[Stochastic Packaging of Cas Proteins into Exosomes].

CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.

Community-based study to describe the epidemiology of dengue infection in a large cantonment during one transmission season.

Background: Dengue is one of the most important vector-borne disease in India. It has been linked to monsoons when Aedes aegypti mosquitoes breed profusely in containers. No study exists in Armed Forces wherein a community-based sero-survey has described the epidemiology of dengue. The present study tries to fill this knowledge gap. Methods: A total of 422 participants were studied for one transmission season of July-December. Blood samples were collected for testing dengue IgG and IgM at the beginning and at end of the study period. The study participants were interviewed at least twice within this period of 6 months to assess clinical condition and follow-up. Point prevalence and incidence were measured. Distribution of presence or absence of symptoms was noted for positive as well as negative cases. Results: All participants were males. Average age was 31.75 years. Point prevalence at the beginning of transmission season was 11.6% (95% CI: 8.4%-14.6%) and 15.6% (95% CI: 12.1%-19.1%) towards the end. Incidence was found to be 147.4 per 1000 for 6 months. Forty percent of incident cases were asymptomatic. Conclusion: Healthcare planners and hospital commanders in stations across Armed Forces can use the prevalence and incidence figures obtained in this study as a general guide while planning for prevention and control of dengue. Also, this study points to the fact that dengue transmission in Delhi may have shifted earlier to months of April/May than the conventionally accepted season of July-December.

Evidence of novel Treponema phylotypes implicated in contagious ovine digital dermatitis and association of treponemes with major lameness causing foot pathogens.

In this study 269 swabs collected from 254 ovine foot lesions and 15 apparently healthy ovine feet were screened by PCR for the presence of major lameness causing foot pathogens viz. Treponema species, D. nodosus, F. necrophorum and T. pyogenes with the presumption that ovine foot lesion positive for Treponema species alone or in association with other three pathogens were categorized as contagious ovine digital dermatitis (CODD). While samples positive for D. nodosus alone or its combination with F. necrophorum and T. pyogenes were considered as footrot (FR) and samples in which F. necrophorum or T. pyogenes was found either alone or in combination were considered as interdigital dermatitis (ID). The overall occurrence of Treponema sp. in ovine foot lesions was 48.0%, and ranged from 33 to 58%. In Treponema positive samples D. nodosus, F. necrophorum and T. pyogenes were present in 34 (27.4%), 66 (54.4%) and 84 (68.5%) in contrast to Treponema negative samples in which these were present in 15 (11.1%), 20 (14.12%) and 17 (12.6%) samples, respectively. The data signifies that Treponema sp. are significantly associated with these foot pathogens and their different combinations with Treponema sp. influence the severity of CODD lesion. The identification of Treponema phylotypes was done by sequencing the 16S rRNA gene fragment of ten representative samples. Out of ten sequences, four (Trep-2, Trep-4, Trep-7 and Trep-10) were identical to Treponema sp. phylotype 1 (PT1) that belongs to phylogroup T. refringens-like, one sequence (Trep-1) was genetically close (90% sequence homology) to Treponema brennaborense while five sequences (Trep-3, Trep-5, Trep-6, Trep-8 and Trep-9) matched with uncultured bacterium clones of treponemes forming separate monophyletic group in phylogenetic tree and could represent new digital dermatitis phylogroup presently containing five ovine specific phylotypes. This is the first report on the presence of Treponema phylotypes other than three digital dermatitis (DD) Treponema phylogroups viz. T. phagedenis-like, T. medium/T. vincentii-like, and T. pedis-like that are frequently detected in CODD lesions. Metagenomic analysis of two representative samples revealed the abundance of genus Treponema in CODD lesion while this genus was absent in swab collected from clinically healthy foot suggesting that it might play primary role in producing CODD. These findings may further aid in understanding the etiopathogenesis of CODD and could help to develop appropriate treatment and mitigation strategies to combat the disease.

Impact of COVID-19 & Response Measures on HIV-HCV Prevention Services and Social Determinants in People Who Inject Drugs in 13 Sites with Recent HIV Outbreaks in Europe, North America and Israel.

HIV/HCV prevention among people who inject drugs (PWID) is of key public health importance. We aimed to assess the impact of COVID-19 and associated response measures on HIV/HCV prevention services and socio-economic status of PWID in high-HIV-risk sites. Sites with recent (2011-2019) HIV outbreaks among PWID in Europe North America and Israel, that had been previously identified, were contacted early May 2020. Out of 17 sites invited to participate, 13 accepted. Semi-structured qualitative site reports were prepared covering data from March to May 2020, analyzed/coded and confirmed with a structured questionnaire, in which all sites explicitly responded to all 103 issues reported in the qualitative reports. Opioid maintenance treatment, needle/syringe programs and antiretroviral treatment /hepatitis C treatment continued, but with important reductions and operational changes. Increases in overdoses, widespread difficulties with food and hygiene needs, disruptions in drug supply, and increased homelessness were reported. Service programs rapidly reformed long established, and politically entrenched, restrictive service delivery policies. Future epidemic control measures should include mitigation of negative side-effects on service provision and socio-economic determinants in PWID.

RESUMEN: La prevención del VIH/VHC entre las personas que se inyectan drogas (PWID) es de vital importancia para la salud pública. Nuestro objetivo fue evaluar el impacto de COVID-19 y las medidas de respuesta asociadas en los servicios de prevención del VIH/VHC y el estado socioeconómico de las PWID en sitios de alto riesgo de VIH. Se contactó con sitios con brotes recientes (2011­2019) de VIH entre PWID en Europa, América del Norte e Israel, que habían sido previamente identificados, a principios de mayo de 2020. De los 17 sitios invitados a participar, 13 aceptaron. Se prepararon informes cualitativos semiestructurados del sitio que cubrían los datos de marzo a mayo de 2020, analizados/codificados y confirmados con un cuestionario estructurado, en el que todos los sitios respondieron explícitamente a los 103 asuntos reportados en los informes cualitativos. El tratamiento de mantenimiento con opiáceos, los programas de agujas/jeringas y el tratamiento antirretroviral/tratamiento de la hepatitis C continuaron, pero con importantes reducciones y cambios operativos. Se reportaron aumentos en las sobredosis, dificultades generalizadas con las necesidades alimentarias y de higiene, interrupciones en el suministro de medicamentos y aumento de personas sin hogar. Los programas de servicios reformaron rápidamente las políticas restrictivas de prestación de servicios, establecidas desde hace mucho tiempo y políticamente arraigadas. Las futuras medidas de control de epidemias deben incluir la mitigación de los efectos secundarios negativos en la prestación de servicios y los determinantes socioeconómicos en las PWID.

Chemically aware unitary coupled cluster with ab initio calculations on an ion trap quantum computer: A refrigerant chemicals' application.

Circuit depth reduction is of critical importance for quantum chemistry simulations on current and near term quantum computers. This issue is tackled by introducing a chemically aware strategy for the unitary coupled cluster ansatz. The objective is to use the chemical description of a system to aid in the synthesis of a quantum circuit. We combine this approach with two flavors of symmetry verification for the reduction of experimental noise. These methods enable the use of Quantinuum's System Model H1 ion trap quantum computer for a 6-qubit quantum subspace expansion calculation. We present (i) calculations to obtain methane's optical spectra; (ii) an atmospheric gas reaction simulation involving [CH3⋅-H-OH]‡. Using our chemically aware unitary coupled cluster state-preparation strategy in tandem with state of the art symmetry verification methods, we improve device yield for CH4 at 6 qubits. This is demonstrated by a 90% improvement in two-qubit gate count and a reduction in relative error to 0.2% for electronic energy calculated on System Model H1.

Optimizing the Agrobacterium tumefaciens-mediated transformation conditions in Colletotrichum lindemuthianum: a step forward to unravel the functions of pathogenicity arsenals.

Colletotrichum lindemuthianum is a hemibiotrophic fungal pathogen that causes bean anthracnose and it is rated among the top 10 important diseases infecting beans. Currently our knowledge on molecular mechanisms underlying C. lindemuthianum pathogenesis is limited. About five pathogenicity genes have been identified in C. lindemuthianum using Restricted Enzyme Mediated Integration and the transformation using Agroinfection has not been optimized. In this study, a series of experiments were conducted to optimize the key parameters affecting the Agrobacterium tumefaciens-mediated transformation for C. lindemuthianum. The transformation efficiency increased with increase in spore concentration and co-cultivation time. However, the optimum conditions that yielded significant number of transformants were 106 ml-1 spore concentration, co-cultivation time of 72 h, incubation at 25°C and using a cellulose membrane filter for the co-cultivation. The optimized protocol resulted in establishment of large mutant library (2400). A few mutants were melanin deficient and a few were unable to produce conidia. To determine the altered pathogenicity, two new approaches such as detached leaf and twig techniques proved reliable and require fewer resources to screen the large mutant libraries in a short time. Among the 1200 transformants tested for virulence, 90% transformants were pathogenically similar to wild type (race 2047), 96 and 24 were reduced and impaired, respectively. The altered avirulent transformants can prove vital for understanding the missing link between growth and developmental stages of pathogen with virulence. This platform will help to develop strategies to determine the potential pathogenicity genes and to decipher molecular mechanisms of host-pathogen interactions in more detail.

Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.

AIM: The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family- and two species-specific (A. faecis and A. lanthieri) loop-mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches. METHODS AND RESULTS: Three sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family-specific) and gyrB (species-specific) genes. Optimized Arcobacteraceae family-specific LAMP assay correctly amplified and detected 24 species, whereas species-specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL-1 (10 fg). Direct DNA-based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family-level (93%) with the concentration ranging from 2·1 × 101 to 2·2 × 105 cells 100 mL-1 . CONCLUSIONS: Overall, these three DNA-based rapid and cost-effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on-site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.

The impact of COVID-19 on emergency general surgery admissions and operative volumes: A single centre experience.

INTRODUCTION: The COVID-19 pandemic has placed a significant strain on healthcare resources and utilisation globally. The appearance of the disease in the Republic of Ireland resulted in a broad postponement of scheduled and routine surgical care. The influence of the novel coronavirus, and the associated imposition of public health measures such as school closures and social distancing, on the burden of emergency surgical disease is less clear. AIM: The aim of this study was to examine the impact of COVID-19 on the number of patients presenting to our institution with emergent surgical illnesses or requiring emergency general surgical procedures. METHODS: All patients attending our service between March 1st 2020 and April 30th 2020 were identified retrospectively by examining electronic handover and electronic discharge summaries, and data were collected relating to demographics, presenting illness, critical care utilisation, length of stay, operative or endoscopic procedure performed, and in-hospital mortality. Similar data were collected March 1st to April 30th 2019, 2018, and 2017 respectively to allow direct comparison. RESULTS: 151 patients were admitted during the study period, compared to a total of 788 during the proceeding three years (mean 2.49 admissions per night versus 4.35 per night, 42.8% reduction, p < 0.001). Median age of admitted patients was 51.8 years, compared to 50.3 years formerly (p = 0.35). 53 emergency procedures were performed, compared to a median of 70 over the same period in the previous years (mean 0.87 per day versus 1.16 per day, 25.4% reduction, p = 0.05). CONCLUSION: A significant overall reduction in the number of patients being admitted to our unit and requiring emergency surgical procedures during March and April 2020 was seen, in line with patterns reported internationally.

COVID-19 pandemic and its positive impacts on environment: an updated review.

In December, 2019 in Wuhan city of China, a novel coronavirus (SARS-CoV-2) has garnered global attention due to its rapid transmission. World Health Organization (WHO) termed the infection as Coronavirus Disease 2019 (COVID-19) after phylogenic studies with SARS-CoV. The virus causes severe respiratory infections with dry cough, high fever, body ache and fatigue. The virus is primarily transmitted among people through respiratory droplets from COVID-19 infected person. WHO declared this COVID-19 outbreak a pandemic and since February, 2020 affected countries have locked down their cities, industries and restricted the movement of their citizens to minimize the spread of the virus. In spite of the negative aspects of coronavirus on the globe, the coronavirus crises brought a positive impact on the natural environment. Countries where the movement of citizens was seized to stop the spread of coronavirus infection have experienced a noticeable decline in pollution and greenhouse gases emission. Recent research also indicated that this COVID-19-induced lockdown has reduced the environmental pollution drastically worldwide. In this review, we have discussed some important positive impacts of coronavirus on environmental quality by compiling the recently published data from research articles, NASA (National Aeronautics and Space Administration) and ESA (European Space Agency).

E-cigarette constituents propylene glycol and vegetable glycerin decrease glucose uptake and its metabolism in airway epithelial cells in vitro.

Electronic nicotine delivery systems, or e-cigarettes, utilize a liquid solution that normally contains propylene glycol (PG) and vegetable glycerin (VG) to generate vapor and act as a carrier for nicotine and flavorings. Evidence indicated these "carriers" reduced growth and survival of epithelial cells including those of the airway. We hypothesized that 3% PG or PG mixed with VG (3% PG/VG, 55:45) inhibited glucose uptake in human airway epithelial cells as a first step to reducing airway cell survival. Exposure of H441 or human bronchiolar epithelial cells (HBECs) to PG and PG/VG (30-60 min) inhibited glucose uptake and mitochondrial ATP synthesis. PG/VG inhibited glycolysis. PG/VG and mannitol reduced cell volume and height of air-liquid interface cultures. Mannitol, but not PG/VG, increased phosphorylation of p38 MAPK. PG/VG reduced transepithelial electrical resistance, which was associated with increased transepithelial solute permeability. PG/VG decreased fluorescence recovery after photobleaching of green fluorescent protein-linked glucose transporters GLUT1 and GLUT10, indicating that glucose transport function was compromised. Puffing PG/VG vapor onto the apical surface of primary HBECs for 10 min to mimic the effect of e-cigarette smoking also reduced glucose transport. In conclusion, short-term exposure to PG/VG, key components of e-cigarettes, decreased glucose transport and metabolism in airway cells. We propose that this was a result of PG/VG reduced cell volume and membrane fluidity, with further consequences on epithelial barrier function. Taking these results together, we suggest these factors contribute to reduced defensive properties of the epithelium. We propose that repeated/chronic exposure to these agents are likely to contribute to airway damage in e-cigarette users.

Neutrophil elastase and endogenous inhibitors in Behçet's disease saliva.

Behçet's disease (BD) is a vasculitis of unknown aetiology typified by chronic recurrent oral ulcers and systemic inflammatory manifestations. Neutrophils, and specifically their protease neutrophil elastase (NE), have been implicated in its pathology. Although NE is an effective anti-microbial, excessive NE can damage host tissue. Recurrent oral ulceration is a primary BD symptom, therefore we hypothesized that excessive neutrophil infiltration evidenced by increased NE and a reduction in specific endogenous inhibitors, secretory leucocyte protease inhibitor (SLPI) and alpha1-anti-trypsin (α1AT) contributes to BD mucosal instability. NE, SLPI and α1AT were quantified in saliva from BD patients with active oral ulcers (BDa) and quiet without ulcers (BDq), recurrent aphthous stomatitis (RASa; RASq) and healthy controls (HC). Although BDq saliva had marginally higher median NE levels (1112 ng/ml) compared to both RASq (1043 ng/ml) and HC (999 ng/ml), SLPI was significantly reduced in BDq (P < 0·01). Despite decreased SLPI protein, mRNA expression was significantly increased in BDq buccal epithelial swabs compared to RASq and HC (P < 0·05, P < 0·001). NE remained enzymatically active, although α1AT levels were at least eight times higher than SLPI in all groups, suggesting that α1AT does not have a primary role in counteracting NE in saliva. Furthermore, NE levels in BDa patients medicated with both azathioprine (AZA) and colchicine (COLC) were significantly lower than those on COLC (P = 0·0008) or neither (P = 0·02), indicating that combining AZA + COLC may help to regulate excessive NE during ulceration. This study showed that enzymatically active NE coupled with reduced SLPI in BD saliva may contribute to recurrent oral ulcerations.

Oral Health Inequalities in 0-17-year-old Children Referred for Dental Extractions Under General Anaesthesia in Wolverhampton, 2013-2017.

OBJECTIVE: Describe the inequalities in oral health in children treated in a hospital located in a deprived urban area in the UK. RESEARCH DESIGN: Case-note review of 1911 0-17-year-olds who underwent dental extractions under a general anaesthetic (DGA). MAIN OUTCOME MEASURES: Associations between Age, Ethnicity, Year-of-Treatment and Index of Multiple Deprivation (IMD) with the number of teeth extracted. Analysis used multilevel modelling assuming a Poisson distribution. RESULTS: Mean number of teeth extracted was higher in the youngest children treated aged 0-5 years (relative risk coefficient, (RR=exp(ß)=1.39; 95% CI 1.24 to 1.56) compared to those aged 6-17 years and in 'Other Whites' (predominantly immigrants from Eastern Europe) (RR=exp(ß)=1.34; 95% CI 1.25 to 1.43), 'South Asians' (RR=exp(ß)=1.15; 95% CI 1.08 to 1.23) but fewer in the 'Black' ethnic group (RR=exp(ß)=0.85; 95% CI 0.76 to 0.95). DGA increased during the study with more teeth extracted in 2015, 2016 and 2017 (RR=exp(ß)=1.12, 95% CI 1.22, 1.25) and with a negative gradient in the rate of DGA's (per decile) in children from the most deprived to most affluent locations (RR=exp(ß)=0.98; 95% CI 0.97 to 0.99). CONCLUSIONS: Significant oral health inequalities exist in children from a deprived urban area in the UK. A preventive approach to children's oral health is needed to reduce such inequalities, including public health and healthcare agencies to informing parents of children whose first language is not English about dental caries.

Sub-ppm level high energy resolution fluorescence detected X-ray absorption spectroscopy of selenium in articular cartilage.

The speciation of highly-diluted elements by X-ray absorption spectroscopy in a diverse range of materials is extremely challenging, especially in biological matrices such as articular cartilage. Here we show that using a high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) technique coupled to an array of crystal analyzers, selenium speciation down to 400 ppb (µg kg-1) within articular cartilage can be demonstrated. This is a major advance in the speciation of highly-diluted elements through X-ray absorption spectroscopy and opens new possibilities to study the metabolic role of selenium and other elements in biological samples.

Inflammation and oxidative stress transcription profiles due to in vitro supply of methionine with or without choline in unstimulated blood polymorphonuclear leukocytes from lactating Holstein cows.

Neutrophils are the most important polymorphonuclear leukocytes (PMNL), representing the front-line defense involved in pathogen clearance upon invasion. As such, they play a pivotal role in immune and inflammatory responses. Isolated PMNL from 5 mid-lactating Holstein dairy cows were used to evaluate the in vitro effect of methionine (Met) and choline (Chol) supplementation on mRNA expression of genes related to the Met cycle and innate immunity. The target genes are associated with the Met cycle, cell signaling, inflammation, antimicrobial and killing mechanisms, and pathogen recognition. Treatments were allocated in a 3 × 3 factorial arrangement, including 3 Lys-to-Met ratios (L:M, 3.6:1, 2.9:1, or 2.4:1) and 3 levels of supplemental Chol (0, 400, or 800 µg/mL). Three replicates per treatment group were incubated for 2 h at 37°C and 5% atmospheric CO2. Both betaine-homocysteine S-methyltransferase and choline dehydrogenase were undetectable, indicating that PMNL (at least in vitro) cannot generate Met from Chol through the betaine pathway. The PMNL incubated without Chol experienced a specific state of inflammatory mediation [greater interleukin-1ß (IL1B), myeloperoxidase (MPO), IL10, and IL6] and oxidative stress [greater cysteine sulfinic acid decarboxylase (CSAD), cystathionine gamma-lyase (CTH), glutathione reductase (GSR), and glutathione synthase (GSS)]. However, data from the interaction L:M × Chol indicated that this negative state could be overcome by supplementing additional Met. This was reflected in the upregulation of methionine synthase (MTR) and toll-like receptor 2 (TLR2); that is, pathogen detection ability. At the lowest level of supplemental Chol, Met downregulated GSS, GSR, IL1B, and IL6, suggesting it could reduce cellular inflammation and enhance antioxidant status. At 400 µg/mL Chol, supplemental Met upregulated PMNL recognition capacity [higher TLR4 and L-selectin (SELL)]. Overall, enhancing the supply of methyl donors to isolated unstimulated PMNL from mid-lactating dairy cows leads to a low level of PMNL activation and upregulates a cytoprotective mechanism against oxidative stress. Enhancing the supply of Met coupled with adequate Chol levels enhances the gene expression of PMNL pathogen-recognition mechanism. These data suggest that Chol supply to PMNL exposed to low levels of Met effectively downregulated the entire repertoire of innate inflammatory-responsive genes. Thus, Met availability in PMNL during an inflammatory challenge may be sufficient for mounting an appropriate biologic response.

Alpha lipoic acid supplementation ameliorates the wrath of simulated tropical heat and humidity stress in male Murrah buffaloes.

A supplement which ameliorates temperature-humidity menace in food producing livestock is a prerequisite to develop climate smart agricultural packages. A study was conducted to investigate the heat stress ameliorative efficacy of alpha lipoic acid (ALA) in male Murrah water buffaloes (Bubalus bubalis). Eighteen animals (293.61 ± 4.66Kg Bwt) were randomly allocated into three groups (n = 6); NHSC (non-heat-stressed control), HS (heat-stressed) and HSLA (heat-stressed-supplemented with ALA@32 mg/kg Bwt orally) based on the temperature humidity index (THI) and ALA supplementation. HS and HSLA were exposed to simulated heat challenge in a climatically controlled chamber (40 °C) for 21 consecutive days, 6 h daily. Physiological responses viz. Respiration rate (RR), Pulse rate (PR) and Rectal temperature (RT) were recorded daily before and after heat exposure. Blood samples were collected at the end of heat exposure on days 1, 6, 11, 16, and 21 and on day 28 (7th day post exposure which is considered as recovery) for peripheral blood mononuclear cells (PBMCs) separation, followed by RNA and Protein extraction for Real time quantitative PCR and Western blot analysis respectively, of heat shock proteins (HSPs). Two-way repeated measure ANOVA was performed between groups at different experimental periods. RR (post exposure) in HS and HSLA was significantly higher (P < 0.05) than NHSC from day 1 onwards but HSLA varied significantly from the HS 8th day onwards. Post exposure RT and PR in both HS and HSLA varied (P < 0.05) from NHSC throughout the study; but between HS and HSLA, RT significantly varied on initial 2 days and last 6 days (from days 16 to 21). HSP70 mRNA expression significantly up regulated in high THI groups with respect to the low THI group throughout the experimental period. During chronic stress (days 16 and 21) HSP70 significantly (P < 0.05) increased in HS but not in HSLA (P > 0.05) with respect to NHSC. ALA supplementation up-regulates and sustains (P < 0.05) the expression of HSP90 in HSLA in comparison to the HS and NHSC. HSP105 expression was significantly up-regulated (P < 0.05) in HS on days 16 and 21 (during long-term exposure) but only on day 21 (P < 0.05) in HSLA. HSP70, HSP90, and HSP105 protein expression dynamics were akin to the mRNA transcript data between the study groups. In conclusion, supplementing ALA ameliorates the deleterious effect of heat stress as reflected by improved physiological and cellular responses. ALA supplementation improved cellular antioxidant status and sustained otherwise easily decaying heat shock responses which concertedly hasten the baton change from a limited window of thermo tolerance to long run acclimatization.

Cryopreservation of Axillary Buds of Cannabis sativa L. by V-Cryoplate Droplet-Vitrification: The Critical Role of Sucrose Preculture.

BACKGROUND: Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of important germplasm. Recent use of the cryo-plate system has proven beneficial in further simplifying the cryopreservation protocols. OBJECTIVE: Developing an efficient protocol for the cryopreservation of axillary buds of Cannabis sativa elite cultivars (MX and V1-20) by the V-cryoplate droplet-vitrification technique. MATERIALS AND METHODS: Stem segments (~5 cm in length) with mature axillary buds collected from indoor-grown plants were surface sterilized and then either precultured on MS basal medium with 0.1 M sucrose (1st step preculture) for 72 h or non-precultured. All mature axillary buds (~1 mm) were aseptically excised from stem segments and precultured for an additional 48 h on MS basal medium with sucrose (0.3 M) and 5% DMSO prior to cryopreservation (2nd step preculture). Biomass samples of fully mature mother plants and regrown cryopreserved plants were analyzed for Δ9-THC and CBD content using gas chromatography-flame ionization detector (GC/FID). RESULTS: The survival and regrowth rates of cryopreserved axillary buds of cultivar MX following this two-step preculture were 45% and 42% respectively, while those of cultivar V1-20 were 47% and 44% respectively. A direct preculture of axillary buds (2nd step preculture) on high sucrose (0.3M sucrose) significantly decreased both the survival and regrowth levels of axillary buds of cultivar MX (5% and 3% respectively) as well as those of cultivar V1-20 (20% and 17% respectively). Δ9-THC and CBD content of mother plants and regrown cryopreserved plants were found to be highly comparable to each other. CONCLUSION: The resulting plants after cryopreservation appeared normal without any callus formation or morphogenetic variation. On maturity, mother plants and re-grown cryopreserved plants were comparable in terms of Δ9-THC and CBD content. This report provides an efficient protocol for cryopreservation of axillary buds of Cannabis sativa cultivars which may be applicable to other important cultivars, plant parts and other related medicinal plants.

Progenitor cells in auricular cartilage demonstrate cartilage-forming capacity in 3D hydrogel culture.

Paramount for the generation of auricular structures of clinically-relevant size is the acquisition of a large number of cells maintaining an elastic cartilage phenotype, which is the key in producing a tissue capable of withstanding forces subjected to the auricle. Current regenerative medicine strategies utilize chondrocytes from various locations or mesenchymal stromal cells (MSCs). However, the quality of neo-tissues resulting from these cell types is inadequate due to inefficient chondrogenic differentiation and endochondral ossification, respectively. Recently, a subpopulation of stem/progenitor cells has been identified within the auricular cartilage tissue, with similarities to MSCs in terms of proliferative capacity and cell surface biomarkers, but their potential for tissue engineering has not yet been explored. This study compared the in vitro cartilage-forming ability of equine auricular cartilage progenitor cells (AuCPCs), bone marrow-derived MSCs and auricular chondrocytes in gelatin methacryloyl (gelMA)-based hydrogels over a period of 56 d, by assessing their ability to undergo chondrogenic differentiation. Neocartilage formation was assessed through gene expression profiling, compression testing, biochemical composition and histology. Similar to MSCs and chondrocytes, AuCPCs displayed a marked ability to generate cartilaginous matrix, although, under the applied culture conditions, MSCs outperformed both cartilage-derived cell types in terms of matrix production and mechanical properties. AuCPCs demonstrated upregulated mRNA expression of elastin, low expression of collagen type X and similar levels of proteoglycan production and mechanical properties as compared to chondrocytes. These results underscored the AuCPCs' tissue-specific differentiation potential, making them an interesting cell source for the next generation of elastic cartilage tissue-engineered constructs.

Dynamics of antibiotic resistance with special reference to Shiga toxin-producing Escherichia coli infections.

The discovery of antibiotics was paralleled by the evolution of antibiotic resistance which is probably the best example of contemporary evolution in action. The selection pressure, imposed by indiscriminate use of antibiotics, has changed the scale, mode and tempo of antibiotic resistance evolution. The presence of multidrug resistance, wide range of adaptability features and the infectivity make antibiotic resistance of Shiga toxin-producing Escherichia coli (STEC) more dangerous. The characterization, prevalence and the virulence factors of STEC have been profusely reported, whereas, the antibiotic resistance has been largely ignored because the antibiotic use in STEC infections is controversial. Thus, the current review has focussed on the source, evolution, persistence, mechanism, dissemination and control of antibiotic resistance viz-a-viz the STEC infections. The resistance development occurs by the inactivation of antibiotics, regulating the membrane permeability, modification of natural antibiotic targets or the use of efflux pumps against antibiotics. And, the dissemination of resistance genes occurs vertically by DNA replication and horizontally by conjugation, transduction and transformation. The prevention of development and dissemination of antibiotic resistance needs international public health bodies to rationalize the antibiotic use, prevent the flux of antibiotics into the environment, develop the rapid diagnostics tests, undertake proper surveillance of antibiotic resistance, promote the research on antibiotic resistance prevention, promote the research and development of novel alternative antibiotics, and encourage the widespread social awareness campaigns against the inappropriate antibiotic usage.

Oligomeric and fibrillar amyloid beta 42 induce platelet aggregation partially through GPVI.

The effects of the Alzheimer's disease (AD)-associated Amyloid-ß (Aß) peptides on platelet aggregation have been previously assessed, but most of these studies focused on Aß40 species. It also remains to be determined which distinct forms of Aß peptides exert differential effects on platelets. In AD, oligomeric Aß42 species is widely thought to be a major contributor to the disease pathogenesis. We, therefore, examine the ability of oligomeric and fibrillary Aß42 to affect platelet aggregation. We show that both forms of Aß42 induced significant platelet aggregation and that it is a novel ligand for the platelet receptor GPVI. Furthermore, a novel binding peptide that reduces the formation of soluble Aß42 oligomers was effective at preventing Aß42-dependent platelet aggregation. These results support a role for Aß42 oligomers in platelet hyperactivity.

Successful stepdown treatment of pulmonary histoplasmosis with thrice-weekly liposomal amphotericin B in a hospital-associated, outpatient infusion centre: A case report.

WHAT IS KNOWN AND OBJECTIVE: Amphotericin is the preferred treatment for pulmonary histoplasmosis during pregnancy. The long half-life of amphotericin supports less than daily administration. CASE SUMMARY: A 28-year-old pregnant woman diagnosed with recurrent pulmonary histoplasmosis was initiated on liposomal amphotericin 250 mg (4 mg/kg) intravenously daily. After 2 weeks, the patient was discharged and successfully received 250 mg thrice weekly at a hospital-associated outpatient infusion centre. After 6 weeks of outpatient treatment, a chest X-ray demonstrated no remaining disease and therapy was discontinued. WHAT IS NEW AND CONCLUSION: Administration of thrice-weekly liposomal amphotericin in a hospital-associated, outpatient infusion centre may be a promising option for stepdown treatment in patients unable to take itraconazole.