacteria in this regard considering the contribution of all ion types. To address this gap in knowledge, we have developed a deterministic and stochastic Brownian dynamics model to simulate in 3D space the motion of ions through pores formed in the plasma membranes of E. coli cells during electroporation. The diffusion coefficient, mobility, and translation time of Ca 2+ , Mg 2+ , Na + , K + , and Cl - ions within the pore region are estimated from the numerical model. Calculations of pore's conductance have been validated with experiments conducted at Gustave Roussy. From the simulations, it was found that the main driving force of ionic uptake during the pulse is the one due to the externally applied electric field. The results from this work provide a better understanding of ion transport during electroporation, aiding in the design of electrical pulses for maximizing ion throughput, primarily for application in cancer treatment., (© 2023. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Background and Aims: The presence of significant liver fibrosis associated with non-alcoholic steatohepatitis (NASH) is regarded as the major prognostic factor in non-alcoholic fatty liver disease (NAFLD). Identification of patients at risk for NASH with significant fibrosis is therefore important. Although the established fibrosis score FIB-4 is suitable to exclude advanced fibrosis, it does not allow the prediction of significant fibrosis in NAFLD patients. We therefore evaluated whether the hepatokine fibroblast growth factor 21 (FGF21), a regulator of glucose and lipid metabolism, might identify 'at-risk NASH' in NAFLD., Methods: FGF21 levels were assessed by enzyme-linked immunosorbent assay in sera from an exploration (n = 137) and a validation (n = 88) cohort of biopsy-proven NAFLD patients with different disease activity and fibrosis stages. In addition, we evaluated whether the use of FGF21 could improve risk stratification in NAFLD patients with low (<1.3) or intermediate (1.3-2.67) FIB-4., Results: FGF21 levels could significantly discriminate between NASH and non-alcoholic fatty liver (NAFL) patients, even in the absence of diabetes. Moreover, patients with NASH and fibrosis ≥F2 showed significantly higher FGF21 levels compared to NAFLD patients without significant fibrosis. Significantly elevated FGF21 levels could even be detected in NAFLD patients with NASH and significant fibrosis despite low or intermediate FIB-4., Conclusion: Serological FGF21 detection might allow the identification of NAFLD patients at risk and improves patient stratification in combination with FIB-4., (© 2023 The Authors. Liver International published by John Wiley & Sons Ltd.)

Postoperative delirium is a serious sequela of surgery and surgery-related anaesthesia. One recommended method to prevent postoperative delirium is using bi-frontal EEG recording. The single, processed index of depth of anaesthesia allows the anaesthetist to avoid episodes of suppression EEG and excessively deep anaesthesia. The study data presented here were based on multichannel (19 channels) EEG recordings during anaesthesia. This enabled the analysis of various parameters of global electrical brain activity. These parameters were used to compare microstate topographies under anaesthesia with those in healthy volunteers and to analyse changes in microstate quantifiers and EEG global state space descriptors with increasing exposure to anaesthesia. Seventy-three patients from the Surgery Depth of Anaesthesia and Cognitive Outcome study (SRCTN 36437985) received intraoperative multichannel EEG recordings. Altogether, 720 min of artefact-free EEG data, including 210 min (29.2%) of suppression EEG, were analysed. EEG microstate topographies, microstate quantifiers (duration, frequency of occurrence and global field power) and the state space descriptors sigma (overall EEG power), phi (generalized frequency) and omega (number of uncorrelated brain processes) were evaluated as a function of duration of exposure to anaesthesia, suppression EEG and subsequent development of postoperative delirium. The major analyses involved covariate-adjusted linear mixed-effects models. The older (71 ± 7 years), predominantly male (60%) patients received a median exposure of 210 (range: 75-675) min of anaesthesia. During seven postoperative days, 21 patients (29%) developed postoperative delirium. Microstate topographies under anaesthesia resembled topographies from healthy and much younger awake persons. With increasing duration of exposure to anaesthesia, single microstate quantifiers progressed differently in suppression or non-suppression EEG and in patients with or without subsequent postoperative delirium. The most pronounced changes occurred during enduring suppression EEG in patients with subsequent postoperative delirium: duration and frequency of occurrence of microstates C and D progressed in opposite directions, and the state space descriptors showed a pattern of declining uncorrelated brain processes (omega) combined with increasing EEG variance (sigma). With increasing exposure to general anaesthesia, multiple changes in the dynamics of microstates and global EEG parameters occurred. These changes varied partly between suppression and non-suppression EEG and between patients with or without subsequent postoperative delirium. Ongoing suppression EEG in patients with subsequent postoperative delirium was associated with reduced network complexity in combination with increased overall EEG power. Additionally, marked changes in quantifiers in microstate C and in microstate D occurred. These putatively adverse intraoperative trajectories in global electrical brain activity may be seen as preceding and ultimately predicting postoperative delirium., Competing Interests: The authors report no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Background and Objective: Knowledge of accuracy for melanoma diagnosis and melanoma discovering-individual in primary care is limited. We describe general practitioner (GP) characteristics and analyse defined diagnostic accuracy metrics for GPs in the current study comparing this with a previous study for GPs common to both, and we analyse the individual first discovering each melanoma as a lesion of concern., Methods: The characteristics and diagnostic accuracy of 27 Australasian GPs documenting 637 melanomas on the Skin Cancer Audit Research Database (SCARD) in 2013 were described and analysed. The number needed to treat (NNT) and percentage of melanomas that were in situ (percentage in situ) were analysed as surrogates for specificity and sensitivity, respectively. The discovering-individual was analysed according to patient age and sex and lesion Breslow thickness., Results: The average NNT and percentage in situ were 5.73% and 65.07%, respectively. For 21 GPs in both a 2008-2010 study and the current study, the NNT was 10.78 and 5.56, respectively (p = 0.0037). A consistent trend of decreasing NNT and increasing percentage in situ through increasingly subspecialised GP categories did not reach statistical significance. NNT trended high at ages and sites for which melanoma was rare. While the patient or family member was more likely to discover thick melanomas and melanomas in patients under 40 years, GPs discovered 73.9% of the melanomas as lesions of concern., Conclusions: GPs were the discovering-individuals for the majority of melanomas in the current study and their accuracy metrics compared favourably with published figures for dermatologists and GPs., (© 2023 The Authors. Australasian Journal of Dermatology published by John Wiley & Sons Australia, Ltd on behalf of Australasian College of Dermatologists.)

This study aimed to demonstrate that severe neurological motor deficits in the context of late tick-borne disease with mixed microorganism involvement are eligible for long-term combined antibiotic/antiparasitic treatments. The inclusion criteria were: 1. neurological limb paralysis with a disability score >4 according to the EDSS Kurtzke disability scale; 2. serological tests pointing to an involvement of the main tick-borne microorganisms Borrelia burgdorferi s.l., Babesia , Anaplasma , and Bartonella ; 3. a general disease for more than 6 months with fatigue, pain and subjective cognitive deficit. The patients were administered long-term treatments with repeated cycles (at least three) of 35-day IV ceftriaxone and repeated oral regimens of azithromycin-doxycycline and azithromycin-doxycycline-rifampicin. For Babesia , repeated courses of atovaquone-azithromycin were administered. Ten patients had intractable or severe motor deficits before treatment in the context of Borrelia (two cases) Borrelia-Babesia (four cases), Borrelia-Babesia-Anaplasma (two cases), Borrelia-Babesia-Anaplasma-Bartonella (one case) and Babesia-Anaplasma (one case). For several months, five had been in wheelchairs, and four had been walking with sticks. Seven patients out of 10 (70%) showed complete remission after a mean active treatment duration of 20.1 + 6.6 months, with a mean number of 4 ceftriaxone cycles. Three patients showed an initial remission but suffered secondary antibiotic/antiparasitic-resistant motor recurrences. Among the nine patients with Borrelia serologic positivity, treatments obtained complete remission in seven cases (77%). The findings of this ten-case series suggest the usefulness of long-term antibiotic/antiparasitic treatments in patients with severe late tick-borne neurological deficits with highly significant elements of tick-borne involvement.

Tau is an intrinsically disordered neuronal protein in the central nervous system. Aggregated Tau is the main component of neurofibrillary tangles observed in Alzheimer's disease. In vitro, Tau aggregation can be triggered by polyanionic co-factors, like RNA or heparin. At different concentration ratios, the same polyanions can induce Tau condensates via liquid-liquid phase separation (LLPS), which over time develop pathological aggregation seeding potential. Data obtained by time resolved Dynamic Light Scattering experiments (trDLS), light and electron microscopy show that intermolecular electrostatic interactions between Tau and the negatively charged drug suramin induce Tau condensation and compete with the interactions driving and stabilizing the formation of Tau:heparin and Tau:RNA coacervates, thus, reducing their potential to induce cellular Tau aggregation. Tau:suramin condensates do not seed Tau aggregation in a HEK cell model for Tau aggregation, even after extended incubation. These observations indicate that electrostatically driven Tau condensation can occur without pathological aggregation when initiated by small anionic molecules. Our results provide a novel avenue for therapeutic intervention of aberrant Tau phase separation, utilizing small anionic compounds., (© 2023. The Author(s).)

The growth of hexagonal boron nitride (hBN) on epitaxial Ge(001)/Si substrates via high-vacuum chemical vapor deposition from borazine is investigated for the first time in a systematic manner. The influences of the process pressure and growth temperature in the range of 10 -7 -10 -3 mbar and 900-980 °C, respectively, are evaluated with respect to morphology, growth rate, and crystalline quality of the hBN films. At 900 °C, nanocrystalline hBN films with a lateral crystallite size of ~2-3 nm are obtained and confirmed by high-resolution transmission electron microscopy images. X-ray photoelectron spectroscopy confirms an atomic N:B ratio of 1 ± 0.1. A three-dimensional growth mode is observed by atomic force microscopy. Increasing the process pressure in the reactor mainly affects the growth rate, with only slight effects on crystalline quality and none on the principle growth mode. Growth of hBN at 980 °C increases the average crystallite size and leads to the formation of 3-10 well-oriented, vertically stacked layers of hBN on the Ge surface. Exploratory ab initio density functional theory simulations indicate that hBN edges are saturated by hydrogen, and it is proposed that partial de-saturation by H radicals produced on hot parts of the set-up is responsible for the growth.

The geographically widespread species Afrixalus laevis (Anura: Hyperoliidae) currently has a disjunct distribution in western Central Africa (Cameroon, Equatorial Guinea, Gabon, and possibly adjacent countries) and the area in and near the Albertine Rift in eastern Democratic Republic of the Congo and neighboring countries. At least two herpetologists have previously suggested that these disjunct populations represent distinct species, and herein, we utilize an integrative taxonomic approach with molecular and morphological data to reconcile the taxonomy of these spiny reed frogs. We sequenced 1554 base pairs of the 16S and RAG1 genes from 34 samples of A. laevis and one sample of A. orophilus (sympatric with eastern populations of A. laevis), and combined these data with previously sequenced GenBank Afrixalus samples via the bioinformatics toolkit SuperCRUNCH. Phylogenetic trees, dated phylogenetic analyses, and species-delimitation analyses were generated with RAxML, BEAST, and BPP, respectively. Eleven mensural characters were taken from multiple specimens of A. laevis and A. orophilus, and compared with paired t-tests and analyses of covariance. These combined results suggested populations of A. laevis in western Central Africa (Cameroon and Bioko Island, Equatorial Guinea) represent one species, whereas populations from the Albertine Rift and nearby forests represent two undescribed taxa that are sister to A. dorsimaculatus. The two new species (A. lacustris sp. nov. and A. phantasma sp. nov.) are distinguished by our phylogenetic and species-delimitation analyses, significant differences in several mensural characters, qualitative morphological differences, and by their non-overlapping elevational distribution.

Secondary sympatry amongst sister lineages is strongly associated with genetic and ecological divergence. This pattern suggests that for closely related species to coexist in secondary sympatry, they must accumulate differences in traits that mediate ecological and/or reproductive isolation. Here, we characterized inter- and intraspecific divergence in three giant tree frog species whose distributions stretch across West and Central Africa. Using genome-wide single-nucleotide polymorphism data, we demonstrated that species-level divergence coincides temporally and geographically with a period of large-scale forest fragmentation during the late Pliocene. Our environmental niche models further supported a dynamic history of climatic suitability and stability, and indicated that all three species occupy distinct environmental niches. We found modest morphological differentiation amongst the species with significant divergence in tympanum diameter and male advertisement call. In addition, we confirmed that two species occur in secondary sympatry in Central Africa but found no evidence of hybridization. These patterns support the hypothesis that cycles of genetic exchange and isolation across West and Central Africa have contributed to globally significant biodiversity. Furthermore, divergence in both ecology and reproductive traits appear to have played important roles in maintaining distinct lineages. At the intraspecific level, we found that climatic refugia, precipitation gradients, marine incursions, and potentially riverine barriers generated phylogeographic structure throughout the Pleistocene and into the Holocene. Further studies examining phenotypic divergence and secondary contact amongst these geographically structured populations may demonstrate how smaller scale and more recent biogeographic barriers contribute to regional diversification., (© 2021 John Wiley & Sons Ltd.)

Background: Non-alcoholic steatohepatitis (NASH) and fibrosis are the main prognostic factors in non-alcoholic fatty liver disease (NAFLD). The FIB-4 score has been suggested as an initial test for the exclusion of progressed fibrosis. However, increasing evidence suggests that also NASH patients with earlier fibrosis stages are at risk of disease progression, emphasizing the need for improved non-invasive risk stratification. Methods: We evaluated whether the apoptosis biomarker M30 can identify patients with fibrotic NASH despite low or intermediate FIB-4 values. Serum M30 levels were assessed by ELISA, and FIB-4 was calculated in an exploration (n = 103) and validation (n = 100) cohort of patients with histologically confirmed NAFLD. Results: The majority of patients with low FIB-4 (cut-off value < 1.3) in the exploration cohort revealed increased M30 levels (>200 U/L) and more than 80% of them had NASH, mostly with fibrosis. NASH was also detected in all patients with intermediate FIB-4 (1.3 to 2.67) and elevated M30, from which ~80% showed fibrosis. Importantly, in the absence of elevated M30, most patients with FIB-4 < 1.3 and NASH showed also no fibrosis. Similar results were obtained in the validation cohort. Conclusions: The combination of FIB-4 with M30 enables a more reliable identification of patients at risk for progressed NAFLD and might, therefore, improve patient stratification., Competing Interests: The authors declare no conflict of interest.

Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate-like density for nuclear-envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Background and Objective: General practitioners manage more melanomas than dermatologists or surgeons in Australia. Previously undescribed, the management and outcomes of melanoma patients treated by multiple Australasian general practitioners are examined., Methods: The characteristics, management and outcomes of 589 melanoma patients, managed by 27 Australasian general practitioners and documented on the Skin Cancer Audit Research Database (SCARD), were analysed., Results: Most patients (58.9%) were males with mean age at diagnosis of 62.7 years (range 18-96), and most melanomas were in situ or thin-invasive. Patients aged under 40 years had fewer melanomas, but a higher proportion (the majority) were invasive, compared with older patients (P < 0.0001). Most (55.9%) melanomas were diagnosed following elliptical excision biopsy, the rate of unintended involved margins being eightfold higher for shave biopsies. Wide re-excision was performed by the treating general practitioner for most (74.9%) melanomas, with thick melanomas preferentially referred to surgeons. The average Breslow thickness of invasive melanomas re-excised by general practitioners was 0.67 mm compared with 1.99 mm for those referred to other specialists (P < 0.0001). Of 205 patients with invasive melanoma, 14 progressed to metastatic disease, 50% of these being associated with nodular melanoma. Nine patients progressed to melanoma-specific death. The 5-year survival rate for patients with invasive melanoma was 95.2% (95% CI: 91.2-98.5%)., Conclusions: Diagnostic and therapeutic management of a series of melanoma patients by Australasian general practitioners were closely aligned with current guidelines and 5-year survival with respect to invasive melanoma was at least as favourable as national population-based metrics., (© 2022 The Authors. Australasian Journal of Dermatology published by John Wiley & Sons Australia, Ltd on behalf of Australasian College of Dermatologists.)

The aim of this exploratory study was to evaluate the gut microbial signatures of distinct trimethylamine N-oxide (TMAO) responses following raspberry consumption. Investigations were carried out in 24 subjects at risk of developing metabolic syndrome who received 280 g/day of frozen raspberries for 8 weeks. Blood and stool samples were collected at weeks 0 and 8. Inter-individual variability in plasma TMAO levels was analyzed, 7 subjects were excluded due to noninformative signals and 17 subjects were kept for analysis and further stratified according to their TMAO response. Whole-metagenome shotgun sequencing analysis was used to determine the impact of raspberry consumption on gut microbial composition. Before the intervention, the relative abundance of Actinobacteriota was significantly higher in participants whose TMAO levels increased after the intervention ( p = 0.03). The delta TMAO (absolute differences of baseline and week 8 levels) was positively associated with the abundance of gut bacteria such as Bilophila wadsworthia ( p = 0.02; r 2 = 0.37), from the genus Granulicatella ( p = 0.03; r 2 = 0.48) or the Erysipelotrichia class ( p = 0.03; r 2 = 0.45). Changes in the gut microbial ecology induced by raspberry consumption over an 8-week period presumably impacted quaternary amines-utilizing activity and thus plasma TMAO levels.

Numerous studies have reported that diets rich in phenolic compounds are beneficial to immune-metabolic health, yet these effects are heterogeneous and the underlying mechanisms are poorly understood. To investigate the inter-individual variability of the immune-metabolic response to raspberry consumption, whole-blood RNAseq data from 24 participants receiving 280 g/d of raspberries for 8 weeks were used for the identification of responsiveness subgroups by using partial least squares-discriminant analysis (PLSDA) and hierarchical clustering. Transcriptomic-based clustering regrouped participants into two distinct subgroups of 13 and 11 participants, so-called responders and non-responders, respectively. Following raspberry consumption, a significant decrease in triglycerides, cholesterol and C-reactive protein levels were found in responders, as compared to non-responders. Two major gene expression components of 100 and 220 genes were identified by sparse PLSDA as those better discriminating responders from non-responders, and functional analysis identified pathways related to cytokine production, leukocyte activation and immune response as significantly enriched with most discriminant genes. As compared to non-responders, the plasma lipidomic profile of responders was characterized by a significant decrease in triglycerides and an increase in phosphatidylcholines following raspberry consumption. Prior to the intervention, a distinct metagenomic profile was identified by PLSDA between responsiveness subgroups, and the Firmicutes-to-Bacteroidota ratio was found significantly lower in responders, as compared to non-responders. Findings point to this transcriptomic-based clustering approach as a suitable tool to identify distinct responsiveness subgroups to raspberry consumption. This approach represents a promising framework to tackle the issue of inter-individual variability in the understanding of the impact of foods on immune-metabolic health., (Copyright © 2022. Published by Elsevier Inc.)

Background: Despite the numerous literature results about biological effects of electromagnetic field (EMF) exposure, the interaction mechanisms of these fields with organisms are still a matter of debate. Extremely low frequency (ELF) MFs can modulate redox homeostasis and we showed that 24 h exposure to 50 Hz-1 mT has a pro-oxidant effect and effects on the epigenome of SH-SY5Y cells, decreasing miR-34b/c expression through the hypermethylation of their promoter., Methods: Here, we investigated the role of the electromagnetic deposited energy density (ED) during exposures lasting 24 h to 1 mT amplitude MFs at a frequency of 50 Hz in inducing the above mentioned effects. To this end, we delivered ultrashort electric pulses, in the range of microsecond and nanosecond duration, with the same ED of the previously performed magnetic exposure to SH-SY5Y cells. Furthermore, we explored the effect of higher deposited energy densities. Analysis of i) gene and microRNA expression, ii) cell morphology, iii) reactive oxygen species (ROS) generation, and iv) apoptosis were carried out., Results: We observed significant changes in egr-1 and c-fos expression at very low deposited ED levels, but no change of the ROS production, miR-34b/c expression, nor the appearance of indicators of apoptosis. We thus sought investigating changes in egr-1 and c-fos expression caused by ultrashort electric pulses at increasing deposited ED levels. The pulses with the higher deposited ED caused cell electroporation and even other morphological changes such as cell fusion. The changes in egr-1 and c-fos expression were more intense, but, again, no change of the ROS production, miR-34b/c expression, nor apoptosis induction was observed., Conclusions: These results, showing that extremely low levels of electric stimulation (never investigated until now) can cause transcriptional changes, also reveal the safety of the electroporating pulses used in biomedical applications and open up the possibility to further therapeutic applications of this technology.

The COVID-19 pandemic has resulted in school closures and reduction of in-person learning (1). In August 2021, the Lake County Health Department (LCHD) in Illinois introduced a Test to Stay (TTS) strategy, whereby unvaccinated students, teachers, and staff members with certain school-related COVID-19 exposures could remain in school and participate in school-related extracurricular activities. Eligibility to participate in TTS required the following conditions to be met: 1) the exposure occurred while both the person with COVID-19 (index patient) and the close contact were masked; 2) the close contact remained asymptomatic, practiced consistent mask wearing, and maintained physical distancing; and 3) the close contact underwent testing for SARS-CoV-2 (the virus that causes COVID-19) on days 1, 3, 5, and 7 after exposure to the index patient. LCHD permitted kindergarten through grade 12 (K-12) schools in Lake County to implement TTS; 90 schools, representing 31 school districts in Lake County, implemented TTS during August 9-October 29, 2021. During the implementation period, 258 COVID-19 cases were reported. Among 1,035 students and staff members enrolled in TTS, the secondary attack risk (number of close contacts who received a positive SARS-CoV-2 test result within 14 days after exposure to an index patient, divided by total number of close contacts) was 1.5% (16 of 1,035). Among the 16 secondary cases identified, all were in students, and none appeared to transmit SARS-CoV-2 to other school-based contacts. However, nine tertiary cases were identified among household contacts of the 16 secondary cases, and four of the nine were fully vaccinated. Assuming a maximum of 8 missed school days for every 10-day quarantine period, up to 8,152 in-person learning days were saved among TTS participants. Implementation of TTS with other concurrent prevention strategies, including masking and physical distancing, limited further spread of SARS-CoV-2 within K-12 schools and allowed students to safely sustain in-person learning. Although vaccination remains the leading public health recommendation to protect against COVID-19 for those aged ≥5 years, schools might consider TTS as an option for allowing close contacts who are not fully vaccinated to remain in the classroom as an alternative to home quarantine., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Introduction: Ticks are frequently polyinfected and can thus transmit numerous microorganisms. A large number of bacteria, parasites and viruses are transmitted by tick bites and could cause different signs and symptoms in patients. The main goal of this study was to search for these numerous microorganisms in patients presenting with persistent polymorphic syndrome possibly due to a tick bite (SPPT)., Patients and Methods: The following microorganisms were searched for in saliva, urine, venous and capillary blood by using real time PCR: Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Borrelia hermsii, Bartonella spp., Bartonella quintana, Bartonella henselae, Ehrlichia spp., Anaplasma spp., Rickettsia spp., Coxiella burnetii, Brucella spp., Francisella tularensis, Mycoplasma spp., Chlamydia spp., Babesia spp., Theileria spp., Results: 104 patients were included. 48% of the patients were poly-infected, and 25% harboured at least three different microorganisms. Borrelia spp. were not the most frequent bacteria observed, observed far behind Mycoplasma spp., Rickettsia spp. and Ehrlichia spp. which were the most frequent microorganisms observed. Piroplasms were found in a significant number of patients. The most sensitive matrix was saliva, followed by urine, capillary blood and venous blood., Conclusion: Our prospective study has shown that patients with SPPT, a syndrome close to fibromyalgia, could harbour several tick borne microorganisms.

Background and Objective: Most melanomas (including melanomas in situ), in Australasia, are treated by general practitioners (GPs). Previously undescribed, the characteristics of a series of melanomas treated by multiple GPs are examined., Patients and Methods: Six hundred and thirty-seven melanomas treated by 27 Australasian GPs during 2013 and documented on the Skin Cancer Audit Research Database (SCARD) were analysed by anatomical site, subtype, Breslow thickness, diameter, associated naevi and linked adverse outcomes., Results: Most melanomas (59.7%) were on males, mean age at diagnosis being 62.7 years (range 18-96). Most (65.0%) were in situ, with a high incidence of lentiginous melanoma (LM) (38.8%) and 32% were naevus associated. Most LM (86.4%) were in situ, compared to 55% of superficial spreading melanoma (SSM) (P < 0.0001). There was male predominance on the head, neck and trunk and female predominance on extremities. There was no significant association between Breslow thickness and diameter, with small melanomas as likely to be thick as large melanomas, and melanomas ≤3 mm diameter, on average, more likely to be invasive than larger melanomas. There was a positive correlation between age and both melanoma diameter and Breslow thickness. Seven cases progressed to melanoma-specific death: Five nodular melanoma (NM) and two SSM, one of which was thin (Breslow thickness 0.5 mm)., Conclusions: A large series of melanomas treated by Australasian GPs were predominantly in situ, with a high proportion of LM subtype. With implications for GP training, NM linked to death was over-represented and there was a novel finding that older patients had larger diameter melanomas., (© 2021 The Australasian College of Dermatologists.)

Gene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture. This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. Indeed, after centrifugation of the pulsed viral suspension and later on addition to cells, the activity was found mainly associated with the aluminum aggregates concentrated in the lower fraction and was proportional to generated quantities. Overall, this work focused on the use of electrotransfer to facilitate the adenovirus entry into cell, demonstrating that modifications of the penetrating agent can be more important than modifications of the target cell for transfer efficacy., (© 2021. The Author(s).)

Sonoporation is the process of cell membrane permeabilization, due to exposure to ultrasounds. There is a lack of consensus concerning the mechanisms of sonoporation: Understanding the mechanisms of sonoporation refines the choice of the ultrasonic parameters to be applied on the cells. Cells' classical exposure systems to ultrasounds have several drawbacks, like the immersion of the cells in large volumes of liquid, the nonhomogeneous acoustic pressure in the large sample, and thus, the necessity for magnetic stirring to somehow homogenize the exposure of the cells. This article reports the development and characterization of a novel system allowing the exposure to ultrasounds of very small volumes and their observation under the microscope. The observation under a microscope imposes the exposure of cells and Giant Unilamellar Vesicles under an oblique incidence, as well as the very unusual presence of rigid walls limiting the sonicated volume. The advantages of this new setup are not only the use of a very small volume of cells culture medium/microbubbles (MB), but the presence of flat walls near the sonicated region that results in a more homogeneous ultrasonic pressure field, and thus, the control of the focal distance and the real exposure time. The setup presented here comprises the ability to survey the geometrical and dynamical aspects of the exposure of cells and MB to ultrasounds, if an ultrafast camera is used. Indeed, the setup thus fulfills all the requirements to apply ultrasounds conveniently, for accurate mechanistic experiments under an inverted fluorescence microscope, and it could have interesting applications in photoacoustic research.

The work here reported analyzes the effect of increased efficiency of brain-derived neurotrophic factor (BDNF) production by electroporated Schwann cells (SCs) on the axonal extension in a coculture system on a biomaterial platform that can be of interest for the treatment of injuries of the nervous system, both central and peripheral. Rat SCs are electrotransfected with a plasmid coding for the BDNF protein in order to achieve an increased expression and release of this protein into the culture medium of the cells, performing the best balance between the level of transfection and the number of living cells. Gene-transfected SCs show an about 100-fold increase in the release of BDNF into the culture medium, compared to nonelectroporated SCs. Cocultivation of electroporated SCs with rat dorsal root ganglia (DRG) is performed on highly aligned substrates of polylactic acid (PLA) microfibers coated with the electroconductive polymer polypyrrol (PPy). The coculture of DRG with electrotransfected SCs increase both the axonal extension and the axonal sprouting from DRG neurons compared to the coculture of DRG with nonelectroporated SCs. Therefore, the use of PLA-PPy highly aligned microfiber substrates preseeded with electrotransfected SCs with an increased BDNF secretion is capable of both guiding and accelerating axonal growth., (© 2021 Wiley-VCH GmbH.)

The genus Toxicodryas, historically included with the renowned Australasian cat-eyed snakes of the colubrid genus Boiga, currently includes two widespread species (T. blandingii and T. pulverulenta) in western, central, and eastern Africa. We leverage findings from a recent phylogenomic and historical demographic analysis of this genus (based on 2848-4471 Rad-seq loci from across the genome), with robust sampling from throughout the ranges of both species, to define two additional taxonomic units, with species boundaries corresponding to river barriers. Additional morphometric data from scores of examined museum specimens and literature records bolster the recognition of these two new cryptic species. We hypothesize that T. blandingii occurs west of the confluence of the Congo and Ubangi rivers, whereas a cryptic new species that is found east of this biogeographic barrier has significantly higher numbers of ventral scale counts in both sexes, additional significant differences in several scale counts, and lower venom toxicity. Toxicodryas pulverulenta occurs west of the Niger Delta in West Africa, whereas a cryptic new species that is found east of this biogeographic barrier has significantly higher numbers of subcaudal scale counts in both sexes. A review of published information regarding morphological variation, ecology, natural history, habitat, and venom is summarized for these four Toxicodryas species.

The use of conductive nanoparticles (NPs) was previously proposed as a way to locally amplify the electric field (EF) intensity at the cell membrane to enhance cell electroporation. To achieve this, a close distance between the NPs and the cell membrane is mandatory. Here, a new method to improve the contact between NPs and cell surface using the effects of electric pulses (electrophoretic forces) is explored. The effects of two types of electric pulses are analyzed alone or combined in a two-pulse-train protocol on Chinese hamster DC-3F cells. Particularly we used 100 µs duration pulses, low intensity-millisecond pulses and combinations of both. Finally, we studied the use of surface coated NPs (PEGylated) for this application. Our results demonstrate that the delivery of an electric field prior to the electroporation pulses increases the accumulation of NPs around the cell membrane suggesting that NPs are pushed towards the cell surface through electrophoretic forces. This allowed reducing the need for long incubations between cells and NPs to observe an enhancement of electroporation mediated by conductive NPs. Thus low intensity-millisecond pulses can be used to increase the accumulation of either aggregated or individual (i.e. PEGylated) NPs supporting the electrophoretic nature of the observed effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Consumption of red raspberries has been reported to exert acute beneficial effects on postprandial glycemia, insulinemia, triglyceridemia, and cytokine levels in metabolically disturbed subjects. In a two-arm parallel-group, randomized, controlled trial, 59 subjects with overweight or abdominal obesity and with slight hyperinsulinemia or hypertriglyceridemia were randomized to consume 280 g/day of frozen raspberries or to maintain their usual diet for 8 weeks. Primary analyses measured metabolic differences between the groups. Secondary analyses performed with omics tools in the intervention group assessed blood gene expression and plasma metabolomic changes following the raspberry supplementation. The intervention did not significantly affect plasma insulin, glucose, inflammatory marker concentrations, nor blood pressure. Following the supplementation, 43 genes were differentially expressed, and several functional pathways were enriched, a major portion of which were involved in the regulation of cytotoxicity, immune cell trafficking, protein signal transduction, and interleukin production. In addition, 10 serum metabolites were found significantly altered, among which β-alanine, trimethylamine N-oxide, and bioactive lipids. Although the supplementation had no meaningful metabolic effects, these results highlight the impact of a diet rich in raspberry on the immune function and phospholipid metabolism, thus providing novel insights into potential immune-metabolic pathways influenced by regular raspberry consumption.

Background: MicroRNAs (miRNAs) have been suggested as biomarkers for malignant diseases including hepatocellular carcinoma (HCC). Specifically, hsa-miR-21-5p (miR-21) is among the most frequently deregulated miRNA in cancer. The diagnostic and prognostic value of miR-21 has been demonstrated in HCC tissue, mostly in the Asian population. Although the impact of various factors has been recently reported for circulating hsa-miR-122-5p (miR-122), at present only limited knowledge is available for miR-21., Aim: To evaluate the value of miR-21 for the assessment of prognosis in HCC patients and to delineate the influence of clinical and preanalytical factors on miR-21 level in sera., Methods: Patients with confirmed HCC from our European cohort with predominantly alcohol-associated liver damage were included in the study. All subjects were characterized according to their clinical and laboratory work-up and overall survival data were obtained. Quantitative real-time polymerase chain reaction was performed for miR-21 and spiked-in cel-miR-39-3p. The results were compared to previously reported miR-122 data., Results: Survival of HCC patients was comparable between patients with low and high serum miR-21 concentration. No association was observed between miR-21 level in sera and Child-Pugh score, Barcelona Clinic Liver Cancer staging system, or etiology of HCC/liver disease. Age, gender, or pretreatment had no association with miR-21 level. A positive correlation was observed between miR-21 and aspartate aminotransferase ( r = 0.2854, P = 0.0061), serum miR-122 ( r = 0.2624, P = 0.0120), and the International Normalized Ratio ( r = 0.2065, P = 0.0496). Negative correlation of miR-21 with serum creatinine ( r = -0.2215, P = 0.0348) suggests renal function as a potential influencing factor in miR-21 biogenesis in blood., Conclusion: The results from this work do not support clinically relevant prognostic value of circulating miR-21 in HCC patients in real-life settings. Following systematic evaluation, we identified renal function and aspartate aminotransferase as potential factors that may affect miR-21 concentration in blood. This knowledge should be considered in future miRNA-based biomarker studies not only for HCC but also for other diseases., Competing Interests: Conflict-of-interest statement: Authors declare that they have no potential conflicts of interest., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: The lymphatic system may play an important role in local immunomodulation in vascularized composite allotransplantation (VCA). Currently, there is no standardized VCA model that includes the regional draining lymphatic tissue. The aim of this study was to develop a rapid and efficient orthotopic hindlimb transplantation model in rats that included the draining lymphatic basin to permit further evaluation of the lymphatic system's role in VCA., Methods: Thirty transplantations from Brown Norway rats to Lewis rats were performed. To include the regional lymphatic tissue, the superficial epigastric vessels were preserved to allow retrieval of the corresponding inguinal lymph nodes, including the inguinal fat pad, with the hindlimb. A cuff technique was used for the vein, whereas the conventional microsurgical technique was used for the arterial anastomosis. Vascular patency was confirmed through laser Doppler analysis at postoperative day 1 and histological analysis after euthanasia., Results: The presence and vascularization of the inguinal lymph nodes were verified with indocyanine green lymphoscintigraphy at the time of transplantation. Mean total ischemia time was 69 ± 24 minutes, and mean recipient operation time was 80 ± 19 minutes. Overall transplant survival rate was 93.3%. Laser Doppler analysis showed vascular (technical) success, indocyanine green lymphoscintigraphy confirmed the presence of lymph nodes and the histological analysis revealed patent anastomoses., Conclusions: We successfully developed an experimental orthotopic hindlimb transplantation model in rats that includes the draining inguinal lymphatic basin, which is an important asset in further research on lymphatic tissue and its role in VCA., Competing Interests: The authors declare no funding or conflicts of interest., (Copyright © 2020 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Background: Borrelia species are divided into three groups depending on the induced disease and the tick vector. Borrelia miyamotoi is a relapsing fever Borrelia but can induce symptoms related to Lyme disease. Discovered in 1995, it is found in ticks around the world. In France, this species of Borrelia has been isolated in ticks and rodents, but was not yet observed in humans. Objective: The aim of the study was to look for B. miyamotoi in symptomatic patients. Methods: Real-time PCR was performed on 824 blood samples from patients presenting symptoms of persistent polymorphic syndrome possibly due to tick bite, a syndrome recognized by the French Authority for Health, which is close to the post-treatment Lyme disease syndrome. PCR was also performed on 24 healthy control persons. The primers were specifically designed for this particular species of Borrelia . The sequence of interest of 94 bp is located on the glpQ gene. Sequencing of amplification products, randomly chosen, confirmed the amplification specificity. To better investigate cases, a clinical questionnaire was sent to the patients PCR-positive for B. miyamotoi and to their physician. Results: This search revealed a positive PCR for B. miyamotoi in the blood from 43 patients out of 824 (5.22%). PCR was negative in all control persons. A clinical chart was obtained from 31 of the 43 patients. A history of erythema migrans was reported in five of these 31 patients (16%). All patients complained about fatigue, joint pain and neuro-cognitive disorders. Some patients complained about respiratory problems (chest tightness and/or lack of air in 41.9%). Episodes of relapsing fever were reported by 11 of the 31 patients (35.5%). Chilliness, hot flushes and/or sweats were reported by around half of the patients. B. miyamotoi may not cross-react with B. burgdorferi serology. Conclusion: This study is the first to detect B. miyamotoi in human blood in France. This series of human B. miyamotoi infection is the largest in patients with long term persistent syndrome. Our data suggest that this infection may be persistent, even on the long term., (Copyright © 2020 Franck, Ghozzi, Pajaud, Lawson-Hogban, Mas, Lacout and Perronne.)

The effectiveness of electrochemotherapy (ECT) in local eradication of tumours in human and veterinary medicine has been proven. ECT consists of increasing the uptake of cytotoxic drugs by means of pulsed electric fields (PEFs) that transiently permeabilise the cell membrane. Still, this tumour treatment includes some drawbacks that are linked to the characteristics of the intense electric pulses (EPs) used. Meanwhile, the emerging field of cancer therapies that are based on the application of non-thermal plasmas (NTP) has recently garnered interest because of their potentialities as rich sources of reactive species. In this work, we investigated the potential capabilities of the combined application of indirect NTP treatment and microsecond PEFs (µsPEFs) to outperform in vitro cell electropermeabilisation, the basis of ECT. Thus, phosphate-buffered saline (PBS) was plasma-treated (pPBS) and used afterwards to explore the effects of its combination with µsPEFs. Analysis of two different cell lines (DC-3F Chinese hamster lung fibroblasts and malignant B16-F10 murine melanoma cells), by flow cytometry, revealed that this combination resulted in significant increases of the level of cell membrane electropermeabilisation, even at very low electric field amplitude. The B16-F10 cells were more sensitive to the combined treatment than DC-3F cells. Importantly, the percentage of permeabilised cells reached values similar to those of cells exposed to classical electroporation field amplitude (1100 V/cm) when the cells were treated with pPBS before and after being exposed only to very low PEF amplitude (600 V/cm). Although the level of permeabilisation of the cells that are treated by the pPBS and the PEFs at 600 V/cm is lower than the level reached after the exposure to µsPEFs alone at 1100 V/cm, the combined treatment opens the possibility to reduce the amplitude of the EPs used in ECT, potentially allowing for a novel ECT with reduced side-effects.

Background: There is ongoing search for new noninvasive biomarkers to improve management of patients with hepatocellular carcinoma (HCC). Studies, mostly from the Asian-Pacific region, demonstrated differential expression of liver-specific microRNA-122 (miR-122) in tissue as well as in sera of patients with hepatitis B virus- and hepatitis C virus-induced HCC., Aim: To evaluate prognostic value of miR-122 in patients with HCC in a European population and determine potential factors related to alteration of miR-122 in sera., Methods: Patients with confirmed HCC ( n = 91) were included in the study over a two-year period. Patients were characterized according to Child-Pugh score, Barcelona clinic liver cancer (BCLC) staging system, etiology of liver disease, laboratory parameters and overall survival. MiR-122 was measured in sera using TaqMan assay normalized to spiked-in cel-miR-39., Results: Serum miR-122 quantity was independent of the Child-Pugh score, the BCLC stage or the underlying etiology. Significant positive correlation was found between miR-122 and alanine aminotransferase ( P < 0.0001), aspartate aminotransferase ( P = 0.0001), alpha-fetoprotein (AFP) ( P = 0.0034) and hemoglobin concentration ( P = 0.076). Negative correlation was observed between miR-122 level and creatinine concentration ( P = 0.0028). AFP, Child-Pugh score and BCLC staging system were associated with survival differences. In overall cohort low miR-122 in sera was only associated with a trend for a better overall survival without reaching statistical significance. Subgroup analysis revealed that low miR-122 was significantly associated with better prognosis in patients with advanced cirrhosis (Child-Pugh class B/C), advanced tumor stage (BCLC B/C/D) and normal AFP (< 7 ng/mL)., Conclusion: Our results strongly support the value of miR-122 as potential biomarker of liver injury and probably prognosis. Nevertheless, the value of miR-122 in prediction of prognosis of HCC patients was limited to certain patients' subgroups. Since circulating miR-122 may be influenced by impaired renal function, AFP and hemoglobin concentration, those factors need to be considered while interpreting miR-122 level., Competing Interests: Conflict-of-interest statement: Alexander Link is principle investigator of the “LiLife”-Project supported by the funds of European Commission through the “Europäischer Fond für regionale Entwicklung” (EFRE) as well as by the regional Ministry of Economy, Science and Digitalization as part of the “Autonomie im Alter” research group. Other authors declare that they have no potential conflicts of interest., (©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.)

Introduction: The consumption of long-chain omega-3 polyunsaturated fatty acids (n-3 PUFA) has been reported to have beneficial health effects, notably, by reducing plasma triglyceride levels. Nonetheless, a concomitant decrease in insulin sensitivity has also been observed, but is highly variable among subjects. Herein, we aimed to determine the importance of the genetic background in the interindividual variability of the insulin sensitivity response following an n-3 PUFA supplementation., Methods: A total of 210 participants completed a 6-week n-3 PUFA supplementation with 5 g/day of fish oil (providing 1.9-2.2 g of eicosapentaenoic acid + 1.1 g of docosahexaenoic acid). Insulin resistance was estimated by the homeostatic model assessment (HOMA-IR), and participants were further classified as high-risk or low-risk depending on their HOMA-IR change following the n-3 PUFA supplementation, as compared to pre-supplementation values. Genome-wide genotyping data were obtained for 138 participants using HumanOmni-5-Quad BeadChips containing 4,301,331 single nucleotide polymorphisms. A genome-wide association analysis (GWAS) was carried out between high-risk and low-risk participants. The population study was split into training (60%) and testing (40%) datasets to assess the predictive accuracy of a genetic risk score (GRS) constructed by summing the number of risk alleles., Results: Following the n-3 PUFA supplementation, 32 participants had increased HOMA-IR as compared to initial values and were classified as high risk (23.2%), whereas remaining subjects were classified as low risk (n = 106, 76.8%). A total of 8 loci had frequency differences between high-risk and low-risk participants at a suggestive GWAS association threshold (p value <1 × 10-5). After applying 10-fold cross validation, the GRS showed a significant association with the risk of increased HOMA-IR in the testing dataset (OR = 3.16 [95% CI, 1.85-7.14]), with a predictive accuracy of 0.85, and explained 40% of variation in HOMA-IR change., Conclusions: These results suggest that the genetic background has a relevant role in the interindividual variability observed in the insulin sensitivity response following an n-3 PUFA supplementation. Subjects being at risk of insulin sensitivity lowering following an n-3 PUFA supplementation may be identified using genetic-based precision nutrition approaches., (© 2019 The Author(s) Published by S. Karger AG, Basel.)

Introduction: Finger proximal interphalangeal joint (PIP) reconstruction after the destruction of parts of the joint remains challenging. Surgical techniques include implant arthroplasty, arthrodesis, free vascularized joint transfer, and non-vascularized bone and joint transfer. This study analyzes our experience after non-vascularized transfer in terms of range of motion, postoperative rehabilitation, and patient satisfaction., Materials and Methods: Between 2009 and 2014, ten patients underwent non-vascularized partial joint transfer for PIP joint reconstruction. One of them was lost to follow-up. Included patients had osteochondral partial joint transplants of 25-50% of the toes (n = 4) and the hand (n = 5). Range of motion (ROM), grip-, and pinch-strength were measured at the last follow-up control and compared to the healthy side. Patients were asked to score the pain at rest/ on load on a visual scale (VAS: 0 = no pain; 10 = excruciating pain). Satisfaction self-assessment was evaluated by asking the patients to grade their postoperative result as excellent, very good, good or poor., Results: Mean follow-up period was 4.0 years (range 1.2-7.9 years). Mean PIP joint flexion was 93 ± 26° at the last follow-up control. Mean grip- and pinch-strength of the operated side at the last control were, respectively, 43 ± 18 kg and 8 ± 5 kg, close to the healthy side values (45 ± 15 kg and 9 ± 4 kg). Mean pain at rest/on load measured on a visual scale was, respectively, 0.3 ± 1 and 1.8 ± 2. Eight patients (89%) rated their operation as excellent, and one as poor., Conclusion: In this study, non-vascularized partial joint transfer provides a mobile and stable PIP joint 4 years after reconstruction. The surgical technique presented herein is complex depending on additional injuries but results in great patient satisfaction.

Objectives/hypothesis: To describe the histopathologic findings and clinical presentation of arachnoid cysts (ACs) within the human temporal bone., Study Design: Retrospective cohort analysis., Methods: An analysis of all medical records of patients diagnosed with an AC was performed. Temporal bones underwent standard processing for histologic examination. The slides were examined by light microscopy. The histologic findings were compared to premortem clinical data., Results: Twenty-seven ACs were identified in 22 patients. Twenty ears (74%) had no identified risk factor for AC development. The median volume was 12.8 mm 3 . The most prevalent location of the ACs was at the fundus (16 ACs) followed by the middle portion of the internal auditory canal (IAC) (six ACs). Nine ACs were asymptomatic. Among the 18 symptomatic ACs, the most common presentation was sensorineural hearing loss (SNHL) (94%), followed by tinnitus (22%). The most affected structure was the cochlear nerve (59%), followed by the vestibular nerve (41%). The average hearing threshold was of moderately severe SNHL and speech discrimination was in the range of 50% on monosyllabic word tests. The median time interval from initial presentation to death was 12 years. No correlation was found between duration of symptoms and AC volume., Conclusions: AC of the IAC is not uncommon. Its presentation is variable, ranging from asymptomatic to SNHL, with poor speech discrimination, tinnitus, and vertigo. This diagnosis should be kept in the differential diagnosis of retrocochlear pathologies., Level of Evidence: 4 Laryngoscope, 129:1667-1674, 2019., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

Exploration of innovative high hydrostatic pressure (HHP)-assisted enzymatic hydrolysis of plant based food proteins may help improve peptide yield and bioactivity of hydrolysates. In this study, we performed enzymatic hydrolysis of flaxseed proteins using trypsin under HHP (100 and 300 MPa for 5 and 10 min) to evaluate the effect of presurization on protein denaturation, degree of hydrolysis (DH), and peptide profile and bioactivity of hydrolysate. Spectrofluorimetric analyses showed that 300 MPa induced the maximum destablization of flaxseed protein structures. The same pressure level drastically improved the DH by 1.7 times as compared to that of control. Applying HHP did not modify the peptide profiles of flaxseed protein hydrolysates but their concentrations increased with severity of treatment. Similarly, peptide molecular weight distributions were affected by pressurization parameters, increasing mainly the relative abundance of 500-1500 Da peptides. Finally, pressurization at 300 MPa for 5 and 10 min improved the antioxidant activity of flaxseed protein hydrolysates by 39 and 55%, respectively, compared to the control., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: The U.S. Affordable Care Act (ACA) of 2010 included the Well-Woman Visit (WWV) as one of the preventive services, which must be covered without cost sharing. Despite concerted efforts to increase access to the WWV, data from the early years of the ACA demonstrated ongoing barriers, including insufficient consumer and provider awareness of the ACA's no cost-sharing provision for preventive services. As such, 2 years after full implementation of the ACA, the Well-Woman Project (WWP) used qualitative methods to learn about women's perceptions of the WWV and barriers that affect their ability to be healthy and seek well-woman care. Methods: Women's voices were captured by Listening Sessions in eight cities and through stories from women across the United States posted to a WWP Website, or reported over a WWP toll-free phone line. Thematic analysis of Listening Sessions and stories was conducted using Dedoose software. Results: In 2016, Listening Sessions (17) were held with 156 women; in addition, stories were collected from 102 women across the United States. Women are aware of the importance of preventive care, but report multiple barriers to seeking such care. However, they are able to articulate a variety of system and policy strategies that mitigate the complexity of navigating the health care system; help women prioritize their health and accessing health care; promote positive relationships with providers; empower women to advocate for themselves and others; promote positive mental health as well as access to safe environments, healthy food, and social support systems; decrease barriers related to lack of transportation and childcare; and support the provision of trauma informed care in the health care delivery system. Conclusion: To improve women's health status and reduce inequities, making the preventive well-care visit available without cost-sharing is necessary, but not a sufficient strategy., Competing Interests: No author has any commercial association that might create a conflict of interests with the submitted article.

Introduction: In Switzerland, collagenase Clostridium histolyticum therapy (CCH) for Dupuytren's disease was introduced in 2011. This study analyzes possible differences between CCH and limited fasciectomy (LF) in terms of range of motion, patient satisfaction and postoperative rehabilitation., Materials and Methods: This retrospective study included 52 patients with Dupuytren's disease stage 1-3 according to Tubiana, treated with CCH or LF between January 2012 and December 2013. Complications were analyzed for each patient. The contracture of each treated joint measured on average at the 3 months and up to 2 years follow-up was compared with the preoperative values. The Michigan Hand score was evaluated at 2 years and the patients were asked to subjectively evaluate the outcome of the treatment and whether they would repeat it if necessary. Postoperative rehabilitation was also precisely quantified., Results: 11 minor complications were reported for a complication rate of 29% in the CCH group. No major complications were reported in both groups. In the CCH group, mean MCP joint contracture was, respectively, 44° ± 20°, 9° ± 2° (gain of mobility compared to the preoperative situation 35°, P < 0.001), and 10° ± 3° (gain 34°, P < 0.001), respectively, before, at the 3 months' control and at the 2-year clinical control. In the LF group, mean MCP joint contracture was, respectively, 30° ± 21°, 2° ± 0.5° (gain 28°, P < 0.001), and 1° ± 0.5° (gain 29°, P < 0.001) for the same control periods. In the CCH group, mean PIP joint contracture was, respectively, 51° ± 21°, 18° ± 3° (gain of mobility compared to the preoperative situation 33°, P < 0.001), and 32° ± 4° (gain 19°, P < 0.001), respectively, before, at the 3 months' control and at the 2-year clinical control. In the LF group, mean PIP joint contracture was, respectively, 30° ± 20°, 2° ± 0.5° (gain of mobility compared to the preoperative situation 28°, P < 0.001), and 11° ± 4° (gain 19°, P < 0.001) for the same control periods. Outcomes were compared across the LF and CCH groups: surgery performed better than collagenase for PIP joint treatment at early (P < 0.001) and 2-year follow-up (P = 0.004) controls. However, patient satisfaction was higher in the CCH group: 92% were satisfied or very satisfied of the treatment compared to 71% in the LF group. All patients would reiterate the treatment in the CCH group if necessary compared to only 71% in the LF group. Rehabilitation was highly reduced in the CCH group compared to the LF group., Conclusion: In this study, surgery performed better than collagenase at early and 2-year follow-up in PIP joints and similar in MCP joints. While surgery seems to achieve better results, collagenase is considered in Switzerland as an off-the-shelf therapy that provides consistent results without scars, with shorter rehabilitation time, minor hand therapy, shorter splinting time, and applicability., Level of Evidence and Study Type: Level III.

Background: Differentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Classical tests to prove this differentiation employ procedures such as cell fixation, cell lysis or cell scraping. Very few studies report gentle dissociation of mesenchymal stem cells undergoing an osteodifferentiation process. Here we used this technique to reveal the presence of several cell layers during osteogenesis and to study their different properties., Methods: Through the sequential enzymatic detachment of the cells, we confirm the presence of several layers of differentiated cells and we compare them in terms of enzymatic sensitivity for dissociation, expression of cluster of differentiation, cytosolic calcium oscillations and osteogenic potential. Adipogenic and neurogenic differentiations were also performed in order to compare the cell layers., Results: The cells undergoing differentiation formed one layer in the neurogenic differentiation, two layers in the adipogenic differentiation and at least four layers in the osteogenic differentiation. In the latter, the upper layers, maintained by a collagen I extracellular matrix, can be dissociated using collagenase I, while the remaining lowest layer, attached to the bottom of the dish, is sensitive only to trypsin-versene. The action of collagenase I is more efficient before the mineralization of the extracellular matrix. The collagenase-sensitive and trypsin-sensitive layers differ in their cluster of differentiation expression. The dissociation of the cells on day 15 reveals that cells could resume their growth (increase in cell number) and rapidly differentiate again in osteoblasts, in 2 weeks (instead of 4 weeks). Cells from the upper layers displayed a higher mineralization., Conclusions: MSCs undergoing osteogenic differentiation form several layers with distinct osteogenic properties. This could allow the investigators to use upper layers to rapidly produce differentiated osteoblasts and the lowest layer to continue growth and differentiation until an ulterior dissociation.

Lyme disease is the most frequently reported zoonotic tick-borne disease worldwide, and the number of infected humans is increasing. Lyme disease (or Lyme borreliosis) is an affection caused by the spirochete Borrelia burgdorferi, sensu lato. Lyme disease is also reported as a variety of misleading clinical symptomatologies. Infected patient's blood serology is the most currently test used for its diagnosis. However, serology has a low sensitivity, which ranges from 34% to 70%. Thus, there are numerous subsequent false-negative diagnoses despite an active clinical infection profile. Therefore, alternative and more sensitive techniques are required to detect the antigens or nucleic acids of Borrelia. Actually, the most appropriate methodological approach seems to be the polymerase chain reaction (PCR). However, PCR will detect the only "visible" part available of the targeted DNA presence in the blood of the infected patients. Consequently PCR alone will not be conclusive enough to reach the final diagnosis. Considering the ability of Borrelia to invade host cells, we hypothesize that a selective lysis of all blood cells should improve the diagnostic sensitivity of the detection of Borrelia by PCR in whole blood, and subsequently reduce the false-negative diagnostic rate, thus improving the patient's diagnosis and therapeutic management., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background : Although thousands of clinical isolates of Plasmodium falciparum are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. Methods : Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15 P. falciparum isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal var genes clusters.   Results : The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates.  We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa. Conclusions : These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions., Competing Interests: No competing interests were disclosed.

Low doses of mercury (Hg) promote deleterious effects on cardiovascular system, but the mechanisms implicated remain unclear. This study analyzed whether angiotensin II AT-1 receptors are involved in the vascular dysfunction caused by chronic exposure to low HgCl 2 doses. For this, rats were divided into four groups and untreated (saline by im injections and tap water by gavage) or treated for 30 days as follows: Mercury (HgCl 2 im, first dose of 4.6 µg kg -1 and subsequent doses of 0.07 µg kg -1 day -1 , and tap water by gavage); Losartan (saline im and losartan, 15 mg kg -1 day -1 , by gavage); Losartan-Mercury (HgCl 2 im and Losartan by gavage). Systolic blood pressure was measured by tail plethysmography, vascular reactivity in aorta by isolated organ bath, oxidative stress by measuring the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and antioxidant capacity (FRAP) and protein expression of AT-1 receptors by Western Blot. As results, co-treatment with losartan prevented the increased aortic vasoconstrictor responses to phenylephrine (Phe), the involvement of ROS and prostanoids on the response to Phe and the reduced negative endothelial modulation by nitric oxide on these responses. Moreover, this co-treatment avoided the increase in plasmatic and vascular oxidative stress and AT-1 protein expression in aorta. In conclusion, these results suggest that AT-1 receptors upregulation might play a key role in the vascular damage induced by Hg exposure by increasing oxidative stress and probably by reducing NO bioavailability., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Microsecond pulsed electric fields (μsPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. For internal membranes permeabilization, nanosecond pulsed electric fields (nsPEF) are applied but this technology is complex to use. Here we report that the endoplasmic reticulum (ER) membrane can also be electropermeabilized by one 100 µs pulse without affecting the cell viability. Indeed, using Ca 2+ as a permeabilization marker, we observed cytosolic Ca 2+ peaks in two different cell types after one 100 µs pulse in a medium without Ca 2+ . Thapsigargin abolished these Ca 2+ peaks demonstrating that the calcium is released from the ER. Moreover, IP3R and RyR inhibitors did not modify these peaks showing that they are due to the electropermeabilization of the ER membrane and not to ER Ca 2+ channels activation. Finally, the comparison of the two cell types suggests that the PM and the ER permeabilization thresholds are affected by the sizes of the cell and the ER. In conclusion, this study demonstrates that µsPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, μsPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca 2+ roles in cell physiology.

Background: Immunosuppressive therapies derived from solid organ transplantation are effective in promoting survival of vascularized composite allotransplantation (VCA), but they cause serious side effects that are difficult to justify for this non-life-saving procedure. Unlike solid organ transplantation, hand and face transplants offer the possibility of site-specific immunosuppression for reducing systemic exposure while increasing intra-graft concentrations of the drug. Therefore, in this study, we tested whether a single intra-graft injection tacrolimus could promote VCA survival., Methods: Brown Norway-to-Lewis hind limb transplantations were performed, and animals were left untreated (group I), treated with a daily injection of 1-mg/kg tacrolimus for 21 days (group 2) or injected with 7-mg tacrolimus directly into the transplanted limb on day 1 (group III). Graft rejection was monitored, and animals were sacrificed at grade 3 rejection or 200 days after transplantation., Results: Intra-graft injection of tacrolimus significantly prolonged allograft survival as compared to untreated animals or animals treated with systemic tacrolimus. Half of the intra-graft-treated rats rejected their graft on average at day 70.5. Interestingly, the other half remained rejection-free for more than 200 days without signs of kidney or liver toxicity. In these animals, tacrolimus was detected in the VCA skin but not in the blood until day 200. Long-term survival was not linked to induction of donor-specific tolerance but to a higher level of lymphocyte chimerism., Conclusions: Intra-graft delivery of tacrolimus may promote VCA survival by increasing tissue drug availability and promoting the establishment of transient chimerism and thus long-term graft acceptance., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mercury is a ubiquitous environmental pollutant and mercury contamination and toxicity are serious hazards to human health. Some studies have shown that mercury impairs male reproductive function, but less is known about its effects following exposure at low doses and the possible mechanisms underlying its toxicity. Herein we show that exposure of rats to mercury chloride for 30 days (first dose 4.6µgkg -1 , subsequent doses 0.07µgkg -1 day -1 ) resulted in mean (±s.e.m.) blood mercury concentrations of 6.8±0.3ngmL -1 , similar to that found in human blood after occupational exposure or released from removal of amalgam fillings. Even at these low concentrations, mercury was deposited in reproductive organs (testis, epididymis and prostate), impaired sperm membrane integrity, reduced the number of mature spermatozoa and, in the testes, promoted disorganisation, empty spaces and loss of germinal epithelium. Mercury increased levels of reactive oxygen species and the expression of glutathione peroxidase (GPx) 1 and GPx4. These results suggest that the toxic effects of mercury on the male reproductive system are due to its accumulation in reproductive organs and that the glutathione system is its potential target. The data also suggest, for the first time, a possible role of the selenoproteins GPx1 and GPx4 in the reproductive toxicity of mercury chloride.

Background: Human mesenchymal stem cells are promising tools for regenerative medicine due to their ability to differentiate into many cellular types such as osteocytes, chondrocytes and adipocytes amongst many other cell types. These cells present spontaneous calcium oscillations implicating calcium channels and pumps of the plasma membrane and the endoplasmic reticulum. These oscillations regulate many basic functions in the cell such as proliferation and differentiation. Therefore, the possibility to mimic or regulate these oscillations might be useful to regulate mesenchymal stem cells biological functions., Methods: One or several electric pulses of 100 μs were used to induce Ca 2+ spikes caused by the penetration of Ca 2+ from the extracellular medium, through the transiently electropermeabilized plasma membrane, in human adipose mesenchymal stem cells from several donors. Attached cells were preloaded with Fluo-4 AM and exposed to the electric pulse(s) under the fluorescence microscope. Viability was also checked., Results: According to the pulse(s) electric field amplitude, it is possible to generate a supplementary calcium spike with properties close to those of calcium spontaneous oscillations, or, on the contrary, to inhibit the spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca 2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. None of the pulses used here, even those with the highest amplitude, caused a loss of cell viability., Conclusions: An easy way to control Ca 2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca 2+ spikes, is reported here. Indeed, the direct link between the microsecond electric pulse(s) delivery and the occurrence/cancellation of cytosolic Ca 2+ spikes allowed us to mimic and regulate the Ca 2+ oscillations in these cells. Since microsecond electric pulse delivery constitutes a simple technology available in many laboratories, this new tool might be useful to further investigate the role of Ca 2+ in human mesenchymal stem cells biological processes such as proliferation and differentiation.