Determination of the optimal conditions for the continuous culture of Candida utilis in sugarcane stillage

Summary Candida utilis was continuously cultured in sugarcane stillage without any supplementation, but the yeast utilized the stillage nitrogen only partially. As a result, the cell biomass production was low and the residual chemical oxygen demand (COD), as well as the total nitrogen content of the effluent were high. The addition of 2–4 g/l ammonium phosphate and small amounts of Fe²⁺, Cu²⁺, and Zn²⁺, markedly enhanced both the everall nitrogen uptake and cell biomass production, decreased COD, and a higher critical dilution rate was attained. No substantial differences were found when pH was kept at 5.0 or allowed to evolve freely. The optimal temperature range was between 30 and 35°C.     

Ketimine rings: useful detectors of enzymatic activities in solution and on polyacrylamide gel. Detection of L-amino acid oxidase, glutamine transaminase, pantetheinase, and acylase I

A simple procedure has been developed for the detection of L-amino acid oxidase, glutamine transaminase, pantetheinase, and acylase I in solution and on polyacrylamide gels. The method is based on the strong absorbance at 296 nm of some ketimine rings which can be directly produced by the enzymatic reaction or formed by the reaction of the enzymatic product with 3-bromopyruvate. The procedure allows one to visualize up to about 1-10 mU of enzyme.     

Detection of glutathione transferase activity on polyacrylamide gels

A simple and sensitive assay for glutathione transferase activity on polyacrylamide gel is described. The method is based on the fast reduction of nitroblue tetrazolium salt by glutathione. Blue insoluble formazan colors the gel except in the glutathione transferase area. The stable and defined colorless zone is still detectable with 0.005 unit enzyme. This technique has been successfully applied with enzyme preparations of human heart and other tissues.     

Effect of some imidazopyrimidines on tobacco callus cultures

Abstract

Several 7-chloro-imidazo(1,2-c)pyrimidines were tested on tobacco callus cultures in order to verify a possible cytokinin and anticytokinin-like activity. These compounds were used alone and in combination with kinetin or zeatin.

The aminofurfuryl derivatives showed a strong inhibitory action on cell multiplication and this effect was enhanced when they were mixed with kinetin or zeatin.

The isopentenyl derivatives on the contrary were able to induce cell division in tobacco callus.     

Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics

Background aimsThe presence of ectopic tissues in the pathologic artery wall raises the issue of whether multipotent stem cells may reside in the vasculature itself. Recently mesenchymal stromal cells (MSC) have been isolated from different human vascular segments (VW MSC), belying the previous view that the vessel wall is a relatively quiescent tissue.MethodsResident multipotent cells were recovered from fresh arterial segments (aortic arches, thoracic and femoral arteries) collected in a tissue-banking facility and used to establish an in situ and in vitro study of the stemness features and multipotency of these multidistrict MSC populations.ResultsNotch-1⁺, Stro-1⁺, Sca-1⁺ and Oct-4⁺ cells were distributed along an arterial wall vasculogenic niche. Multidistrict VW MSC homogeneously expressed markers of stemness (Stro-1, Notch-1 and Oct-4) and MSC lineages (CD44, CD90, CD105, CD73, CD29 and CD166) whilst they were negative for hematopoietic and endothelial markers (CD34, CD45, CD31 and vWF). Each VW MSC population had characteristics of stem cells, i.e. a high efflux capability for Hoechst 33342 dye and the ability to form spheroids when grown in suspension and generate colonies when seeded at low density. Again, VW MSC cultured in induction media exhibited adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic differentiation, as documented by histochemical, immunohistochemical, molecular and electron microscopy analysis.ConclusionsOverall, these findings may enlighten the physiopathologic mechanisms of vascular wall diseases as well as having potential implications for cellular, genetic and tissue engineering approaches to treating vascular pathologies when these are unresponsive to medical and surgical therapies.     

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (K_m) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.     

Bighorn sheep show similar in‐host responses to the same pathogen strain in two contrasting environments

Ecological context—the biotic and abiotic environment, along with its influence on population mixing dynamics and individual susceptibility—is thought to have major bearing on epidemic outcomes. However, direct comparisons of wildlife disease events in contrasting ecological contexts are often confounded by concurrent differences in host genetics, exposure histories, or pathogen strains. Here, we compare disease dynamics of a Mycoplasma ovipneumoniae spillover event that affected bighorn sheep populations in two contrasting ecological contexts. One event occurred on the herd''s home range near the Rio Grande Gorge in New Mexico, while the other occurred in a captive facility at Hardware Ranch in Utah. While data collection regimens varied, general patterns of antibody signal strength and symptom emergence were conserved between the two sites. Symptoms appeared in the captive setting an average of 12.9 days postexposure, average time to seroconversion was 24.9 days, and clinical signs peaked at approximately 36 days postinfection. These patterns were consistent with serological testing and subsequent declines in symptom intensity in the free‐ranging herd. At the captive site, older animals exhibited more severe declines in body condition and loin thickness, higher symptom burdens, and slower antibody response to the pathogen than younger animals. Younger animals were more likely than older animals to clear infection by the time of sampling at both sites. The patterns presented here suggest that environment may not be a major determinant of epidemiological outcomes in the bighorn sheep—M. ovipneumoniae system, elevating the possibility that host‐ or pathogen‐factors may be responsible for observed variation.