A rapid non-radioactive procedure for plaque hybridization using biotinylated probes prepared by random primed labeling

A simple, non-radioactive method has been developed for the rapid screening of phage libraries. In the present study, nanogram amounts of a small restriction fragment (135 bp) were biotinylated via random primed labeling and used to probe cDNA libraries using a modified plaque hybridization protocol. The high backgrounds that are seen typically with avidin/biotin-based methods for plaque hybridization were eliminated by incubation of filters with one of several different proteases prior to hybridization. A comparison of several detection systems indicated that streptavidin conjugated to calf intestinal alkaline phosphatase (AP) was the most sensitive, yielding signals comparable to those obtained with 32P-labeled probes. The times required for phage growth and pre-hybridization were reduced substantially, permitting a convenient one-day screening protocol. Nitrocellulose filters gave the best signal to noise ratio, although "streaking" of plaque DNA was observed occasionally; this problem can be overcome by using nylon-based membranes, which allows exact visualization of the positive plaques. The method was highly reliable; 29 out of 33 putative clones retested positive and the authenticity of these was confirmed by DNA sequence analysis. The random primed biotinylation procedure has been utilized successfully with several different cDNA fragments and has proven useful for other hybridization-based methods (Northern and Southern blots), without the problems associated with the use of radiolabeled probes.     

Circulating anti-Tax cytotoxic T lymphocytes from human T-cell leukemia virus type I-infected people, with and without tropical spastic paraparesis, recognize multiple epitopes simultaneously.

CD8+ T cells were freshly isolated from a human T-cell leukemia virus type I (HTLV-I)-infected patient with tropical spastic paraparesis. These cells, which were specific for HTLV-I Tax, simultaneously recognized a minimum of five, and possibly as many as seven, distinct peptide epitopes within the protein. A further Tax epitope was recognized after a short period of culture without exogenous peptide stimulation. All but one of these epitopes were clustered in the N-terminal third of Tax, and one of the epitopes was clearly immunodominant on two separate occasions of testing. Recognition of the immunodominant epitope was restricted by human leukocyte antigen (HLA) B15, and recognition of all the others was by HLA A2. Similar patterns of cytotoxic T lymphocyte recognition of the HLA A2-restricted Tax peptides in two healthy HTLV-I-seropositive individuals, each of whom carried the HLA A2 allele, were observed.     

A shared cryoglobulin antigen in familial cryoglobulinemia

Ten members of 3 generations of a family have IgM-IgG cryoglobulins and rheumatoid factors in their sera; one additional member has rheumatoid factor but not cryoglobulins. The disorder occurs in an autosomal dominant pattern. Here we describe an antigen, first identified on the cryoglobulin IgM of the index case, which is present in the sera of all 11 members of this kindred with rheumatoid factor. This antigen has the serologic properties of an IgM rheumatoid factor idiotype.     

A highly sensitive molecular structural probe applied to in situ biosensing of metabolites using PEDOT:PSS

A large amount of research within organic biosensors is dominated by organic electrochemical transistors (OECTs) that use conducting polymers such as poly(3,4-ethylene dioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS). Despite the recent advances in OECT-based biosensors, the sensing is solely reliant on the amperometric detection of the bioanalytes. This is typically accompanied by large undesirable parasitic electrical signals from the electroactive components in the electrolyte. Herein, we present the use of in situ resonance Raman spectroscopy to probe subtle molecular structural changes of PEDOT:PSS associated with its doping level. We demonstrate how such doping level changes of PEDOT:PSS can be used, for the first time, on operational OECTs for sensitive and selective metabolite sensing while simultaneously performing amperometric detection of the analyte. We test the sensitivity by molecularly sensing a lowest glucose concentration of 0.02 mM in phosphate-buffered saline solution. By changing the electrolyte to cell culture media, the selectivity of in situ resonance Raman spectroscopy is emphasized as it remains unaffected by other electroactive components in the electrolyte. The application of this molecular structural probe highlights the importance of developing biosensing probes that benefit from high sensitivity of the material's structural and electrical properties while being complimentary with the electronic methods of detection.     

Generic Escherichia coli Contamination of Spinach at the Preharvest Stage: Effects of Farm Management and Environmental Factors

The objective of this study was to determine the effects of farm management and environmental factors on preharvest spinach contamination with generic Escherichia coli as an indicator of fecal contamination. A repeated cross-sectional study was conducted by visiting spinach farms up to four times per growing season over a period of 2 years (2010 to 2011). Spinach samples (n = 955) were collected from 12 spinach farms in Colorado and Texas as representative states of the Western and Southwestern United States, respectively. During each farm visit, farmers were surveyed about farm-related management and environmental factors using a questionnaire. Associations between the prevalence of generic E. coli in spinach and farm-related factors were assessed by using a multivariable logistic regression model including random effects for farm and farm visit. Overall, 6.6% of spinach samples were positive for generic E. coli. Significant risk factors for spinach contamination with generic E. coli were the proximity (within 10 miles) of a poultry farm, the use of pond water for irrigation, a >66-day period since the planting of spinach, farming on fields previously used for grazing, the production of hay before spinach planting, and the farm location in the Southwestern United States. Contamination with generic E. coli was significantly reduced with an irrigation lapse time of >5 days as well as by several factors related to field workers, including the use of portable toilets, training to use portable toilets, and the use of hand-washing stations. To our knowledge, this is the first report of an association between field workers'' personal hygiene and produce contamination with generic E. coli at the preharvest level. Collectively, our findings support that practice of good personal hygiene and other good farm management practices may reduce produce contamination with generic E. coli at the preharvest level.     

Gradients in microbial methanol uptake: productive coastal upwelling waters to oligotrophic gyres in the Atlantic Ocean

Methanol biogeochemistry and its importance as a carbon source in seawater is relatively unexplored. We report the first microbial methanol carbon assimilation rates (k) in productive coastal upwelling waters of up to 0.117±0.002 d⁻¹ (∼10 nmol l⁻¹d⁻¹). On average, coastal upwelling waters were 11 times greater than open ocean northern temperate (NT) waters, eight times greater than gyre waters and four times greater than equatorial upwelling (EU) waters; suggesting that all upwelling waters upon reaching the surface (⩽20 m), contain a microbial population that uses a relatively high amount of carbon (0.3–10 nmol l⁻¹d⁻¹), derived from methanol, to support their growth. In open ocean Atlantic regions, microbial uptake of methanol into biomass was significantly lower, ranging between 0.04–0.68 nmol l⁻¹d⁻¹. Microbes in the Mauritanian coastal upwelling used up to 57% of the total methanol for assimilation of the carbon into cells, compared with an average of 12% in the EU, and 1% in NT and gyre waters. Several methylotrophic bacterial species were identified from open ocean Atlantic waters using PCR amplification of mxaF encoding methanol dehydrogenase, the key enzyme in bacterial methanol oxidation. These included Methylophaga sp., Burkholderiales sp., Methylococcaceae sp., Ancylobacter aquaticus, Paracoccus denitrificans, Methylophilus methylotrophus, Methylobacterium oryzae, Hyphomicrobium sp. and Methylosulfonomonas methylovora. Statistically significant correlations for upwelling waters between methanol uptake into cells and both chlorophyll a concentrations and methanol oxidation rates suggest that remotely sensed chlorophyll a images, in these productive areas, could be used to derive total methanol biological loss rates, a useful tool for atmospheric and marine climatically active gas modellers, and air–sea exchange scientists.     

Metformin modifies the exercise training effects on risk factors for cardiovascular disease in impaired glucose tolerant adults

Impaired glucose tolerant (IGT) adults are at elevated risk for cardiovascular disease (CVD). Exercise or metformin reduce CVD risk, but the efficacy of combining treatments is unclear.

Objective:

To determine the effects of exercise training plus metformin (EM), compared with each treatment alone, on CVD risk factors in IGT adults.

Design and Methods:

Subjects were assigned to placebo (P), metformin (M), exercise training plus placebo (EP), or EM (8/group). In a double‐blind design, P or 2,000 mg/d of M were administered for 12 weeks and half performed aerobic and resistance training 3 days/week for ～60 min/day at 70% pretraining heart rate peak. Outcomes included adiposity, blood pressure (BP), lipids, and high sensitivity C‐reactive protein (hs‐CRP). Z‐scores were calculated to determine metabolic syndrome severity.

Results:

M and EM, but not EP, decreased body weight compared with P (P < 0.05). M and EP lowered systolic blood pressure by 6% (P < 0.05), diastolic blood pressure by 6% (P < 0.05), and hs‐CRP by 20% (M: trend P = 0.06; EP: P < 0.05) compared with P. Treatments raised high‐density lipoprotein cholesterol (P < 0.05; EM: trend P = 0.06) compared with P and lowered triacyglycerol (P < 0.05) and metabolic syndrome Z‐score compared with baseline (EP; trend P = 0.07 and EM or M; P < 0.05).

Conclusions:

Although exercise and/or metformin improve some CVD risk factors, only training or metformin alone lowered hs‐CRP and BP. Thus, metformin may attenuate the effects of training on some CVD risk factors and metabolic syndrome severity in IGT adults.     

The influence of surface padding properties on head and neck injury risk

A validated computational head-neck model was used to understand the mechanical relationships between surface padding characteristics and injury risk during impacts near the head vertex. The study demonstrated that injury risk can be decreased by maximizing the energy-dissipating ability of the pad, choosing a pad stiffness that maximizes pad deformation without bottoming out, maximizing pad thickness, and minimizing surface friction. That increasing pad thickness protected the head without increasing neck loads suggests that the increased cervical spine injury incidence previously observed in cadaveric impacts to padded surfaces relative to lubricated rigid surfaces was due to increased surface friction rather than pocketing of the head in the pad.