Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.     

Microbial hydroxylation of progesterone with Acremonium strictum

Microbial hydroxylation of progesterone occurred in the culture of Acremonium strictum PTCC 5282 to produce two hydroxylated pregnene-like steroids. The metabolites were purified and characterized using spectroscopic methods and identified as 15alpha-hydroxyprogesterone and 15alpha-hydroxydeoxycorticosterone.     

Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons

Mesencephalic dopaminergic (MesDA) neurons play crucial roles in motor and behavioral processes; their loss in Parkinson's disease (PD) results in striatal dopamine (DA) deficiency and hypokinetic movement disorder. The Pitx3 homeobox gene is expressed in the MesDA system. We now show that only a subset of MesDA neurons express Pitx3 and that in Pitx3-deficient aphakia mice, this subset is progressively lost by apoptosis during fetal (substantia nigra, SN) and postnatal (ventral tegmental area) development, resulting in very low striatal DA and akinesia. Similar to human PD, dorsal SN neurons (which are Pitx3 negative) are spared in mutant mice. Thus, Pitx3 defines a pathway for survival of neurons that are implicated in PD and that are required for spontaneous locomotor activity.     

Adjunctive Corticotherapy for Community Acquired Pneumonia: A Systematic Review and Meta-Analysis

Background

Community-acquired pneumonia (CAP) induces lung and systemic inflammation, leading to high morbidity and mortality. We systematically reviewed the risks and benefits of adjunctive corticotherapy in the management of patients with CAP.

Methods

We systematically searched Pubmed, Embase and the Cochrane Library for randomized controlled trials comparing adjunctive corticotherapy and antimicrobial therapy with antimicrobial therapy alone in patients with CAP. The primary outcome was 30-day mortality. Secondary outcomes were length of hospital stay, time to clinical stability and severe complications.

Results

14 trials (2077 patients) were included. The reported 30-day mortality was 7.9% (80/1018) among patients treated with adjunctive corticotherapy versus 8.3% (85/1028) among patients treated with antimicrobial therapy alone (RR 0.84; 95%CI 0.55 to1.29). Adjunctive corticotherapy was associated with a reduction of severe complications (RR 0.36; 95%CI 0.23 to 0.56), a shorter length of stay (9.0 days; 95%CI 7.6 to 10.7 vs 10.6 days; 95%CI 7.4 to 15.3) and a shorter time to clinical stability (3.3 days; 95% CI 2.8 to 4.1 vs 4.3 days; 95%CI 3.6 to 5.1). The risk of hyperglycemia was higher among patients treated with adjunctive corticotherapy (RR 1.59; 95%CI 1.06 to 2.38), whereas the risk of gastro-intestinal bleeding was similar (RR 0.83; 95%CI 0.35 to 1.93). In the subgroup analysis based on CAP severity, a survival benefit was found among patients with severe CAP (RR 0.47; 95%CI 0.23 to 0.96).

Conclusion

Adjunctive corticotherapy is associated with a reduction of length of stay, time to clinical stability, and severe complications among patients with CAP, but the effect on mortality remains uncertain.     

Problem-Solving Skills Appraisal Mediates Hardiness and Suicidal Ideation among Malaysian Undergraduate Students

Objectives

Recent evidence suggests that suicidal ideation is increased among university students, it is essential to increase our knowledge concerning the etiology of suicidal ideation among university students. This study was conducted to examine the relationships between problem-solving skills appraisal, hardiness, and suicidal ideation among university students. In addition, this study was conducted to examine problem-solving skills appraisal (including the three components of problem-solving confidence, approach-avoidance style, and personal control of emotion) as a potential mediator between hardiness and suicidal ideation.

Methods

The participants consisted of 500 undergraduate students from Malaysian public universities.

Results

Structural Equation Modelling (SEM) estimated that undergraduate students with lower hardiness, poor problem-solving confidence, external personal control of emotion, and avoiding style was associated with higher suicidal ideation. Problem-solving skills appraisal (including the three components of problem-solving confidence, approach-avoidance style, and personal control of emotion) partially mediated the relationship between hardiness and suicidal ideation.

Conclusion

These findings underline the importance of studying mediating processes that explain how hardiness affects suicidal ideation.     

Variability of United States Isolates of Macrophomina phaseolina Based on Simple Sequence Repeats and Cross Genus Transferability to Related Genera Within Botryosphaeriaceae

Twelve simple sequence repeat (SSRs) loci were used to evaluate genetic diversity of 109 isolates of Macrophomina phaseolina collected from different geographical regions and host species throughout the United States (US). Genetic diversity was assessed using Nei’s minimum genetic distance, and the usefulness of each locus was determined by calculating the polymorphism information content (PIC). A total of 98 alleles were detected and of these 31 were unique to individual genotypes. Eight of twelve loci were highly informative with PIC values greater than 0.50. The majority of pairwise comparisons of genetic distance were greater than 0.60 indicating moderate to high genetic diversity. Dendrograms based on the genetic dissimilarities were created for the 109 isolates of which 79 were from soybean. Some clustering by host and geography was noted, but, the dendrograms generally grouped isolates independent of host or geography. Additionally, sequencing of the internal transcribed spacer region (ITS) for 10 isolates revealed that all of these isolates were 99% similar. Three SSR loci from M. phaseolina were cross amplified in other genera in the Botryosphaeriaceae. This was the first study of genotyping and assessing genetic diversity of M. phaseolina isolates collected from a widespread host and geographic range across the US with SSRs. With an additional 34 loci publically available for M. phaseolina, the results indicate that previously developed SSRs from one species can be used in future population, ecological, and genetic studies of M. phaseolina and other genera within the Botryosphaeriaceae.     

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.