Single-tube classical PCR for Candida auris and Candida haemulonii identification

Background

Candida auris and Candida haemulonii are emerging and multiresistant pathogens. C. auris has produced hospital outbreaks and is misidentified by phenotypic-based methods. The only reliable identification methods are DNA sequencing and MALDI-TOF.

Cell‐free production of a therapeutic protein: Expression,purification, and characterization of recombinant streptokinase using a CHO lysate

The use of cell‐free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell‐free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell‐free system using instrumented mini‐bioreactors for highly reproducible protein production. We achieved recombinant protein production (～600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell‐free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag‐free self‐cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose‐level production of therapeutic proteins at the point‐of‐care.     

All Trans Retinoic Acid Attenuates Markers of Neuroinflammation in Rat Brain by Modulation of SIRT1 and NFκB

Alcohol abuse affects several neurological pathways and causes significant alterations in the brain. Abstention from alcohol causes only a marginal decrease in oxidative stress and neuro inflammation. Our previous studies had shown that an active metabolite of vitamin A, all trans retinoic acid (ATRA), ameliorates alcohol induced toxicity. Hence in the present study we investigated whether ATRA regressed alcohol induced neuroinflammation. We focused on the role of silent mating type information regulation 2 homolog 1(SIRT1) and nuclear factor kappa-B (NFκB). Animals were administered with ethanol at a daily dose of (4 g/kg body weight) for 90 days. On the 91st day ethanol administration was stopped and animals were divided into ethanol abstention (A) and ATRA supplementation group (ATRA?+?A) (100 µg/kg body weight) and maintained for 30 days. Ethanol exposure increased markers of oxidative stress, inflammation and the activities of alcohol and acetaldehyde dehydrogenases and reduced the expression of SIRT1 in the whole brain.The ethanol induced altered expressions of NFκB and SIRT1 were modulated by supplementation of ATRA. Abstention also reduced toxicity, but to a lower extent in comparison with supplementation of ATRA. Our results seemed to suggest that ATRA regressed the mediators of ethanol induced neuroinflammation by reducing oxidative stress and by regulating the expression of NFκB and SIRT1. The ameliorative potential of ATRA was much higher than abstention.     

A rapid method to monitor DNA precipitation onto microcarriers before particle bombardment

The binding or precipitation of DNA onto gold or tungsten microcarriers represents one of the most crucial steps for gene transfer via the particle bombardment process. We have developed a simple and rapid method to monitor DNA precipitation onto microcarriers before delivery to intact cells or tissues. Binding of DNA constructs to different microcarriers was evaluated with relative fluorescence values using a dedicated fluorometer. Significantly greater precipitation was detected using gold vs. tungsten microcarriers. Addition of glycerol resulted in a 46% increase in precipitation. A 42% difference in precipitation was observed using two different brands of polyproplyene tubes. Fluorescence values dropped 10–50% 3 hr after initial precipitation. Fluorescence values were correlated with the number of transient GUS transformants of rice (Oryza sativa, L.) cells. Precipitation with PEG gave higher fluorescent values and GUS transformants than a similar method without PEG. Results from these experiments indicate that fluorescence measurements are an effective and rapid method to monitor DNA precipitation for particle bombardment experiments.Communicated by C. Quiros     

Electron Transfer Reactions in RB90745, A Bioreductive Drug Having Both Aromatic N-Oxide and Nitroarene Moieties

The bifunctional hypoxia-specific cytotoxin RB90745, has a nitroimidazole moiety attached to an imidazo[1,2,-a]quinoxaline mono-N-oxide with a spacer/linking group. The reduction chemistry of the drug was studied by pulse radiolysis using the one electron reductant CO₂^˙-. As N-oxides and nitro compounds react with CO₂^˙- at diffusion controlled rates, initial reaction produced a mixture of the nitro radical (λ_max 410 nm) and the N-oxide radical (λ_max 550 nm) in a few microseconds. Subsequently an intramolecular electron transfer (IET) was observed (k = 1.0 ± 0.25 × 10³ s^-1 at pH 5-9), from the N-oxide to the more electron-affinic nitro group. This was confirmed by the first order decay rate of the radical at 550 nm and formation at 410 nm, which was independent of both the concentration of the parent compound and the radicals. The rates of electron transfer and the decay kinetics of the nitro anion radicals were pH dependent and three different pK_aS could be estimated for the one electron reduced species: 5.6 (nitroimidazole group) and 4.3, and 7.6 (N-oxide function). The radicals react with oxygen with rate constants of 3.1 × 10⁷ and 2.8 × 10⁶ dm³ mol^-1 s^-1 observed at 575 nm and 410 nm respectively. Steady state radiolysis studies indicated four electron stoichiometry for the reduction of the compound.     

Modification of Delivery System Enhances MHC Nonrestricted Immunogenicity of V3 Loop Region of HIV-1 gp120

A successful peptide vaccine for AIDS is desired to elicit T-helper and cytotoxic T lymphocyte responses besides neutralizing antibodies. The V3 loop peptide of HIV-1 has been shown to contain the principal neutralizing domain, one of the most immunodominant regions, having both B-cell and T-cell determinants. In this study, the tip of the V3 loop region was mutated from GPGR to GPGQ based on the sequence of Indian isolates (CKRKIHIGPGQAFYT). To further enhance the immunogenicity of this epitope, two delivery systems of immune stimulating complexes (ISCOMs) and liposomes were used to incorporate the peptide. Mice of differing haplotypes, H-2^b, H-2^d, H-2^k and H-2^S, showed no MHC restriction when immunized with these formulations. The IgG levels as assessed by ELISA were found to be significantly higher (P<0.05 to P< 0.001) for even five-fold lower doses of the peptide in ISCOMs and liposomes as compared to the conventional alum-based preparation. The major subtype elicited was IgG2a/IgG2b, suggestive of a Th1-like response for all the formulations. Thus, it would appear that the same peptide incorporated in ISCOMs and liposomes selects a Th1 response and may therefore be important not only for neutralization but also for virus clearance.     

LEAF TRICHOMES OF SOME PARTHENIUM SPECIES

Guayule (Parthenium argentatum), a native shrub of the Chihuahuan desert, contains rubber. Guayule has been crossed with other Parthenium species in an attempt to improve its agronomic characteristics. The resulting hybrids show intermediate morphologies. Each Parthenium species has a characteristic combination of leaf trichomes. In order to recognize the contribution of each parent in future studies of hybrids, characteristics of leaf trichomes of the following Parthenium species were studied: P. tomentosum, P. fruticosum, P. Schottii and P. rollinsianum. All species studied had two or more types of trichomes, and, in some species, trichomes of upper and lower epidermal surfaces were different. The prominent trichomes on upper epidermis of P. tomentosum and P. fruticosum were simple, uniseriate, conical trichomes, which also were observed on both epidermal surfaces of P. Schottii. Extremely long, narrow, simple, whiplike, trichomes formed a dense cover on both surfaces of P. rollinsianum and on the lower surfaces of P. tomentosum and P. fruticosum. Simple, uniseriate, cylindrical trichomes, and biseriate, glandular trichomes were observed in all four species.