Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.

CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature.

Multivalent tyrosine kinase inhibition promotes T cell recruitment to immune-desert gastric cancers by restricting epithelial-mesenchymal transition via tumour-intrinsic IFN-γ signalling.

OBJECTIVE: Gastric cancer (GC) ranks fifth in incidence and fourth for mortality worldwide. The response to immune checkpoint blockade (ICB) therapy in GC is heterogeneous due to tumour-intrinsic and acquired immunotherapy resistance. We developed an immunophenotype-based subtyping of human GC based on immune cells infiltration to develop a novel treatment option. DESIGN: A algorithm was developed to reclassify GC into immune inflamed, excluded and desert subtypes. Bioinformatics, human and mouse GC cell lines, syngeneic murine gastric tumour model, and CTLA4 blockade were used to investigate the immunotherapeutic effects by restricting receptor tyrosine kinase (RTK) signalling in immune desert (ICB-resistant) type GC. RESULTS: Our algorithm restratified subtypes of human GC in public databases and showed that immune desert-type and excluded-type tumours are ICB-resistant compared with immune-inflamed GC. Moreover, epithelial-mesenchymal transition (EMT) signalling was highly enriched in immune desert-type GC, and syngeneic murine tumours exhibiting mesenchymal-like, compared with epithelial-like, properties are T cell-excluded and resistant to CTLA4 blockade. Our analysis further identified a panel of RTKs as potential druggable targets in the immune desert-type GC. Dovitinib, an inhibitor of multiple RTKs, strikingly repressed EMT programming in mesenchymal-like immune desert syngeneic GC models. Dovitinib activated the tumour-intrinsic SNAI1/2-IFN-γ signalling axis and impeded the EMT programme, converting immune desert-type tumours to immune inflamed-type tumours, sensitising these mesenchymal-like 'cold' tumours to CTLA4 blockade. CONCLUSION: Our findings identified potential druggable targets relevant to patient groups, especially for refractory immune desert-type/ 'cold' GC. Dovitinib, an RTK inhibitor, sensitised desert-type immune-cold GC to CTLA4 blockade by restricting EMT and recruiting T cells.

A randomized-controlled trial of SOF/VEL/VOX with or without ribavirin for retreatment of chronic hepatitis C.

BACKGROUND & AIMS: The combination of sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) is recommended for the retreatment of patients with HCV infection in whom previous direct-acting antiviral (DAA) treatment failed. However, whether ribavirin further increases the therapeutic efficacy of SOF/VEL/VOX retreatment remains unclear. We aimed to test this hypothesis in a randomized-controlled trial. METHODS: We randomly assigned 315 patients with DAA treatment failure from five Egyptian sites into two groups. Group A (n = 158) received SOF/VEL/VOX for 12 weeks, and group B (n = 157) received SOF/VEL/VOX + weight-based ribavirin for 12 weeks. Therapeutic efficacy was defined as SVR12 (sustained virologic response 12 weeks after treatment end). Safety and tolerability were evaluated by monitoring treatment-related adverse events (AEs) and laboratory abnormalities. RESULTS: Males comprised 53.9% of group A and 57.1% of group B (p = 0.58); mean ages were 51.8 and 47.3 years in group A and B, respectively. Seventeen patients in each group were lost to follow-up. SVR12 rates were 87.3% (138/158) by intention-to-treat analysis and 97.8% (138/141) by per-protocol analysis in group A; and 87.9% (138/157) and 98.5% (138/140), respectively, in group B (p = n.s. for intention-to-treat and per-protocol analyses). Both regimens were well-tolerated, with no deaths and only one serious AE (anemia) in group B, which required ribavirin discontinuation. Fifty-five patients in group A vs. 77 in group B experienced any AE (p = 0.002). CONCLUSION: This randomized-controlled trial showed equal, high efficacy of both regimens for the retreatment of previous DAA failures, although ribavirin was associated with more AEs. Therefore SOF/VEL/VOX monotherapy should be the preferred retreatment strategy. CLINCIALTRIALS. GOV NUMBER: NCT04695769. IMPACT AND IMPLICATIONS: HCV treatment guidelines recommend retreatment of direct-acting antiviral (DAA) treatment failures with the combination of sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) for 12 weeks. However, whether ribavirin exerts an additional/synergistic effect remains unclear. The present study confirmed that SOF/VEL/VOX without ribavirin is the best regimen for retreatment of DAA treatment failures, and thus will help guide clinicians caring for patients who are not cured with a first course of DAA therapy.

Efficacy of Cryotherapy Combined With Intralesional Purified Protein Derivative (PPD) Versus Intralesional PPD Monotherapy in the Treatment of Multiple Common Warts.

INTRODUCTION: Intralesional antigen immunotherapy represents a promising therapeutic approach for the treatment of different types of warts, particularly if multiple and/or recalcitrant. AIM: to investigate the efficacy and safety of combined cryotherapy with intralesional purified protein derivative (PPD) immunotherapy in the treatment of multiple common warts. METHODS: Fifty patients were randomly divided into two groups (25 patients each): Group A: receiving intralesional PPD immunotherapy for the largest wart, while group B: receiving cryotherapy for all warts plus intralesional PPD for the largest wart. Treatments were performed every 2 weeks for a maximum of four sessions. Photographs were taken at baseline and at each visit and clinical response was evaluated by the reduction in number and size of warts. Adverse effects were recorded. RESULTS: There was a significant reduction in size and number of warts in both groups (P < .001), with no significant difference between the two groups. Complete clearance of the lesions was observed in 48% of patients in group A and 44% in group B (P = .39). Higher rates of near complete/complete response were achieved after fewer sessions (2, 3 sessions) in group B (P = .002). Blistering was common after cryotherapy. Higher rate of hypopigmentation was noticed after combined treatment than after PPD monotherapy (56%, 8% respectively; P < .001), which resolved gradually. CONCLUSION: Both intralesional PPD alone and combined cryotherapy with PPD are safe and effective in clearing of common warts. Cryotherapy may be a successful adjunct to intralesional PPD immunotherapy that helps in reducing the number of treatment sessions.The study protocol was registered at ClinicalTrials.gov with ID: NCT04288817.

Bioengineered Wheat Arabinoxylan - Fostering Next-Generation Prebiotics Targeting Health-Related Gut Microbes.

Dietary prebiotic fibers play an important role in modulating gut microbiota by enhancing the abundance of beneficial microorganisms and their bioactive metabolites. However, dietary fibers are a structurally heterogeneous class of polysaccharides, varying in molar mass, branching patterns, and monosaccharide composition, which could influence their utilization by various gut microorganisms. The present study aimed to investigate the effects of molar mass and chemical structure of wheat arabinoxylan fiber (AX) on the growth and metabolism of two key gut resident bacteria (Faecalibacterium prausnitzii and Lacticaseibacillus rhamnosus LGG), which are linked to human health. For this purpose, low, medium, and high molar masses of AX (LAX, MAX, and HAX, respectively) were modified with specific α-arabinofuranosidases to leave only singly substituted, only doubly substituted, or unsubstituted xylose units. Almost all the modified AX samples showed a better prebiotic score than unmodified AX for different molar masses. The modified LAX exhibited a better prebiotic effect than HAX and MAX. In addition, LAX, with doubly substituted xylose units, exhibited the highest prebiotic potential and SCFA production by both microorganisms. Furthermore, AX, either singly or doubly substituted, had a consistent impact on L. rhamnosus growth, whereas AX, with all arabinose residues removed, had a greater impact on F. prausnitzii. These findings support the potential of bioengineered AX as next-generation prebiotics targeting health-related gut microbes.

Screening, characterization and growth of γ-aminobutyric acid-producing probiotic candidates from food origin under simulated colonic conditions.

AIMS: This study aims to isolate probiotic bacteria candidates from various starter cultures and fermented foods and characterize their ability to produce γ-aminobutyric acid (GABA). GABA is a major inhibitory neuromediator of the central and enteric nervous systems with a role in several health disorders. METHODS AND RESULTS: Fourteen strains of lactic acid bacteria were isolated from food environment and screened for the presence of the glutamate decarboxylase (gadB) gene using PCR and GAD enzymatic assay. The identified potent GABA-producers included Strep. thermophilus, Lactiplantibacillus plantarum and Lact. delbrueckii subsp. bulgaricus. GC-FID analyses confirmed the high GABA production capacity of Strep. thermophilus ST16 (1641.5 ± 154.15 µmol l-1 ), Strep. thermophilus ST8 (1724.5 ± 48.08 µmol/L). To a lesser extent, Bif. animalis ST20, Lact. acidophilus LP16-2 and Ent. faecium ST3 produced 947.5 ± 70.71, 918.0 ± 121.42, and 907.83 ± 55.15 µmol/L of GABA, respectively. These potent strains were able to grow and produce GABA in MRS broth and pre-fermented Macfarlane broth, the latter medium mimicking the nutrient and metabolome composition encountered in the colon. The identified bioactive strains exhibited strong biological safety and probiotic potential profiles as indicated by sensitivity to antibiotics, absence of virulence factors and survival in gastrointestinal conditions. CONCLUSIONS: Several GABA producing probiotic candidates, including Bif. animals ST20, Strep. thermophilus ST8, Lact. acidophilus LP16-2, L. plantarum LP6 & LP9, and Ent. faecium ST3, have shown potential to grow under simulated colonic conditions. SIGNIFICANCE AND IMPACT OF STUDY: Findings from this study provide evidence of the suitability of the isolated GABA-producing probiotic candidates for the development of health-oriented functional food products.

Guide RNA functional modules direct Cas9 activity and orthogonality.

The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9:guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA:tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.

Editorial: The Metabolic (Dysfunction)-Associated Fatty Liver Disease (MAFLD) and Non-Alcoholic Fatty Liver Disease (NAFLD) Debate: Why the American Association for the Study of Liver Diseases (AASLD) and European Association for the Study of the Liver (EASL) Consensus Process is Not Representative.

A debate has recently arisen in hepatology on the redefinition of fatty liver disease associated with metabolic dysfunction. The definition of metabolic (dysfunction)-associated fatty liver disease (MAFLD) has been widely endorsed by multiple stakeholders and societies. More importantly, although robust evidence supports the utility of the definition of MAFLD in clinical practice and research, and for increasing awareness of liver disease, controversy still abounds. Recently, the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) have undertaken similar consensus approaches for MAFLD. However, there are serious concerns with these regional consensus approaches. The views of hepatologists from the Middle East, North Africa, and sub-Saharan Africa are not represented. Also, the selection of experts raises concerns regarding the validity of the outcomes of the expert consensus process. We conclude that unless the process has global involvement, there will be no incentive for global adherence to these regional recommendations. This Editorial aims to highlight these concerns and to call for those involved in leading the AASLD and EASL consensus process to be more inclusive, which may facilitate the adoption of more unified recommendations that have global clinical importance.

The NAFLD-MAFLD debate: Eminence vs evidence.

Debates are inevitable in science and could be a powerful tool for addressing controversial topics as it promotes critical thinking and inspires individuals to consider alternate viewpoints. However, debates can help only to identify the issues that need to be clarified to address this question, but it can never help resolve the controversy itself. In the era of evidence-based medicine, the need for an evidence-based debate is mandatory. Polarising opinions and major debate have recently arisen in hepatology on the nomenclature and diagnostic criteria for fatty liver disease associated with metabolic dysfunction (non alcoholic fatty liver disease [NAFLD]-metabolic (dysfunction) associated fatty liver disease [MAFLD] debate). The aim of this viewpoint is to suggest a way to settle the debate through evidence. Descriptive review using PubMed to identify literature on the evidence and eminence-based medicine and studies comparing MAFLD and NAFLD criteria. The emerging studies comparing the performance of diagnostic criteria of NAFLD and MAFLD represent the dawn of a new era for reframing the ongoing debate by acquisition of the mandatory evidence that will both resolve the debate and lead to novel avenues of research. In conclusion, the time has come to hold debate and focus on gathering and building the evidence to settle it. It does not matter who wins the debate and once there is robust evidence, we should all follow it wherever it leads.

Pregnancy outcome of anti-HCV direct-acting antivirals: Real-life data from an Egyptian cohort.

We aimed to assess the pregnancy outcome in women with chronic HCV who had negative pregnancy test prior to the anti-HCV course and had unintended pregnancy while on HCV treatment. Hundred patients with a mean age of 30 ± 6.7 y were included and advised to withhold antivirals and continue follow-up in viral hepatitis and obstetrics centres till delivery. All patients received a 12-weeks regimen of anti-HCV [sofosbuvir plus daclatasvir (SOF/DCV): n = 95, SOF/DCV plus ribavirin: n = 3, and paritaprevir/ritonavir/ombitasvir plus ribavirin: n = 2]. Only nine patients completed the full antiviral course against medical advice, and 91 stopped between on-treatment weeks 4 and 8. Eighty-eight patients delivered full-term babies, eight had preterm babies and two had abortions. Of the nine patients who completed the full course of DAAs, seven (77.8%) delivered normal babies, attended their post-treatment week 12 visit, and all (100%) achieved sustained virological response. No major antiviral-related adverse events were reported.

Split-leg modified lateral versus prone position in percutaneous nephrolithotomy: a prospective, randomized trial.

PURPOSE: Standard prone position (PP) during percutaneous nephrolithotomy (PNL) has multiple drawbacks. We aimed to compare PNLs performed in split-leg (SL) modified lateral position (MLP) and those performed in standard PP. METHODS: A prospective, randomized, unblind, double arm trial was conducted at a tertiary care academic medical center in Egypt, between November 2017 and October 2019. Adult patients with renal stones undergoing PNL were included. According to renal anatomy and stone complexity, stratified randomization was performed and study participants were allocated into either SL-MLP group or PP group. The stone free rate (SFR), total operative time, track formation time, fluoroscopy time, auxiliary procedures, and complications were compared. RESULTS: There were 61 patients in SL-MLP group and 63 patients in PP group. Both groups had similar baseline characteristics. The SFR was comparable between groups: 75.4% in SL-MLP group and 77.8% in PP group (p = 0.755). The mean total operative time was shorter and mean track formation time was longer in SL-MLP group (55.33 ± 20.73 vs. 98.49 ± 9.23, p < 0.001 and 7.89 ± 3.68 vs. 6.52 ± 1.77, p = 0.002). There was no significant difference in fluoroscopy time, total complication rates, hemoglobin reduction and need for blood transfusion between the groups. In SL-MLP group, all PNL procedures as well all the associated procedures were performed with the patients in the same position. CONCLUSION: SL-MLP PNL has a short operative time and similar SFR and complication rate compared to PP PNL. SL-MLP allowed antegrade and retrograde access to the urinary tract without patient repositioning.

Efficacy of topical versus intradermal injection of Tranexamic Acid In Egyptian melasma Patients: A randomised clinical trial.

BACKGROUND: Melasma is one of the common pigmentary problems affecting females in our community, owing to the frequent use of hormonal contraceptives as well as our sunny climate. A lot of treatment options are available but none of them is completely satisfactory. Many patients prefer the use of topical preparations and minimally invasive methods. Tranexamic acid (TA) is a potential treatment option for hyperpigmentation with different delivery routes. AIM: We designed the study in order to evaluate the efficacy of TA in melasma using 2 different routes of delivery. PATIENTS AND METHODS: A randomised clinical trial was performed on 60 female patients with melasma, they randomly divided into three groups; A, B and C. Group (A) patients received TA (4 mg/mL) intradermal injections every 2 weeks with, group B received TA (10 mg/mL) intradermal injections every 2 weeks, group C received TA cream (10% concentration) twice daily, treatment continued for 12 weeks in all groups. Melasma Area and Severity Index (MASI) scores were measured for each patient before and after completion of treatment. RESULTS: The percentage of MASI score reduction was highest in group B (62.7%) versus (39.1%) in group A, while the percentage of MASI reduction was the lowest in group C (4.2%). CONCLUSION: Tranexamic acid is a safe effective and well-tolerated treatment option for melasma patients. Intradermal injection of TA leads to better results than the topical application. Topical TA cream (even in a high concentration) produce fair improvement of melasma.

Serum level of S100B in vitiligo patients: Is it a marker of disease activity?

BACKGROUND: Vitiligo is a chronic depigmentary skin disorder, characterised clinically by the development of white macules and or patches caused by loss of epidermal melanocytes. S100B is a dual function protein released from epidermal melanocytes in response to injury. It considered a possible marker of disease activity in both malignant melanoma and vitiligo. AIM OF THE STUDY: To estimate the serum level of S100B level in vitiligo patients and correlate its level with disease activity and various disease parameters. PATIENTS AND METHODS: Sixty vitiligo patients and 60 healthy volunteers as controls were included in the study. Vitiligo Area Severity Index (VASI) and Vitiligo Disease Activity (VIDA) scores were estimated for each patient. Quantitative assessment of S100B level using ELISA technique was done for all participants. RESULTS: S100B level was significantly correlated with the presence of vitiligo (P = 0.01), while it showed no correlation with the disease activity using VASI or VIDA scores. As regards the receiver operating characteristic (ROC) curve analysis of S100B for diagnosis and discrimination of vitiligo, serum S100B showed area under the curve (AUC) of 0.781 with 73.3% sensitivity and 80% specificity. CONCLUSION: The serum level of S100B was related to the presence of vitiligo, but its level did not show any relation to the disease activity using either VASI and VIDA scores or various disease parameters.

Inhibition of AURKA Reduces Proliferation and Survival of Gastrointestinal Cancer Cells With Activated KRAS by Preventing Activation of RPS6KB1.

BACKGROUND & AIMS: Activation of KRAS signaling and overexpression of the aurora kinase A (AURKA) are often detected in luminal gastrointestinal cancers. We investigated regulation of ribosomal protein S6 kinase B1 (RPS6KB1) by AURKA and the effects of alisertib, an AURKA inhibitor, in mice xenograft tumors grown from human gastrointestinal cancer cells with mutant, activated forms of KRAS. METHODS: We tested the effects of alisertib or AURKA overexpression or knockdown in 10 upper gastrointestinal or colon cancer cell lines with KRAS mutations or amplifications using the CellTiter-Glo luminescence and clonogenic cell survival assays. We used the proximity ligation in situ assay to evaluate protein co-localization and immunoprecipitation to study protein interactions. Nude mice with xenograft tumors grown from HCT116, SNU-601, SW480, or SNU-1 cells were given oral alisertib (40 mg/kg, 5 times/wk) for 4 weeks. Tumor samples were collected and analyzed by immunoblots and immunohistochemistry. Tissue microarrays from 151 paraffin-embedded human colon tumors, with adjacent normal and adenoma tissues, were analyzed by immunohistochemistry for levels of AURKA. RESULTS: Alisertib reduced proliferation and survival of the cell lines tested. AURKA knockdown or inhibition with alisertib reduced levels of phosphorylated RPS6KB1 (at T389) and increased levels of proteins that induce apoptosis, including BIM, cleaved PARP, and cleaved caspase 3. AURKA co-localized and interacted with RPS6KB1, mediating RPS6KB1 phosphorylation at T389. We detected AURKA-dependent phosphorylation of RPS6KB1 in cell lines with mutations in KRAS but not in cells with wild-type KRAS. Administration of alisertib to mice with xenograft tumors significantly reduced tumor volumes (P < .001). Alisertib reduced phosphorylation of RPS6KB1 and Ki-67 and increased levels of cleaved caspase 3 in tumor tissues. In analyses of tissue microarrays, we found significant overexpression of AURKA in gastrointestinal tumor tissues compared with non-tumor tissues (P = .0003). CONCLUSION: In studies of gastrointestinal cancer cell lines with activated KRAS, we found AURKA to phosphorylate RPS6KB1, promoting cell proliferation and survival and growth of xenograft tumors in mice. Agents that inhibit AURKA might slow the growth of gastrointestinal tumors with activation of KRAS.

What's in a name? Renaming 'NAFLD' to 'MAFLD'.

In medicine, language matters and the words used to name and describe a disease can have a profound impact on patients and their families. Over the last two decades, many criticisms have been voiced about the nomenclature and definition of non-alcoholic fatty liver disease (NAFLD) in regards not only to the prominent role that alcohol plays in the definition but also on the negative impacts of the nomenclature including trivialization, stigmatization and less consideration of the disease in health policy. Recently, a consensus of international experts proposed that the disease acronym be changed from NAFLD to metabolic (dysfunction) associated fatty liver disease or 'MAFLD'. This change goes far beyond a mere semantic revision and may be the first step that catalyses the process to better conceptualize the disease for health promotion, patient orientation, case identification, ongoing clinical trials and for health services delivery. Here we review the history of, and definitions of MAFLD in the context of advancing our understanding of the pathogenesis of the disease. We also address the reasons, signals, promises, challenges and the way going forward from the name change from various stakeholder perspectives.

The course of infection with Toxoplasma gondii RH strain in mice pre-vaccinated with gamma irradiated tachyzoites.

Toxoplasma gondii is a ubiquitous protozoan of major medical and veterinary importance. Its treatment is difficult since the available drugs have severe side effects and reactivation may occur anytime. Vaccination with irradiated parasites exhibits ideal characteristics for vaccine development. In our experimental mice model, the protection against challenge with the virulent RH strain was assessed, using 255Gy irradiated tachyzoites. Eighty mice were allocated into 3 groups: naive control group, challenged with virulent RH tachyzoites group and a third group which is challenged with 1 × 106 irradiated tachyzoites, administered as two biweekly doses intraperitoneally. Protection was tested by challenging vaccinated mice with the virulent type RH tachyzoites 30 days after the 2nd vaccination dose. The assessment was built on qualitative clinical, quantitative parasitological, histopathological parameters and measurement of serum Nitric Oxide (NO). The results showed prolonged survival rate, absence of tachyzoites in the peritoneal aspirate by counting, absence of tachyzoites in all examined organs by impression smears, amelioration of histopathological changes in the liver, spleen, brain and lung specimens and increase of the serum NO level in the vaccinated group. Therefore, we propose that irradiated Toxoplasma tachyzoites confer protection for challenged mice and could be an alternative immunization schedule for vaccine development especially for who are at risk of severe immunosuppression.

Robust Vehicle Detection and Counting Algorithm Employing a Convolution Neural Network and Optical Flow.

Automatic vehicle detection and counting are considered vital in improving traffic control and management. This work presents an effective algorithm for vehicle detection and counting in complex traffic scenes by combining both convolution neural network (CNN) and the optical flow feature tracking-based methods. In this algorithm, both the detection and tracking procedures have been linked together to get robust feature points that are updated regularly every fixed number of frames. The proposed algorithm detects moving vehicles based on a background subtraction method using CNN. Then, the vehicle's robust features are refined and clustered by motion feature points analysis using a combined technique between KLT tracker and K-means clustering. Finally, an efficient strategy is presented using the detected and tracked points information to assign each vehicle label with its corresponding one in the vehicle's trajectories and truly counted it. The proposed method is evaluated on videos representing challenging environments, and the experimental results showed an average detection and counting precision of 96.3% and 96.8%, respectively, which outperforms other existing approaches.

Bacteriocinogenic properties of Escherichia coli P2C isolated from pig gastrointestinal tract: purification and characterization of microcin V.

The aim of this study was to isolate and investigate the bacteriocinogenic and probiotic potential of new Gram-negative isolates. Of 22 bacterial isolates from pig intestine and chicken crops, ten isolates had demonstrated a good activity, and the most potent five strains were identified as four E. coli and one as Proteus sp. No virulence factors were detected for E. coli strains isolated from pig intestine. The semi-purified microcins proved to be resistant to temperature and pH variation, but sensitive to proteolytic enzymes. Of particular interest, strain E. coli P2C was the most potent, free of virulence genes and sensitive to tested antibiotics. Purification procedure revealed the presence of a single pure peak having a molecular mass of 8733.94 Da and matching microcin V (MccV). The sequence obtained by LC-MS/MS confirmed the presence of MccV. Purified MccV showed a good activity against pathogenic coliforms, especially E. coli O1K1H7 involved in avian colibacillosis. The present study provides evidence that E. coli strains isolated from pig intestine produce microcin-like substances. E. coli P2C is a safe MccV producer that could be a good candidate for its application as novel probiotic strain to protect livestock and enhance growth performance.

Workplace violence injury in 106 US hospitals participating in the Occupational Health Safety Network (OHSN), 2012-2015.

BACKGROUND: Workplace violence is a substantial occupational hazard for healthcare workers in the United States. METHODS: We analyzed workplace violence injury surveillance data submitted by hospitals participating in the Occupational Health Safety Network (OHSN) from 2012 to 2015. RESULTS: Data were frequently missing for several important variables. Nursing assistants (14.89, 95%CI 10.12-21.91) and nurses (8.05, 95%CI 6.14-10.55) had the highest crude workplace violence injury rates per 1000 full-time equivalent (FTE) workers. Nursing assistants' (IRR 2.82, 95%CI 2.36-3.36) and nurses' (IRR 1.70, 95%CI 1.45-1.99) adjusted workplace violence injury rates were significantly higher than those of non-patient care personnel. On average, the overall rate of workplace violence injury among OHSN-participating hospitals increased by 23% annually during the study period. CONCLUSION: Improved data collection is needed for OHSN to realize its full potential. Workplace violence is a serious, increasingly common problem in OHSN-participating hospitals. Nursing assistants and nurses have the highest injury risk.