Giselda Bucca

Comparison of vitamin D2 and vitamin D3 supplementation in raising serum 25-hydroxyvitamin D status: a systematic review and meta-analysis

Background: Currently, there is a lack of clarity in the literature as to whether there is a definitive difference between the effects of vitamins D2 and D3 in the raising of serum 25-hydroxyvitamin D [25(OH)D]. Objective: The objective of this article was to report a systematic review and meta-analysis of randomized controlled trials (RCTs) that have directly compared the effects of vitamin D2 and vitamin D3 on serum 25(OH)D concentrations in humans. Design: The ISI Web of Knowledge (January 1966 to July 2011) database was searched electronically for all relevant studies in adults that directly compared vitamin D3 with vitamin D2. The Cochrane Clinical Trials Registry, International Standard Randomized Controlled Trials Number register, and clinicaltrials.gov were also searched for any unpublished trials. Results: A meta-analysis of RCTs indicated that supplementation with vitamin D3 had a significant and positive effect in the raising of serum 25(OH)D concentrations compared with the effect of vitamin D2 (P = 0.001). When the frequency of dosage administration was compared, there was a significant response for vitamin D3 when given as a bolus dose (P = 0.0002) compared with administration of vitamin D2, but the effect was lost with daily supplementation. Conclusions: This meta-analysis indicates that vitamin D3 is more efficacious at raising serum 25(OH)D concentrations than is vitamin D2, and thus vitamin D3 could potentially become the preferred choice for supplementation. However, additional research is required to examine the metabolic pathways involved in oral and intramuscular administration of vitamin D and the effects across age, sex, and ethnicity, which this review was unable to verify.

Effects of insufficient sleep on circadian rhythmicity and expression amplitude of the human blood transcriptome

Significance Insufficient sleep and circadian rhythm disruption are associated with negative health outcomes, but the mechanisms involved remain largely unexplored. We show that one wk of insufficient sleep alters gene expression in human blood cells, reduces the amplitude of circadian rhythms in gene expression, and intensifies the effects of subsequent acute total sleep loss on gene expression. The affected genes are involved in chromatin remodeling, regulation of gene expression, and immune and stress responses. The data imply molecular mechanisms mediating the effects of sleep loss on health and highlight the interrelationships between sleep homeostasis, circadian rhythmicity, and metabolism. Insufficient sleep and circadian rhythm disruption are associated with negative health outcomes, including obesity, cardiovascular disease, and cognitive impairment, but the mechanisms involved remain largely unexplored. Twenty-six participants were exposed to 1 wk of insufficient sleep (sleep-restriction condition 5.70 h, SEM = 0.03 sleep per 24 h) and 1 wk of sufficient sleep (control condition 8.50 h sleep, SEM = 0.11). Immediately following each condition, 10 whole-blood RNA samples were collected from each participant, while controlling for the effects of light, activity, and food, during a period of total sleep deprivation. Transcriptome analysis revealed that 711 genes were up- or down-regulated by insufficient sleep. Insufficient sleep also reduced the number of genes with a circadian expression profile from 1,855 to 1,481, reduced the circadian amplitude of these genes, and led to an increase in the number of genes that responded to subsequent total sleep deprivation from 122 to 856. Genes affected by insufficient sleep were associated with circadian rhythms (PER1, PER2, PER3, CRY2, CLOCK, NR1D1, NR1D2, RORA, DEC1, CSNK1E), sleep homeostasis (IL6, STAT3, KCNV2, CAMK2D), oxidative stress (PRDX2, PRDX5), and metabolism (SLC2A3, SLC2A5, GHRL, ABCA1). Biological processes affected included chromatin modification, gene-expression regulation, macromolecular metabolism, and inflammatory, immune and stress responses. Thus, insufficient sleep affects the human blood transcriptome, disrupts its circadian regulation, and intensifies the effects of acute total sleep deprivation. The identified biological processes may be involved with the negative effects of sleep loss on health, and highlight the interrelatedness of sleep homeostasis, circadian rhythmicity, and metabolism.

Mistimed sleep disrupts circadian regulation of the human transcriptome

Significance Disruption of the timing of the sleep–wake cycle and circadian rhythms, such as occurs during jet lag and shift work, leads to disordered physiological rhythms, but to what extent the molecular elements of circadian rhythm generation are affected is not known. Here, we show that delaying sleep by 4 h for 3 consecutive days leads to a sixfold reduction of circadian transcripts in the human blood transcriptome to just 1%, whereas, at the same time, the centrally driven circadian rhythm of melatonin is not affected. Genes and processes affected included those at the core of circadian rhythm generation and gene expression. The data have implications for understanding the negative health outcomes of disruption of the sleep–wake cycle. Circadian organization of the mammalian transcriptome is achieved by rhythmic recruitment of key modifiers of chromatin structure and transcriptional and translational processes. These rhythmic processes, together with posttranslational modification, constitute circadian oscillators in the brain and peripheral tissues, which drive rhythms in physiology and behavior, including the sleep–wake cycle. In humans, sleep is normally timed to occur during the biological night, when body temperature is low and melatonin is synthesized. Desynchrony of sleep–wake timing and other circadian rhythms, such as occurs in shift work and jet lag, is associated with disruption of rhythmicity in physiology and endocrinology. However, to what extent mistimed sleep affects the molecular regulators of circadian rhythmicity remains to be established. Here, we show that mistimed sleep leads to a reduction of rhythmic transcripts in the human blood transcriptome from 6.4% at baseline to 1.0% during forced desynchrony of sleep and centrally driven circadian rhythms. Transcripts affected are key regulators of gene expression, including those associated with chromatin modification (methylases and acetylases), transcription (RNA polymerase II), translation (ribosomal proteins, initiation, and elongation factors), temperature-regulated transcription (cold inducible RNA-binding proteins), and core clock genes including CLOCK and ARNTL (BMAL1). We also estimated the separate contribution of sleep and circadian rhythmicity and found that the sleep–wake cycle coordinates the timing of transcription and translation in particular. The data show that mistimed sleep affects molecular processes at the core of circadian rhythm generation and imply that appropriate timing of sleep contributes significantly to the overall temporal organization of the human transcriptome.

A bacterial hormone (the SCB1) directly controls the expression of a pathway‐specific regulatory gene in the cryptic type I polyketide biosynthetic gene cluster of Streptomyces coelicolor

Gamma‐butyrolactone signalling molecules are produced by many Streptomyces species, and several have been shown to regulate antibiotic production. In Streptomyces coelicolor A3(2) at least one γ‐butyrolactone (SCB1) has been shown to stimulate antibiotic production, and genes encoding proteins that are involved in its synthesis (scbA) and binding (scbR) have been characterized. Expression of these genes is autoregulated by a complex mechanism involving the γ‐butyrolactone. In this study, additional genes influenced by ScbR were identified by DNA microarray analysis, and included a cryptic cluster of genes for a hypothetical type I polyketide. Further analysis of this gene cluster revealed that the pathway‐specific regulatory gene, kasO, is a direct target for regulation by ScbR. Gel retardation and DNase I footprinting analyses identified two potential binding sites for ScbR, one at −3 to −35 nt and the other at −222 to −244 nt upstream of the kasO transcriptional start site. Addition of SCB1 eliminated the DNA binding activity of ScbR at both sites. The expression of kasO was growth phase regulated in the parent (maximal during transition phase), undetectable in a scbA null mutant, and constitutively expressed in a scbR null mutant. Addition of SCB1 to the scbA mutant restored the expression of kasO, indicating that ScbR represses kasO until transition phase, when presumably SCB1 accumulates in sufficient quantity to relieve kasO repression. Expression of the cryptic antibiotic gene cluster was undetectable in a kasO deletion mutant. This is the first report with comprehensive in vivo and in vitro data to show that a γ‐butyrolactone‐binding protein directly regulates a secondary metabolite pathway‐specific regulatory gene in Streptomyces.

THE DNAK OPERON OF STREPTOMYCES-COELICOLOR ENCODES A NOVEL HEAT-SHOCK PROTEIN WHICH BINDS TO THE PROMOTER REGION OF THE OPERON

Transcriptional studies have demonstrated that the dnaK gene of Streptomyces coelicolor A3(2) is contained within a 4.3 kb operon. The operon is transcribed from a single (transiently) heat‐inducible promoter, dnaKp, that resembles the typical vegetative (σ70‐recognized) eubacterial consensus promoter sequence. dnaK transcription was found to be heat‐inducible at all stages of development in surface‐grown cultures. In addition, at the normal growth temperature of 30°C, dnaK transcript levels were shown to vary at different stages of development, being more abundant in young germinating cultures and in mycelium undergoing sporogenesis. The nucleotide sequence of the dnaK operon has been completed, revealing the gene organization 5′‐dnaK‐grpE‐dnaJ‐orfX. orfX represents a novel heat‐shock gene. Its predicted product displays high similarity to the GlnR repressor proteins of Bacillus spp. and to the MerR family of eubacterial transcriptional regulators. The S. coelicolor OrfX protein has been over‐produced in Escherichia coli, and DNA‐binding experiments indicate that it interacts specifically with the dnaKp region, binding to three partially related inverted repeat sequences; they are centred at −75, −49 and +4, respectively, relative to the transcription start site of the operon. These results suggest that OrfX plays a direct role in the regulation of the dnaK operon.

The HspR regulon of Streptomyces coelicolor: a role for the DnaK chaperone as a transcriptional co‐repressor†

The dnaK operon of Streptomyces coelicolor encodes the DnaK chaperone machine and HspR, the transcriptional repressor of the operon; HspR confers repression by binding to several inverted repeat sequences in the promoter region, dnaKp. Here, we demonstrate that HspR specifically requires the presence of DnaK protein to retard a dnaKp fragment in gel‐shift assays. This requirement is independent of the co‐chaperones, DnaJ and GrpE, and it is ATP independent. Furthermore the retarded protein–DNA complex can be ‘supershifted’ by anti‐DnaK monoclonal antibody, demonstrating that DnaK forms an integral component of the complex. It was shown in DNase I footprinting experiments that refolding and specific binding of HspR to its DNA target does not require DnaK. We conclude that the formation of the stable DnaK–HspR–DNA ternary complex does not depend on the chaperoning activity of DnaK. In affinity chromatography experiments using whole‐cell extracts, DnaK was shown to co‐purify with HspR, providing additional evidence that the two proteins interact in vivo; it was not possible to purify HspR away from DnaK in any experiments unless a powerful denaturant was used. The level of heat shock induction of chromosomal DnaK could be partially suppressed by expressing dnaK extrachromosomally from a heterologous promoter. In addition, it is shown that DnaK confers enhanced HspR‐mediated repression of transcription in vitro. Taken together, these results suggest that DnaK functions as a transcriptional co‐repressor by binding to HspR at its operator sites. In this model, the DnaK–HspR system would represent a novel example of feedback regulation of gene expression by a molecular chaperone, in which DnaK directly activates a repressor, rather than inactivates an activator (as is the case in the DnaK–σ32 and Hsp70–HSF systems of other organisms).

Negative feedback regulation of dnaK, clpB and lon expression by the DnaK chaperone machine in Streptomyces coelicolor, identified by transcriptome and in vivo DnaK-depletion analysis.

The dnaK operon of Streptomyces coelicolor encodes the DnaK chaperone machine and the negative autoregulator HspR, which confers repression of the operon by binding to several inverted repeat sequences in the promoter region, dnaKp. Previous in vitro studies demonstrated that DnaK forms a specific complex with HspR bound to its operator sequences in dnaKp, and a model was proposed in which DnaK functions as a corepressor of the dnaK operon (Bucca, G., Brassington, A., Schonfeld, H.J., and Smith, C.P. (2000) Mol Microbiol 38: 1093–1103). Here we report in vivo DnaK depletion experiments which demonstrate that DnaK is a negative regulator of the dnaK operon. Cellular depletion of the DnaK chaperone leads to high‐level transcription from dnaKp at the normal growth temperature. DNA microarray‐based analysis of gene expression in wild‐type and hspR‐disruption mutant strains has identified a core cluster of genes regulated by HspR: the dnaK and clpB‐SCO3660 operons and lon. These three transcription units are considered to be the direct targets of HspR. Significantly, analysis of the entire genome sequence revealed that the promoter regions of dnaK, clpB and lon are the only sequences that contain the HspR consensus binding sequence 5′‐TTGAGY‐N7‐ACTCAA. S1 nuclease mapping confirmed that transcription of both clpB and lon is substantially enhanced at ambient temperature in strains depleted of DnaK, providing further evidence that these genes are members of the DnaK‐HspR regulon. From transcriptome analysis, 17 genes were shown to be upregulated more than twofold in an hspR disruption mutant. This included the seven genes encoded by the dnaK, clpB and lon transcription units. Significantly, the other 10 genes are not heat‐shock inducible in the wild type and their upregulation in the hspR mutant is considered to be an indirect consequence of enhanced synthesis of one or more components of the HspR regulon (the DnaK chaperone machine, ClpB and Lon protease).

Gene Expression Profiling of Human Cancers

Abstract: DNA microarrays allow us to visualize simultaneously the expression of potentially all genes within a cell population or tissue sample—revealing the “transcriptome.” The analysis of this type of data is commonly called “gene expression profiling” (GEP) because it provides a comprehensive picture of the pattern of gene expression in a particular biological sample. For this reason microarrays are revolutionizing life sciences research and are leading to the development of novel and powerful methods for investigating cancer biology, classifying cancers, and predicting clinical outcome of cancers. Several recent high‐profile reports have revealed how clustering of GEP data can clearly identify clinically (and prognostically) important subtypes of cancer among patients considered by established clinicopathological criteria to have similar tumors. Accurate “prognostic signatures” can be obtained from GEP data, which represent relatively small numbers of genes. These signatures can be valuable in directing appropriate treatment and in predicting clinical outcome, and they generally outperform other systems based on clinical and histological criteria. In this paper the basic principles of DNA microarray technology and the different types of microarray platforms available will be introduced, and the power of the technique will be illustrated by reviewing some recent GEP studies on selected cancers, including a preliminary analysis of hepatocellular carcinoma from our Palermo laboratory. GEP is likely to be adopted in the future as a key decision‐making tool in the clinical arena. However, several issues relating to data analysis, reproducibility, cross‐comparability, validation, and cost need to be resolved before the technology can be adopted broadly in this context.

Diverse control of metabolism and other cellular processes in Streptomyces coelicolor by the PhoP transcription factor: genome-wide identification of in vivo targets

Streptomycetes sense and respond to the stress of phosphate starvation via the two-component PhoR–PhoP signal transduction system. To identify the in vivo targets of PhoP we have undertaken a chromatin-immunoprecipitation-on-microarray analysis of wild-type and phoP mutant cultures and, in parallel, have quantified their transcriptomes. Most (ca. 80%) of the previously in vitro characterized PhoP targets were identified in this study among several hundred other putative novel PhoP targets. In addition to activating genes for phosphate scavenging systems PhoP was shown to target two gene clusters for cell wall/extracellular polymer biosynthesis. Furthermore PhoP was found to repress an unprecedented range of pathways upon entering phosphate limitation including nitrogen assimilation, oxidative phosphorylation, nucleotide biosynthesis and glycogen catabolism. Moreover, PhoP was shown to target many key genes involved in antibiotic production and morphological differentiation, including afsS, atrA, bldA, bldC, bldD, bldK, bldM, cdaR, cdgA, cdgB and scbR-scbA. Intriguingly, in the PhoP-dependent cpk polyketide gene cluster, PhoP accumulates substantially at three specific sites within the giant polyketide synthase-encoding genes. This study suggests that, following phosphate limitation, Streptomyces coelicolor PhoP functions as a ‘master’ regulator, suppressing central metabolism, secondary metabolism and developmental pathways until sufficient phosphate is salvaged to support further growth and, ultimately, morphological development.

The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2)

Individual Streptomyces species have the genetic potential to produce a diverse array of natural products of commercial, medical and veterinary interest. However, these products are often not detectable under laboratory culture conditions. To harness their full biosynthetic potential, it is important to develop a detailed understanding of the regulatory networks that orchestrate their metabolism. Here we integrate nucleotide resolution genome-scale measurements of the transcriptome and translatome of Streptomyces coelicolor, the model antibiotic-producing actinomycete. Our systematic study determines 3,570 transcription start sites and identifies 230 small RNAs and a considerable proportion (∼21%) of leaderless mRNAs; this enables deduction of genome-wide promoter architecture. Ribosome profiling reveals that the translation efficiency of secondary metabolic genes is negatively correlated with transcription and that several key antibiotic regulatory genes are translationally induced at transition growth phase. These findings might facilitate the design of new approaches to antibiotic discovery and development.