Validation of a Lysis Buffer Containing 4 M Guanidinium Thiocyanate (GITC)/ Triton X-100 for Extraction of SARS-CoV-2 RNA for COVID-19 Testing: Comparison of Formulated Lysis Buffers Containing 4 to 6 M GITC, Roche External Lysis Buffer and Qiagen RTL Lysis Buffer

The COVID-19 pandemic has resulted in increased need for diagnostic testing using reverse transcriptase real-time PCR (RT-PCR). An exponential increase in demand has resulted in a shortage of numerous reagents in particular those associated with the lysis buffer required to extract the viral RNA. Herein, we describe a rapid collective effort by hospital laboratory scientists, academic researchers and the biopharma industry to generate a validated lysis buffer. We have formulated a 4M Guanidinium thiocyanate (GITC)/ Triton X-100 Lysis buffer which provides comparable results with the recommended reagents. This buffer will ease the burden on hospital labs in their heroic efforts diagnose a large population of patients

Down-Modulation of Cockroach (CR) Allergen-specific Th2 Cell Responses Following Subcutaneous German Cockroach Allergen Immunotherapy (SCIT)

Rationale: The responses of T cells to subcutaneous allergen immunotherapy (SCIT) are not fully elucidated. We conducted a functional immunological evaluation of cockroach (CR) allergen-specific CD4+ T cell reactivity in the double-blinded, placebo-controlled, multi-center CRITICAL study. Methods: Participants (8-17 years of age) with mild to moderate, well-controlled asthma received 12 months of maintenance dosing of CR SCIT (n=20) or placebo (n=26). Peripheral blood mononuclear cells (PBMC) were isolated prior to, and after 12 months of therapy. CD4+ T cell responses at baseline and after treatment were assessed using overlapping peptide pools derived from 11 well-defined CR allergens and intracellular cytokine staining for IL-4, IFNg, and IL-10 production. T cell responses were further evaluated in terms of magnitude, cytokine polarization, and allergen immunodominance. Results: Significant down-modulation of the total magnitude of CD4+ T cell responses was observed with SCIT but not placebo, with a significant change between groups (-4.46±0.82 vs. −1.81±0.72, respectively, p = 0.020). Responses were driven by a decrease in IL-4 (-4.87±0.86 vs. −1.09±0.75, p = 0.002) with unaltered IFNg and IL-10 production, reflecting a shift towards a Th1 polarization profile (1.35±0.58 vs. −0.37±0.50, in SCIT and placebo respectively, p = 0.031). The largest effects were observed against the allergens Bla g 5 and Bla g 9, which are dominantly recognized, suggesting that dominant responses are susceptible to modulation. Conclusions: Our results demonstrate a significant down-regulation of CR-specific Th2 cell responses in urban children with asthma who received SCIT, compared with those who received placebo

The Effect of Subcutaneous German Cockroach Immunotherapy (SCIT) on Nasal Allergen Challenge (NAC) and Cockroach-specific Antibody Responses Among Urban Children and Adolescents

Rationale: Cockroach allergy contributes to asthma and rhinitis morbidity among many urban children. Treatment with cockroach SCIT could be beneficial. Methods: 8-17 year-old children with mild-moderate asthma from 11 urban sites participated in a randomized double-blind placebo-controlled SCIT trial using non-standardized, glycerinated German cockroach extract. Positive cockroach skin tests, cockroach-specific IgE, and nasal challenge response with total nasal symptom scores (TNSS) ≥6 or maximal sneeze scores of 3 during a graded NAC were required for enrollment. Following dose escalation, 0.4 ml of undiluted extract was targeted for maintenance dosing (∼7 mcg Bla g2/dose). The primary endpoint was change in NAC-induced mean TNSS from baseline to one year post randomization. Changes in cockroach-specific IgE (CRsIgE) and IgG4 (CRsIgG4) were also analyzed. Results: Mean TNSS did not significantly change from baseline in either group (placebo n=29, SCIT n=28). There was no significant difference in the change in mean TNSS between placebo and SCIT [−0.79±0.35 vs. −1.02±0.37, respectively, difference=0.2(−1.15, 0.70), p=0.63]. Baseline CRsIgE and CRsIgG4 didn’t differ between groups. Mean CRsIgE decreased in both groups following treatment: 3.6 to 2.3 kU/L (0.64 fold change), p=0.015 and 8.3 to 4.2 kU/L (0.51 fold change), p\u3c0.001 in placebo and SCIT respectively, but did not differ between groups [p=0.33]. Significant increases in CRsIgG4 post-treatment were observed among SCIT recipients only: 0.07 to 12.3 mg/L (176 fold change), p\u3c0.001. Conclusions: Cockroach SCIT increased CRsIgG4 levels but did not significantly alter NAC-induced TNSS responses. The extent to which NAC in these children may reflect clinical efficacy for rhinitis or asthma is uncertain

Heterogeneity of magnitude, allergen immunodominance, and cytokine polarization of cockroach allergen-specific T cell responses in allergic sensitized children.

Background: Characterization of allergic responses to cockroach (CR), a common aeroallergen associated with asthma, has focused mainly on IgE reactivity, but little is known about T cell responses, particularly in children. We conducted a functional evaluation of CR allergen-specific T cell reactivity in a cohort of CR allergic children with asthma. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 71 children, with mild-to-moderate asthma who were enrolled in a CR immunotherapy (IT) clinical trial, prior to treatment initiation. PBMC were stimulated with peptide pools derived from 11 CR allergens, and CD4+ T cell responses assessed by intracellular cytokine staining. Results: Highly heterogeneous responses in T cell reactivity were observed among participants, both in terms of the magnitude of cytokine response and allergen immunodominance. Reactivity against Bla g 9 and Bla g 5 was most frequent. The phenotype of the T cell response was dominated by IL-4 production and a Th2 polarized profile in 54.9% of participants, but IFNγ production and Th1 polarization was observed in 25.3% of the participants. The numbers of regulatory CD4+ T cells were also highly variable and the magnitude of effector responses and Th2 polarization were positively correlated with serum IgE levels specific to a clinical CR extract. Conclusions: Our results demonstrate that in children with mild-to-moderate asthma, CR-specific T cell responses display a wide range of magnitude, allergen dominance, and polarization. These results will enable examination of whether any of the variables measured are affected by IT and/or are predictive of clinical outcomes

Development of Technologies for Separation and Functional Improvement of Individual Milk Protein Fractions

End of Project ReportMilk proteins can be hydrolysed (i.e. fragmented) using proteolytic enzymes to give enhanced functional and nutritional properties. There is an increasing demand for hydrolysed protein ingredients with specific properties for nutrition of individuals with specialised dietary requirements including infants, the critically ill, the immuno-compromised and athletes. Such hydrolysed proteins can be specifically designed to provide distinctive tailor-made solutions to meet customer needs in these areas. This project explored the technologies for the production of two types of hydrolysates i.e. acid-soluble and glutamine-rich. Acid-soluble protein hydrolysates have potential in the fortification of acidic beverages, including soft drinks. Glutamine-rich hydrolysates are suggested as an optimal glutamine source for administration during periods of stress, such as recovery from strenuous exercise, or from surgery. Casein was selected as the protein for development of acid-soluble product and cereal protein for the glutamine-rich product. The main conclusions were as follows: A number of protein hydrolysate products with value added properties and the processes required for their manufacture have been developed and are available for uptake by the food industry. Laboratory investigations identified conditions for the generation of two casein hydrolysates with desirable functional properties. Scale-up conditions for the manufacture of these hydrolysates in the pilot plant were successfully developed. Both hydrolystates were 100% soluble at pH 4.6, exhibited clarity in solution at low pH in clear soft drinks and in caramelised beverages and were stable in solution over a wide temperature range (from 4 to 30ºC) for extended periods. Solutions containing these hydrolysates exhibited no foaming properties and had acceptable sensory properties, being considered as weakly bitter compared to unsupplemented solutions. These performance characteristics make the acid-soluble hydrolysates useful supplements for caramelised beverages, such as colas, and clear soft drinks. Six glutamine-enriched peptide products were produced at laboratory scale using two commercially available enzyme preparations. These products had desirable characteristics such as increased levels of peptide bound glutamine, low free amino acid and free pyroglutamate levels. Pilot plant processes were developed for manufacture of the two glutamine-rich hydrolysates with most suitable compositional properties and these were fully characterised chemically. The manufacturing process was modified to enable industrial scale batches (5,000 litres) to be produced.Department of Agriculture, Food and the Marin

Human skin microbiota is a rich source of bacteriocin-producing staphylococci that kill human pathogens

The demand for novel antimicrobial therapies due to the threat posed by antimicrobial resistance has resulted in a growing interest in the protective role of our skin bacteria and the importance of competition among bacteria on the skin. A survey of the cultivable bacteria on human skin was undertaken to identify the capacity of the skin microbiota to produce bacteriocins with activity against skin pathogens. Twenty-one bacteriocins produced by bacteria isolated from seven sites on the human body of each subject exhibited inhibition spectra ranging from broad to narrow range, inhibiting many Gram-positive bacteria, including opportunistic skin pathogens such as Propionibacterium acnes (recently renamed Cutibacterium acnes), Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus (MRSA). Sequencing indicated that the antimicrobial-producing isolates were predominately species/strains of the Staphylococcus genus. Colony mass spectrometry revealed peptide masses that do not correspond to known bacteriocins. In an era where antibiotic resistance is of major concern, the inhibitory effect of novel bacteriocins from the bacteria of skin origin demonstrates the antimicrobial potential that could be harnessed from within the human skin microbiota

Comparison of the principal proteins in bovine, caprine, buffalo, equine and camel milk

Proteomic analysis of bovine, caprine, buffalo, equine and camel milk highlighted significant interspecies differences. Camel milk was found to be devoid of β-lactoglobulin, whereas β-lactoglobulin was the major whey protein in bovine, buffalo, caprine, and equine milk. Five different isoforms of κ-casein were found in camel milk, analogous to the micro-heterogeneity observed for bovine κ-casein. Several spots observed in 2D-electrophoretograms of milk of all species could tentatively be identified as polypeptides arising from the enzymatic hydrolysis of caseins. The understanding gained from the proteomic comparison of these milks may be of relevance both in terms of identifying sources of hypoallergenic alternatives to bovine milk and detection of adulteration of milk samples and products

Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

It is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.This work was supported by the Irish Government under the National Development Plan; by the Irish Research Council for Science Engineering and Technology (IRCSET); by Enterprise Ireland; and by Science Foundation Ireland (SFI), through the Alimentary Pharmabiotic Centre (APC) at University College Cork, Ireland, which is supported by the SFI-funded Centre for Science, Engineering and Technology (SFI-CSET) and provided P.D.C., C.H. and R.P.R. with SFI Principal Investigator funding

Commentary: Defining Disability in Health Care Education

According to the 2008 American Community Survey, about 12% of the population of the United States is living with one or more disabling conditions. These conditions impact lives in a variety of ways, some with more or less direct impact on an individual\u27s health and access to health care services. Although it has been 20 years since the passage of the Americans with Disabilities Act, people with disabilities still experience health disparities and a lack of access to the appropriate care. This commentary is part of a collection of articles that describe various aspects of incorporating content into the medical school curriculum to enhance the preparation of today\u27s medical students to meet the needs of people with disabilities. The authors briefly describe the scope of the problem and define the population of people with disabilities that constitutes the focus of the work described in the other articles in this collection

Control of Cheese Microflora using Bacteriocins.

End of Project ReportBacteriocins are proteins, produced by some bacteria which are capable of inhibiting other bacteria. The overall aim of this project was the development and exploitation of bacteriocins such as Lacticin 3147 (produced by a food-grade microorganism), as biological tools to control the microflora of foods. Lacticin 3147-producing strains were evaluated for their ability to improve the microbial quality of a variety of dairy products and in particular, Cheddar cheese. The manipulation of cheese flora using bacteriocins should offer manufacturers greater control in the consistency and quality of the final product, in addition to improving its safety. In concert with these studies, Lacticin 3147 was studied in detail at the molecular level resulting in its biochemical and genetic analysis. These studies have demonstrated the complexity and uniqueness of this potent antimicrobial.Department of Agriculture, Food and the Marin