Closed-loop parameter optimization for patient-specific phrenic nerve stimulation.

BACKGROUND: Ventilator-induced diaphragm dysfunction occurs rapidly following the onset of mechanical ventilation and has significant clinical consequences. Phrenic nerve stimulation has shown promise in maintaining diaphragm function by inducing diaphragm contractions. Non-invasive stimulation is an attractive option as it minimizes the procedural risks associated with invasive approaches. However, this method is limited by sensitivity to electrode position and inter-individual variability in stimulation thresholds. This makes clinical application challenging due to potentially time-consuming calibration processes to achieve reliable stimulation. METHODS: We applied non-invasive electrical stimulation to the phrenic nerve in the neck in healthy volunteers. A closed-loop system recorded the respiratory flow produced by stimulation and automatically adjusted the electrode position and stimulation amplitude based on the respiratory response. By iterating over electrodes, the optimal electrode was selected. A binary search method over stimulation amplitudes was then employed to determine an individualized stimulation threshold. Pulse trains above this threshold were delivered to produce diaphragm contraction. RESULTS: Nine healthy volunteers were recruited. Mean threshold stimulation amplitude was 36.17 ± 14.34 mA (range 19.38-59.06 mA). The threshold amplitude for reliable nerve capture was moderately correlated with BMI (Pearson's r = 0.66, p = 0.049). Repeating threshold measurements within subjects demonstrated low intra-subject variability of 2.15 ± 1.61 mA between maximum and minimum thresholds on repeated trials. Bilateral stimulation with individually optimized parameters generated reliable diaphragm contraction, resulting in significant inhaled volumes following stimulation. CONCLUSION: We demonstrate the feasibility of a system for automatic optimization of electrode position and stimulation parameters using a closed-loop system. This opens the possibility of easily deployable individualized stimulation in the intensive care setting to reduce ventilator-induced diaphragm dysfunction.

Rescue of Mycobacterium bovis DNA Obtained from Cultured Samples during Official Surveillance of Animal TB: Key Steps for Robust Whole Genome Sequence Data Generation.

Epidemiological surveillance of animal tuberculosis (TB) based on whole genome sequencing (WGS) of Mycobacterium bovis has recently gained track due to its high resolution to identify infection sources, characterize the pathogen population structure, and facilitate contact tracing. However, the workflow from bacterial isolation to sequence data analysis has several technical challenges that may severely impact the power to understand the epidemiological scenario and inform outbreak response. While trying to use archived DNA from cultured samples obtained during routine official surveillance of animal TB in Portugal, we struggled against three major challenges: the low amount of M. bovis DNA obtained from routinely processed animal samples; the lack of purity of M. bovis DNA, i.e., high levels of contamination with DNA from other organisms; and the co-occurrence of more than one M. bovis strain per sample (within-host mixed infection). The loss of isolated genomes generates missed links in transmission chain reconstruction, hampering the biological and epidemiological interpretation of data as a whole. Upon identification of these challenges, we implemented an integrated solution framework based on whole genome amplification and a dedicated computational pipeline to minimize their effects and recover as many genomes as possible. With the approaches described herein, we were able to recover 62 out of 100 samples that would have otherwise been lost. Based on these results, we discuss adjustments that should be made in official and research laboratories to facilitate the sequential implementation of bacteriological culture, PCR, downstream genomics, and computational-based methods. All of this in a time frame supporting data-driven intervention.

Hepatic Alterations in a BTBR T + Itpr3tf/J Mouse Model of Autism and Improvement Using Melatonin via Mitigation Oxidative Stress, Inflammation and Ferroptosis.

Autism spectrum disorder (ASD) is a complicated neurodevelopmental disorder, and its etiology is not well understood. It is known that genetic and nongenetic factors determine alterations in several organs, such as the liver, in individuals with this disorder. The aims of the present study were to analyze morphological and biological alterations in the liver of an autistic mouse model, BTBR T + Itpr3tf/J (BTBR) mice, and to identify therapeutic strategies for alleviating hepatic impairments using melatonin administration. We studied hepatic cytoarchitecture, oxidative stress, inflammation and ferroptosis in BTBR mice and used C57BL6/J mice as healthy control subjects. The mice were divided into four groups and then treated and not treated with melatonin, respectively. BTBR mice showed (a) a retarded development of livers and (b) iron accumulation and elevated oxidative stress and inflammation. We demonstrated that the expression of ferroptosis markers, the transcription factor nuclear factor erythroid-related factor 2 (NFR2), was upregulated, and the Kelch-like ECH-associated protein 1 (KEAP1) was downregulated in BTBR mice. Then, we evaluated the effects of melatonin on the hepatic alterations of BTBR mice; melatonin has a positive effect on liver cytoarchitecture and metabolic functions.

Do nomadic lactobacilli fit as potential vaginal probiotics? The answer lies in a successful selective multi-step and scoring approach.

BACKGROUND: The goal of this study was to create a multi-strain probiotic gel that would foster a lactobacilli-dominated vaginal microbiota in pregnant women and ensure appropriate eubiosis for the newborn. Nomadic lactobacilli (95 strains), mostly isolated from food sources, were preliminarily screened for functional traits before being characterized for their capability to inhibit the two vaginal pathogens Streptococcus agalactiae and Candida albicans, which may lead to adverse pregnancy-related outcomes. Eight best-performing strains were chosen and furtherly investigated for their ability to produce biofilm. Lastly, the two selected potential probiotic candidates were analyzed in vitro for their ability to reduce the inflammation caused by C. albicans infection on the reconstituted human vaginal epithelium (HVE). RESULTS: Lactiplantibacillus plantarum produced both isomers of lactic acid, while Lacticaseibacillus paracasei produced only L-isomer. The production of hydrogen peroxide was strain-dependent, with the highest concentrations found within Lact. paracasei strains. The auto-aggregation capacity and hydrophobicity traits were species-independent. S. agalactiae 88II3 was strongly inhibited both at pH 7.0 and 4.0, whereas the inhibition of C. albicans UNIBZ54 was less frequent. Overall, L. plantarum strains had the highest pathogen inhibition and functional scoring. L. plantarum C5 and POM1, which were selected as potential probiotic candidates also based on their ability to form biofilms, were able to counteract the inflammation process caused by C. albicans infection in the HVE model. CONCLUSIONS: Our multi-step and cumulative scoring-based approach was proven successful in mining and highlighting the probiotic potential of two nomadic lactobacilli strains (L. plantarum C5 and POM1), being applicable to preserve and improve human vaginal health.

Application of Topical Sandalore® Increases Epidermal Dermcidin Synthesis in Organ-Cultured Human Skin ex vivo.

INTRODUCTION: Several olfactory receptors (ORs) are expressed in human skin, where they regulate skin pigmentation, barrier function, wound healing, and hair growth. Previously, we found that the selective activation of OR family 2 subfamily AT member 4 (OR2AT4) by the synthetic, sandalwood-like odorant Sandalore® differentially stimulates the expression of antimicrobial peptides (AMPs) in human scalp hair follicle epithelium ex vivo. As OR2AT4 is also expressed by epidermal keratinocytes, we hypothesized that it may modulate intraepidermal AMP synthesis, thereby contributing to skin microbiome management. METHODS: We investigated this hypothesis in organ-cultured human skin in the presence of Sandalore® and antibiotics and evaluated epidermal production of two AMPs, LL37 (cathelicidin) and dermcidin (DCD), as well as OR2AT4, by quantitative immunohistomorphometry. Moreover, we quantified DCD secretion into the culture medium by ELISA and studied the effect of culture medium on selected bacterial and fungal strains. RESULTS: Topical application of Sandalore®to organ-cultured human skin increased OR2AT4 protein expression, the number of DCD-positive intraepidermal cells, and DCD secretion into culture media, without significantly affecting epidermal LL37 expression. In line with the significantly increased secretion of DCD into the culture medium, we demonstrated, in a spectrophotometric assay, that application of conditioned media from Sandalore®-treated skin promotes Staphylococcus epidermidis, Malassezia restricta, and, minimally, Cutibacterium acnes and inhibits Staphylococcus aureus growth. CONCLUSION: In addition to demonstrating for the first time that DCD can be expressed by epidermal keratinocytes, our pilot study suggests that topical treatment of human skin with a cosmetic odorant (Sandalore®) has the potential to alter the composition of the human skin microbiome through the selective upregulation of DCD. If confirmed, Sandalore® could become an attractive adjuvant, nondrug treatment for dermatoses characterized by dysbiosis due to overgrowth of S. aureus and Malassezia, such as atopic dermatitis and seborrheic dermatitis.

Cellular and Enzymatic Determinants Impacting the Exolytic Action of an Anti-Staphylococcal Enzybiotic.

Bacteriophage endolysins are bacteriolytic enzymes that have been explored as potential weapons to fight antibiotic-resistant bacteria. Despite several studies support the application of endolysins as enzybiotics, detailed knowledge on cellular and enzymatic factors affecting their lytic activity is still missing. The bacterial membrane proton motive force (PMF) and certain cell wall glycopolymers of Gram-positive bacteria have been implicated in some tolerance to endolysins. Here, we studied how the anti-staphylococcal endolysin Lys11, a modular enzyme with two catalytic domains (peptidase and amidase) and a cell binding domain (CBD11), responded to changes in the chemical and/or electric gradients of the PMF (ΔpH and Δψ, respectively). We show that simultaneous dissipation of both gradients enhances endolysin binding to cells and lytic activity. The collapse of ΔpH is preponderant in the stimulation of Lys11 lytic action, while the dissipation of Δψ is mainly associated with higher endolysin binding. Interestingly, this binding depends on the amidase domain. The peptidase domain is responsible for most of the Lys11 bacteriolytic activity. Wall teichoic acids (WTAs) are confirmed as major determinants of endolysin tolerance, in part by severely hindering CBD11 binding activity. In conclusion, the PMF and WTA interfere differently with the endolysin functional domains, affecting both the binding and catalytic efficiencies.

Non-invasive phrenic nerve stimulation to avoid ventilator-induced diaphragm dysfunction in critical care.

BACKGROUND: Diaphragm muscle atrophy during mechanical ventilation begins within 24 h and progresses rapidly with significant clinical consequences. Electrical stimulation of the phrenic nerves using invasive electrodes has shown promise in maintaining diaphragm condition by inducing intermittent diaphragm muscle contraction. However, the widespread application of these methods may be limited by their risks as well as the technical and environmental requirements of placement and care. Non-invasive stimulation would offer a valuable alternative method to maintain diaphragm health while overcoming these limitations. METHODS: We applied non-invasive electrical stimulation to the phrenic nerve in the neck in healthy volunteers. Respiratory pressure and flow, diaphragm electromyography and mechanomyography, and ultrasound visualization were used to assess the diaphragmatic response to stimulation. The electrode positions and stimulation parameters were systematically varied in order to investigate the influence of these parameters on the ability to induce diaphragm contraction with non-invasive stimulation. RESULTS: We demonstrate that non-invasive capture of the phrenic nerve is feasible using surface electrodes without the application of pressure, and characterize the stimulation parameters required to achieve therapeutic diaphragm contractions in healthy volunteers. We show that an optimal electrode position for phrenic nerve capture can be identified and that this position does not vary as head orientation is changed. The stimulation parameters required to produce a diaphragm response at this site are characterized and we show that burst stimulation above the activation threshold reliably produces diaphragm contractions sufficient to drive an inspired volume of over 600 ml, indicating the ability to produce significant diaphragmatic work using non-invasive stimulation. CONCLUSION: This opens the possibility of non-invasive systems, requiring minimal specialist skills to set up, for maintaining diaphragm function in the intensive care setting.

The First Evidence of Bacterial Foci in the Hair Part and Dermal Papilla of Scalp Hair Follicles: A Pilot Comparative Study in Alopecia Areata.

The role of the microbiome in hair follicle (HF) growth represents a growing field of research. Here, we studied the bacterial population in the scalp hair follicles of subjects with alopecia areata (AA). Two Healthy and two AA subjects, respectively (20−60 years old), were enrolled and studied regarding the microbial community in the subepidermal scalp compartments by means of a 4-mm biopsy punch. Samples were examined by 16S sequencing, histochemical staining (Gram's method), and transmission electron microscopy (TEM). Bacterial foci were observed in the AA subjects' follicles with both the two adopted complementary approaches (electron microscopy and Gram staining). Significant (p < 0.05) differences were also found in the three-layer biopsy samples (p < 0.05) regarding the bacterial population. In particular, in the deep epidermis and dermis levels, a significant (p < 0.05) lower abundance of Firmicutes and a higher abundance of Proteobacteria were found in AA samples compared to the healthy control. Firmicutes also showed a significant (p < 0.05) lower abundance in hypodermis in AA subjects. In addition, Enterobacteriaceae and the genera Streptococcus, Gemella, Porphyromonas, and Granulicatella were relatively more abundant in AA groups at the deep epidermis level. The Staphylococcus and Flavobacterium genera were significantly less abundant in AA samples than in controls in all three-layer biopsy samples (p < 0.05). In contrast, Veillonella and Neisseriaceae were relatively more abundant in the healthy control group compared to the AA sample. Therefore, higher alpha diversity was observed in all three-layer biopsy samples of AA patients compared to the control. In conclusion, our data suggest that tAA could be defined as a "hair disease associated with dysregulated microbiome-immunity axis of hair follicles".

Effect of CB2 Stimulation on Gene Expression in Pediatric B-Acute Lymphoblastic Leukemia: New Possible Targets.

Acute lymphoblastic leukemia type B (B-ALL) is the most common kind of pediatric leukemia, characterized by the clonal proliferation of type B lymphoid stem cells. Important progress in ALL treatments led to improvements in long-term survival; nevertheless, many adverse long-term consequences still concern the medical community. Molecular and cellular target therapies, together with immunotherapy, are promising strategies to overcome these concerns. Cannabinoids, enzymes involved in their metabolism, and cannabinoid receptors type 1 (CB1) and type 2 (CB2) constitute the endocannabinoid system, involved in inflammation, immune response, and cancer. CB2 receptor stimulation exerts anti-proliferative and anti-invasive effects in many tumors. In this study, we evaluated the effects of CB2 stimulation on B-ALL cell lines, SUP-B15, by RNA sequencing, Western blotting, and ELISA. We observe a lower expression of CB2 in SUP-B15 cells compared to lymphocytes from healthy subjects, hypothesizing its involvement in B-ALL pathogenesis. CB2 stimulation reduces the expression of CD9, SEC61G, TBX21, and TMSB4X genes involved in tumor growth and progression, and also negatively affects downstream intracellular pathways. Our findings suggest an antitumor role of CB2 stimulation in B-ALL, and highlight a functional correlation between CB2 receptors and specific anti-tumoral pathways, even though further investigations are needed.

Histomorphometry Changes and Decreased Reactivity to Angiotensin II in the Ileum and Colon of Streptozotocin-Induced Diabetic Rats.

Diabetes mellitus (DM) is a chronic progressive metabolic disorder associated with several gastrointestinal complications, affecting up to 75% of patients. Knowing that Angiotensin II (AngII) also regulates intestinal contraction, we decided to evaluate changes in ileum and colon histomorphometry and AngII reactivity in a rat model of DM. Streptozotocin (STZ, 55 mg/kg) was administered to induce DM to 24 adult male Wistar rats. Diabetic rats displayed all the characteristic signs of type 1 DM (T1DM) and fecal excretion increased about 4-fold over 14 days, while the excretion of controls remained unaltered. Compared to controls, diabetic ileum and colon presented an increase in both macroscopic (length, perimeter and weight) and microscopic (muscular wall thickness) parameters. Functionally, AngII-induced smooth muscle contraction was lower in diabetic rats, except in the distal colon. These differences in the contractile response to AngII may result from an imbalance between AngII type 1 (antagonized by candesartan, 10 nM) and type 2 receptors activation (antagonized by PD123319, 100 nM). Taken together, these results indicate that an early and refined STZ-induced T1DM rat model already shows structural remodelling of the gut wall and decreased contractile response to AngII, findings that may help to explain diabetic dysmotility.

Interaction between the Renin-Angiotensin System and Enteric Neurotransmission Contributes to Colonic Dysmotility in the TNBS-Induced Model of Colitis.

Angiotensin II (Ang II) regulates colon contraction, acting not only directly on smooth muscle but also indirectly, interfering with myenteric neuromodulation mediated by the activation of AT1 /AT2 receptors. In this article, we aimed to explore which mediators and cells were involved in Ang II-mediated colonic contraction in the TNBS-induced rat model of colitis. The contractile responses to Ang II were evaluated in distinct regions of the colon of control animals or animals with colitis in the absence and presence of different antagonists/inhibitors. Endogenous levels of Ang II in the colon were assessed by ELISA and the number of AT1/AT2 receptors by qPCR. Ang II caused AT1 receptor-mediated colonic contraction that was markedly decreased along the colons of TNBS-induced rats, consistent with reduced AT1 mRNA expression. However, the effect mediated by Ang II is much more intricate, involving (in addition to smooth muscle cells and nerve terminals) ICC and EGC, which communicate by releasing ACh and NO in a complex mechanism that changes colitis, unveiling new therapeutic targets.

ECF σ factors with regulatory extensions: the one-component systems of the σ universe.

The σ subunit of the bacterial RNA polymerase determines promoter specificity. The extracytoplasmic function σ factors (ECFs) represent the most abundant and diverse group of alternative σ factors and are present in the vast majority of bacterial genomes. Typically, ECFs are regulated by anti-σ factors that sequester their cognate ECFs, thereby preventing their interaction with the RNA polymerase. Beyond these ECF paradigms, a number of distinct modes of regulation have been proposed and experimentally investigated. Regulatory extensions represent one such alternative mechanism of ECF regulation that can be found in 18 phylogenetically distinct ECF groups. Here, the σ factors contain additional domains that are fused to the ECF core domains and are involved in stimulus perception and modulation of σ factor activity. We will summarize the current state of knowledge on regulating ECF activity by C-terminal extensions. We will also discuss newly identified ECF groups containing either N- or C-terminal extensions and propose possible mechanisms by which these extensions have been generated and affect ECF σ factor activity. Based on their modular architecture and the resulting physical connection between stimulus perception and transcriptional output, these ECFs are analogous to one-component systems, the primary mechanism of bacterial signal transduction.

Defining the regulon of genes controlled by σE , a key regulator of the cell envelope stress response in Streptomyces coelicolor.

The extracytoplasmic function (ECF) σ factor, σE , is a key regulator of the cell envelope stress response in Streptomyces coelicolor. Although its role in maintaining cell wall integrity has been known for over a decade, a comprehensive analysis of the genes under its control has not been undertaken. Here, using a combination of chromatin immunoprecipitation-sequencing (ChIP-seq), microarray transcriptional profiling and bioinformatic analysis, we attempt to define the σE regulon. Approximately half of the genes identified encode proteins implicated in cell envelope function. Seventeen novel targets were validated by S1 nuclease mapping or in vitro transcription, establishing a σE -binding consensus. Subsequently, we used bioinformatic analysis to look for conservation of the σE target promoters identified in S. coelicolor across 19 Streptomyces species. Key proteins under σE control across the genus include the actin homolog MreB, three penicillin-binding proteins, two L,D-transpeptidases, a LytR-CpsA-Psr-family protein predicted to be involved in cell wall teichoic acid deposition and a predicted MprF protein, which adds lysyl groups to phosphatidylglycerol to neutralize membrane surface charge. Taken together, these analyses provide biological insight into the σE -mediated cell envelope stress response in the genus Streptomyces.

Microbial Diversity in the Eukaryotic SAR Clade: Illuminating the Darkness Between Morphology and Molecular Data.

Despite their diversity and ecological importance, many areas of the SAR-Stramenopila, Alveolata, and Rhizaria-clade are poorly understood as the majority (90%) of SAR species lack molecular data and only 5% of species are from well-sampled families. Here, we review and summarize the state of knowledge about the three major clades of SAR, describing the diversity within each clade and identifying synapomorphies when possible. We also assess the "dark area" of SAR: the morphologically described species that are missing molecular data. The majority of molecular data for SAR lineages are characterized from marine samples and vertebrate hosts, highlighting the need for additional research effort in areas such as freshwater and terrestrial habitats and "non-vertebrate" hosts. We also describe the paucity of data on the biogeography of SAR species, and point to opportunities to illuminate diversity in this major eukaryotic clade. See also the video abstract here: https://youtu.be/_VUXqaX19Rw.

Engineering orthogonal synthetic timer circuits based on extracytoplasmic function σ factors.

The rational design of synthetic regulatory circuits critically hinges on the availability of orthogonal and well-characterized building blocks. Here, we focus on extracytoplasmic function (ECF) σ factors, which are the largest group of alternative σ factors and hold extensive potential as synthetic orthogonal regulators. By assembling multiple ECF σ factors into regulatory cascades of varying length, we benchmark the scalability of the approach, showing that these 'autonomous timer circuits' feature a tuneable time delay between inducer addition and target gene activation. The implementation of similar timers in Escherichia coli and Bacillus subtilis shows strikingly convergent circuit behavior, which can be rationalized by a computational model. These findings not only reveal ECF σ factors as powerful building blocks for a rational, multi-layered circuit design, but also suggest that ECF σ factors are universally applicable as orthogonal regulators in a variety of bacterial species.

Childhood Head and Neck Lymphadenopathy: A Report by a Single Institution (2003-2017).

Actually, there is still no consensus related to diagnostic and management algorithms in case of head and neck lymphadenopathy in children. The aim of our study was to analyze the causes of head and neck lymphadenopathy in children to determine a systematic diagnostic approach. We enrolled all cases of head and neck lymphadenopathy in children under the age of 18 diagnosed at the Unit of Hemato-Oncology, Pediatric Department of University "Luigi Vanvitelli," Naples, over a 15-year period (January 2003-December 2017). In total, 405 patients (271 males) were enrolled in the study. Thirteen cases due to other causes, were left off the study. Therefore, the study was performed on 392 cases. A total of 220 patients (56.1%) had a history of infection, 66 cases (16.8%) a diagnosis of neoplasia, and 101 (24.9%) cases a diagnosis of reactive inflammatory changes of nonspecific origin. We have observed the following from our study: (1) the acute infections are the most common causes of head and neck lymphadenopathy in the pediatric population; (2) in about a quarter of patients, the lymphadenopathy resulted by nonspecific origin; (3) the supraclavicular nodes should be regarded with a high index of suspicion of malignancy.

Characterization of the Widely Distributed Novel ECF42 Group of Extracytoplasmic Function σ Factors in Streptomyces venezuelae.

Extracytoplasmic function σ factors (ECFs) represent the third most abundant fundamental principle of bacterial signal transduction, outranked only by one- and two-component systems. A recent census of ECFs revealed a large number of novel groups whose functions and regulatory mechanisms have not yet been elucidated. Here, we report the characterization of members of the novel group ECF42. ECF42 is a highly abundant and widely distributed ECF group that is present in 11 phyla but is predominantly found in Actinobacteria Analysis of the genomic context conservation did not identify a putative anti-σ factor. Instead, ECF42 genes are cotranscribed with genes encoding a conserved DGPF protein. We have experimentally verified the target promoter of these ECFs (TGTCGA in the -35 region and CGA/TC in the -10 region), which was found upstream of the ECF42-encoding operons in Streptomyces venezuelae, suggesting that ECF42s are positively autoregulated. RNA sequencing (RNA-seq) was performed to define the regulons of the three ECF42 proteins in S. venezuelae, which identified mostly genes encoding DGPF proteins. In contrast to typical ECFs, ECF42 proteins harbor a long C-terminal extension, which is crucial for their activity. Our work provides the first analysis of the function and regulatory mechanism of this novel ECF group that contains a regulatory C-terminal extension.IMPORTANCE In contrast to the one- and two-component signal transduction systems in bacteria, the importance and diversity of ECFs have only recently been recognized in the course of comprehensive phylogenetic and comparative genomics studies. Thus, most of the ECFs still have not been experimentally characterized regarding their physiological functions and regulation mechanisms so far. The physiological roles, target promoter, and target regulons of a novel group of ECFs, ECF42, in S. venezuelae have been investigated in this work. More importantly, members of this group are characterized by a C-terminal extension, which has been verified to harbor a regulatory role in ECF42s. Hence, our work provides an important source for further research on such C-terminal extension containing ECFs.

The three-component system EsrISR regulates a cell envelope stress response in Corynebacterium glutamicum.

When the cell envelope integrity is compromised, bacteria trigger signaling cascades resulting in the production of proteins that counteract these extracytoplasmic stresses. Here, we show that the two-component system EsrSR regulates a cell envelope stress response in the Actinobacterium Corynebacterium glutamicum. The sensor kinase EsrS possesses an amino-terminal phage shock protein C (PspC) domain, a property that sets EsrSR apart from all other two-component systems characterized so far. An integral membrane protein, EsrI, whose gene is divergently transcribed to the esrSR gene locus and which interestingly also possesses a PspC domain, acts as an inhibitor of EsrSR under non-stress conditions. The resulting EsrISR three-component system is activated among others by antibiotics inhibiting the lipid II cycle, such as bacitracin and vancomycin, and it orchestrates a broad regulon including the esrI-esrSR gene locus itself, genes encoding heat shock proteins, ABC transporters, and several putative membrane-associated or secreted proteins of unknown function. Among those, the ABC transporter encoded by cg3322-3320 was shown to be directly involved in bacitracin resistance of C. glutamicum. Since similar esrI-esrSR loci are present in a large number of actinobacterial genomes, EsrISR represents a novel type of stress-responsive system whose components are highly conserved in the phylum Actinobacteria.

Bortezomib and endocannabinoid/endovanilloid system: a synergism in osteosarcoma.

Osteosarcoma is the most common primary malignant tumor of bone in children and adolescents. Bortezomib (BTZ) is an approved anticancer drug, classified as a selective reversible inhibitor of the ubiquitin-dependent proteasome system, that leads to cancer cell cycle arrest and apoptosis reducing the invasion ability of Osteosarcoma cells in vitro. It also regulates the RANK/RANKL/OPG system, involved in the pathogenesis of bone tumors and in cell migration. A side effect of BTZ is to induce painful sensory peripheral neuropathy which lead to cessation of therapy or dose reduction. Recently BTZ has been evaluated in combination with Cannabinoids targeting CB1 receptor, demonstrating a promising synergic effect. The Endocannabinoid/Endovanilloid (EC/EV) system includes two G protein-coupled receptors (CB1 and CB2), the Transient Potential Vanilloid 1 (TRPV1) channel and their endogenous ligands and enzymes. CB1 and CB2 are expressed mainly in Central Nervous System and Immune Peripheral cells respectively. TRPV1 is also expressed in primary sensory neurons and is involved in pain modulation. EC/EV system induces apoptosis, reduces invasion and cell proliferation in Osteosarcoma cell lines and is involved in bone metabolism. We analyzed the effects of BTZ, alone and in combination with selective agonists at CB2 (JWH-133) and TRPV1 (RTX) receptors, in the Osteosarcoma cell line (HOS) on Apoptosis, Cell Cycle progression, migration and bone balance. We observed that the stimulation of CB2 and TRPV1 receptors increase the efficacy of BTZ in inducing apoptosis and reducing invasion, cell cycle progression and by modulating bone balance. These data suggest the possibility to use BTZ, in combination with EC/EV agonists, in Osteosarcoma therapy reducing its dose and its side effects.

Exploitation of grape marc as functional substrate for lactic acid bacteria and bifidobacteria growth and enhanced antioxidant activity.

This study aimed at using grape marc for the growth of lactic acid bacteria and bifidobacteria with the perspective of producing a functional ingredient having antioxidant activity. Lactobacillus plantarum 12A and PU1, Lactobacillus paracasei 14A, and Bifidobacterium breve 15A showed the ability to grow on grape marc (GM) based media. The highest bacterial cell density (>9.0 CFU/g) was found in GM added of 1% of glucose (GMG). Compared to un-inoculated and incubated control fermented GMG showed a decrease of carbohydrates and citric acid together with an increase of lactic acid. The content of several free amino acids and phenol compounds differed between samples. Based on the survival under simulated gastro-intestinal conditions, GMG was a suitable carrier of lactic acid bacteria and bifidobacteria strains. Compared to the control, cell-free supernatant (CFS) of fermented GMG exhibited a marked antioxidant activity in vitro. The increased antioxidant activity was confirmed using Caco-2 cell line after inducing oxidative stress, and determining cell viability and radical scavenging activity through MTT and DCFH-DA assays, respectively. Supporting these founding, the SOD-2 gene expression of Caco-2 cells also showed a lowest pro-oxidant effect induced by the four CFS of GMG fermented by lactic acid bacteria and bifidobacteria.