Phylogenetic analysis of full-length, early infection, hepatitis C virus genomes among people with intravenous drug use: the InC3 Study.

Cross-continental phylogenetic analysis is important to understand subtle molecular differences of currently circulating hepatitis C virus (HCV) subtypes. Existence of such differences can be crucial in pursuing a universal hepatitis C vaccine. We characterized molecular epidemiology of early HCV infections identified across nine cohorts [North America (n=4), Australia (n=4) and Europe (n=1)] in the International Collaborative of Incident HIV and Hepatitis C in Injecting Cohorts (InC3 ). One hundred and ninety-two full-length HCV genomes were amplified from plasma of incident infections and subjected to next generation sequencing to establish the largest cross-continental, full-length acute HCV genomic data set available to date. Genomes from the most common subtypes (1a: n=94, 2b: n=15 and 3a: n=68) were used in phylogenetic analysis. Using full genome trees, 78 sequences (44%) were found to lie within 29 phylogenetic clusters/pairs defined on the basis of molecular similarity of consensus sequences. Of these, 26 each had exclusively Australian or North American sequences indicating a strong geographical bias for molecular similarity. On further analysis of behavioural and demographic associations, binary logistic regression analysis showed that older age and non-Caucasian ethnicity were significantly associated with clustering. HCV probably evolves in micro-epidemics within geographically isolated communities.

Anti-inflammatory cytokines, pro-fibrogenic chemokines and persistence of acute HCV infection.

Chemokines and cytokines play a vital role in directing and regulating immune responses to viral infections. Persistent hepatitis C virus (HCV) infection is characterized by the loss of anti-HCV cellular immune responses, while control of HCV infection is associated with maintenance of anti-HCV cellular immune responses. To determine whether plasma concentrations of 19 chemokines and cytokines controlling T-cell trafficking and function differed based on infection outcome, we compared them in at-risk subjects followed prospectively for HCV infection. Levels were compared over time in subjects who controlled HCV infection (Clearance) and subjects who developed persistent HCV infection (Persistence) at two time points during acute infection: (i) first viraemic sample (initial viraemia) and (ii) last viraemic sample in Clearance subjects and time-matched samples in Persistence subjects. At initial viraemia, increased pro-inflammatory tumour necrosis factor α (TNFα) plasma concentrations were observed in the Clearance group, while the plasma levels of anti-inflammatory interleukin (IL)-2, IL-10 and IL-13 were higher in the Persistence group. IL-13 was positively correlated with IL-2 and IL-10 at initial viraemia in the Persistence group. At the time of last viraemia, plasma levels of eotaxin, macrophage chemoattractant protein-4 (MCP-4), IL-5 and IL-10 were higher in the Persistence group and IL-10 and IL-5 levels were positively correlated. Collectively, these results suggest that the development of persistent infection is associated with an anti-inflammatory and pro-fibrogenic chemokine and cytokine profile that is evident at the onset of infection and maintained throughout acute infection.

Evaluation of additional cooking procedures to achieve lethality microbiological performance standards for large, intact meat products.

The U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) has a specific lethality performance standard for ready-to-eat products. To assist meat processing establishments in meeting the performance standard, USDA-FSIS developed Appendix A, which provides guidelines for cooking temperatures, times, and relative humidity. This project determined whether the USDA-FSIS performance standards for lethality were met when using parameters other than those identified in Appendix A to cook large hams and beef inside rounds. The effects of alternative lethality parameters on the reduction of Salmonella Typhimurium and coliforms and on the toxin production of Staphylococcus aureus were evaluated. Large (9- to 12-kg) cured bone-in hams (n = 80) and large (8- to 13-kg) uncured beef inside rounds (n = 80) were used in this study. The products were subjected to 1 of 10 treatments defined by combinations of final internal product temperatures (48.9, 54.4, 60.0, 65.6, or 71.1°C) and batch oven relative humidities (50 or 90 % ). For all treatments, at least a 6.5-log reduction in Salmonella Typhimurium was achieved. The coliform counts were also substantially reduced for both hams and rounds. Across all treatments for both products, S. aureus toxin production was not detected. The relative humidity did not alter the lethality effectiveness for any of the treatments. The final internal temperatures and relative humidity combinations used in this project achieved the lethality performance standard established by USDA-FSIS for fully cooked, ready-to-eat products.

Inclusion of blended lipid solutions as functional ingredients to alter the fatty acid profile of beef patties.

UNLABELLED: Beef patties formulated to contain beef fat, plant oil, and a rosemary extract to increase unsaturated fatty acid content and maintain desirable sensory attributes were compared to control beef patties formulated without plant oils. Treatment patties were formulated to a fat content of 10% or 20% by combining beef trimmings (6% fat) with 4% or 14% addition of a lipid blend. Blends contained 57% beef tallow, 0.3% rosemary extract, and 43% of high oleic safflower oil (SO), olive oil (OO), or corn oil (CO). Lipid oxidation, as measured by TBA values, of treatment patties were similar to control patties after 0 and 3 d of refrigerated (2 °C) storage and up to 56 d of frozen (-10 °C) storage. Cooked lipid blend patties having a fat content of 10% or 20% were similar to or higher than control patties for juiciness and were no different for other sensory attributes evaluated. At fat levels of 10% or 20%, oleic acid (18: 1) in cooked SO patties (46.1% and 50.3%, respectively) and OO patties (43.8% and 48.1%, respectively) was higher than the control (37.3% and 37.6%, respectively). Unsaturated to saturated fatty acid ratios at the 10% or 20% fat levels were higher in SO (1.37 and 1.60, respectively) and CO (1.40 and 1.48, respectively) patties than the control (0.97 and 0.94, respectively). Beef patties manufactured with varying lipid blends increased unsaturated fatty acid content and were similar in physical characteristics and sensory attributes of all beef patties formulated without lipid blends. PRACTICAL APPLICATION: The development of healthier beef products that will be more appealing to consumers has long been an industry goal. The authors believe that lipid blends such as the one used in this study could be used to create such products, not only in the form of beef patties, but any number of processed meat products. Because the materials and equipment used to create the lipid blends in this study are widely available, their incorporation into meat products would represent a small capital investment. This is an important factor in bringing a reasonably priced, healthier product to consumers.

Identifying muscle and processing combinations suitable for use as beef for fajitas.

Four different treatments-control, papain, blade tenderization, and papain+blade tenderization-were applied to sixty USDA Choice M. diaphragma pars costalis, M. transversus abdominis, M. obliquus abdominis internus, M. rhomboideus, M. trapezius, M. latissimus, and M. serratus ventralis. Trained (n=6) and consumer (n=81) panelists scored papain samples higher for most sensory traits. Treatment tended not to affect the palatability scores of the M. diaphragma pars costalis and M. serratus ventralis, which tended to receive higher scores in comparison to the other muscles. Consumers were willing to purchase the M. latissimus and M. serratus ventralis treated with papain+blade tenderization and papain, respectively, and these muscles performed well enough to be considered as alternatives in the beef fajita market.

In-home consumer evaluations of individual muscles from beef rounds subjected to tenderization treatments.

In-home evaluations of the M. vastus lateralis, M. rectus femoris, M. semimembranosus, and M. adductor (n=266) muscles that were either blade tenderized, enhanced with a salt and phosphate solution, or served as a control (no tenderization or enhancement treatment) were conducted. Consumers (n=261) cooked these steaks and were asked to document cooking method and degree of doneness, and provide palatability ratings for each steak. Enhancing round muscles with a salt and phosphate solution improved most palatability traits compared to blade tenderized or control steaks. For M. semimembranosus and M. vastus lateralis, the enhanced steaks received higher (P<0.05) ratings for all palatability traits. Cooking method and degree of doneness had little influence on consumer palatability ratings, and where differences occurred, they were muscle specific. This may allow limited recommendations for the most appropriate cooking method and degree of doneness for specific beef round muscles.

Evaluation of low-fat sausage containing desinewed lamb and konjac gel.

Ground (GR) or desinewed (DS) lamb trimmings were combined with a konjac flour (KF) gel (0%, 10% or 20%) to produce a low-fat (8%) cured sausage. Physiochemical, sensory and shelf-life analyses were performed to determine the efficacy of the fat mimetic. Desinewing reduced collagen content (2.3 mg/g tissue) and cook yield (0.6%) when compared to GR trimmings. Grinding increased aerobic plate counts (APCs) ∼0.4log(10)/cm(2) more than DS, but APCs were not affected by inclusion of KF. Sausages with 20% KF lowered cook yield ∼1% and slightly reduced sensory panel and texture profile analysis texture values. KF gel incorporated at 10% had similar properties to a control low-fat desinewed lamb sausage, while KF at 20% could reduce "toughening" in low-fat sausage products. Use of konjac gel as a fat mimetic could reduce total caloric energy by replacing a portion of the meat in a sausage formulation.

Experimental use of a gas sensor-based instrument for differentiation of Escherichia coli O157:H7 from non-O157:H7 Escherichia coli field isolates.

The rapid and economical detection of human pathogens in animal and food production systems would enhance food safety efforts. An instrument based on gas sensors coupled with an artificial neural network (ANN) was developed for the detection of and differentiation between laboratory isolates of Escherichia coli O157:H7 and non-O157:H7 E. coli. The purpose of this study was to use field isolates of E. coli to further evaluate the sensor system. This gas sensor-based, computer-controlled detection system was used to monitor gas emissions from 12 isolates of E. coli O157:H7 and 8 non-O157:H7 E. coli isolates. A standard concentration of each isolate was grown in 10 ml of nutrient broth at 37 degrees C for 16 h, and gas sampling was carried out every 5 min. Readings were continuously plotted to generate gas signatures. A back-propagation ANN algorithm was used to interpret the gas patterns. By analysis of the response of the ANN, the sensitivity and specificity of the instrument were calculated. Detectable differences between the gas signatures of the E. coli O157:H7 isolates and the non-O157:H7 isolates were observed. The instruments degree of sensitivity was high for E. coli O157:H7 isolates, but a lower degree of accuracy was observed for non-O157:H7 isolates because of increased strain variation. The sensitivity of the detection system was improved by the normalization of the data generated from the gas sensors. Because of its ability to detect differences in gas patterns, this instrument has a broad range of potential food safety applications.

Evaluation of Duroc- vs. Pietrain-sired pigs for carcass and meat quality measures.

Crossbred progeny sired by either Duroc or Pietrain boars, normal for the ryanodine receptor gene, were evaluated for carcass composition and meat quality. Boars from each breed were mated to Yorkshire or F1 Yorkshire-Landrace females. A total of 162 off-spring was evaluated for carcass and meat quality traits at a common age (approximately 26 wk of age). Duroc-sired progeny had heavier (108.0 vs. 103.0 kg, P < 0.001) and longer carcasses (86.9 vs. 84.8 cm, P < 0.01), whereas Pietrain-sired pigs had less backfat at the first rib (44.6 vs. 47.7 mm, P < 0.01), last lumbar vertebrae (20.9 vs. 23.0 mm, P < 0.05), and 10th rib (23.0 vs. 25.5 mm, P < 0.01). No difference between Pietrain and Duroc progeny was detected for fat depth at the last rib (27.8 vs. 28.8 mm, respectively). Pietrain progeny had a higher percentage of lean at slaughter (52.6 vs. 50.7, P < 0.05) and higher dressing percentage (74.0 vs. 73.1, P < 0.01). Primal cut weights were collected with Pietrain progeny having a greater percentage of carcass as ham (23.0 vs. 22.4, P < 0.01) and loin (21.6 vs. 21.2, P < 0.05), whereas Duroc progeny had a higher percentage of belly weight (12.0 vs. 11.7, P < 0.05). Percentages of Boston butt (8.8 vs. 9.0) and picnic shoulder (9.9 vs. 9.9) were similar for Duroc vs. Pietrain progeny. Total weight of these five primal cuts, as a percentage of carcass weight, was higher for Pietrain progeny (75.2 vs. 74.3, P < 0.01). With heavier carcass weight, Duroc progeny had greater primal cut weights as a function of age. Subjective meat quality scores for color, marbling, and firmness (1 to 5 scale) were more favorable for Duroc-sired progeny. Furthermore, chops from Duroc progeny had higher 24-h pH (5.53 vs. 5.48, P < 0.001) and Minolta a* (17.33 vs. 17.04, P < 0.05) with less percentage drip loss (2.88 vs. 3.80, P < 0.001). No differences were detected between Duroc- and Pietrain-sired progeny for Minolta L* (54.77 vs. 55.37) or b* (7.58 vs. 7.58) objective color scores, percentage cooking loss (28.63 vs. 29.23), or Warner-Bratzler shear force (6.94 vs. 7.11 kg). Both sire breeds have beneficial traits that can be utilized in commercial pork production and merit further study.

Reduced-fat bologna manufactured with poultry skin connective tissue gel.

The objectives of this study were to determine temperature (50, 60, 70, and 80 C) and time (0.5, 1.0, 1.5, and 2.0 h) effects on the water binding ability of chicken skin connective tissue (CCT) and its ability to form model gels; to develop and determine the functionality of added water (AW, 100, 200, and 300%) CCT gels; and to evaluate the attributes of reduced-fat bologna containing 10 to 30% addition of 100 to 300% AW CCT gels. Determination of water binding and holding capacities, and objective textural and color attributes provided data suggesting the practicality of developing and incorporating AW CCT gels as water binders in reduced-fat bologna. Processing qualities, and textural and sensory attributes were analyzed to assess the feasibility of manufacturing a reduced-fat processed poultry product containing a modified poultry by-product. Heating (60 C) CCT for 0.5 h allowed the formation of model CCT gels containing 100 to 300% AW. Added water decreased CCT gel fat, protein, collagen content, and hardness due to a protein (collagen) dilution. Hydration values were sufficient to allow CCT to bind up to 300% AW. Gel fragility and syneresis were observed in higher AW CCT gels due to protein dilution, a result of the high fat content of raw CCT (approximately 40%) and added water. Percentage gel addition and AW decreased (P < 0.05) the hardness of reduced-fat CCT gel bologna. All bologna treatments exhibited acceptable sensory attributes. This study indicated the feasibility of using lower AW CCT gels as texture-modifying agents in reduced-fat comminuted meat products.

Ex vivo liposome-mediated gene transfer to lung isografts.

OBJECTIVE: Gene therapy is a promising strategy to modify ischemia-reperfusion injury and rejection after transplantation. We evaluated variables that may affect ex vivo gene transfer to rat lung isografts. METHODS: Left lungs were harvested and perfused via the pulmonary vein with chloramphenicol acetyltransferase complementary deoxyribonucleic acid complexed with cationic liposomes. Several variables were examined: (1) Influence of temperature: In group I (n = 4), grafts were stored for 4 hours at 23 degrees C and transplanted. Chloramphenicol acetyltransferase activity was assessed on postoperative day 2. In groups II and III (n = 4), grafts were stored at 10 degrees and 4 degrees C, respectively. Arterial oxygen tension and inflammatory infiltrate were also determined. (2) Influence of storage time: Grafts were preserved at 10 degrees C for 1, 2, 3, 4 (n = 4), and 10 hours (n = 5). chloramphenicol acetyltransferase activity was assessed on postoperative day 2. (3) Rapidity and duration of transgene expression: Grafts were preserved at 10 degrees C for 1 hour and then transplanted. Chloramphenicol acetyltransferase activity was assessed 2, 4, 6, 12, and 24 hours and 2, 7, 14, 21, and 28 days after implantation. RESULTS: Chloramphenicol acetyltransferase expression was apparently less in lungs transfected at 4 degrees C than in those transfected at 10 degrees and 23 degrees C. Storage for 1 hour at 10 degrees C was sufficient to yield significant expression. Increasing the exposure time to 10 hours did not increase toxicity. There were no differences in arterial oxygen tension between transfected and nontransfected lungs. Chloramphenicol acetyltransferase expression was detected for at least 28 days. CONCLUSION: Ex vivo liposome-mediated transfection of lung isografts can be achieved after a short time of cold storage, with minimal toxicity.

Liposome-mediated gene transfer to lung isografts.

OBJECTIVES: Our objective were to determine the feasibility, efficacy, and safety of in vivo and ex vivo liposome-mediated gene transfer to lung isografts. METHODS: Fischer rats were divided into three main groups: (1) Nontransplant setting: Liposome-chloramphenicol acetyl transferase cDNA was intravenously injected, and lungs were harvested at different time points: 2, 6, 12, and 24 hours; 2, 5, 8, and 21 days (n = 3). Chloramphenicol acetyl transferase activity was determined in lungs, hearts, livers, and kidneys. The distribution and type of transfected cells were evaluated by in situ hybridization. Lung toxicity was assessed by arterial oxygen tension, histology, and tumor necrosis factor-alpha levels. (2) In vivo graft transfection: Left lungs were transplanted 6 hours, 4 hours, and 15 minutes after intravenous injection and were assessed for chloramphenicol acetyl transferase activity and arterial oxygen tension on postoperative day 2. (3) Ex vivo graft transfection: Grafts were infused ex vivo with either 660 micrograms (n = 3) or 330 micrograms (n = 3) of DNA complexed to liposomes and stored at 10 degrees C for 4 hours. Chloramphenicol acetyl transferase activity was assessed 44 hours after transplantation. RESULTS: Transgene expression was detected in endothelial cells, macrophages, and interstitial cells. Chloramphenicol acetyl transferase activity was present as early as 2 hours, increased significantly between 6 hours and 8 days, and then decreased to minimal levels by 21 days. Chloramphenicol acetyl transferase activity was greatest in donor lungs and hearts and minimal in livers and kidneys. Arterial oxygen tension was normal in treated animals. Inflammation was minimal, and tumor necrosis factor-alpha levels increased only sevenfold in treated animals. CONCLUSION: In vivo and ex vivo liposome-mediated gene transfer to lung isografts allows significant transgene expression with minimal effects on graft function.

Optimization of plasmid vectors for high-level expression in lung epithelial cells.

Nonviral gene therapy approaches use a plasmid vector to express the desired transgene. We have systematically examined several regulatory elements within plasmid vectors that govern gene expression, e.g., the promoter, enhancer, intron, and polyadenylation signal, by constructing a series of plasmids that differed only in the particular sequence element being evaluated. Of the several promoters and polyadenylation signal sequences that were tested, the human cytomegalovirus (CMV) immediate early gene promoter and the addition of polyadenylation signal sequences from the bovine growth hormone (BGH) gene or rabbit beta-globin gene produced the highest levels of expression in vitro. The inclusion of a hybrid intron 3 to the promoter further increased expression 1.6-fold. The addition of a region of the CMV enhancer 5' to several weak promoters increased expression 8- to 67-fold, and co-transfection with a second plasmid encoding a chimeric transcription factor also enhanced expression. On the basis of these results, the CMV promoter, the hybrid intron, and the BGH polyadenylation signal were selected for consistent high level expression in vitro and in the mouse lung. However, expression was transient, with greater than 60% loss of activity in the first 7 days. This transient expression was not specific to CMV promoter-containing plasmids, because plasmids containing other heterologous promoters showed a similar profile of transient expression in vivo. These comparative analyses begin to provide a basis for the development of optimized expression plasmids for gene therapy of lung diseases.