Genomic description of acquired fluconazole- and echinocandin-resistance in patients with serial Candida glabrata isolates.

Candida glabrata is one of the most common causes of systemic candidiasis, often resistant to antifungal medications. To describe the genomic context of emerging resistance, we conducted a retrospective analysis of 82 serially collected isolates from 33 patients from population-based candidemia surveillance in the United States. We used whole-genome sequencing to determine the genetic relationships between isolates obtained from the same patient. Phylogenetic analysis demonstrated that isolates from 29 patients were clustered by patient. The median SNPs between isolates from the same patient was 30 (range: 7-96 SNPs), while unrelated strains infected four patients. Twenty-one isolates were resistant to echinocandins, and 24 were resistant to fluconazole. All echinocandin-resistant isolates carried a mutation either in the FKS1 or FKS2 HS1 region. Of the 24 fluconazole-resistant isolates, 17 (71%) had non-synonymous polymorphisms in the PDR1 gene, which were absent in susceptible isolates. In 11 patients, a genetically related resistant isolate was collected after recovering susceptible isolates, indicating in vivo acquisition of resistance. These findings allowed us to estimate the intra-host diversity of C. glabrata and propose an upper boundary of 96 SNPs for defining genetically related isolates, which can be used to assess donor-to-host transmission, nosocomial transmission, or acquired resistance. IMPORTANCE In our study, mutations associated to azole resistance and echinocandin resistance were detected in Candida glabrata isolates using a whole-genome sequence. C. glabrata is the second most common cause of candidemia in the United States, which rapidly acquires resistance to antifungals, in vitro and in vivo.

Epidemiology and risk factors for hepatitis C virus infection in a high-prevalence population.

To understand increasing rates of hepatitis C virus (HCV) infection in Tennessee, we conducted testing, risk factor analysis and a nested case-control study among persons who use drugs. During June-October 2016, HCV testing with risk factor assessment was conducted in sexually transmitted disease clinics, family planning clinics and an addiction treatment facility in eastern Tennessee; data were analysed by using multivariable logistic regression. A nested case-control study was conducted to assess drug-using risks and behaviours among persons who reported intranasal or injection drug use (IDU). Of 4753 persons tested, 397 (8.4%) were HCV-antibody positive. HCV infection was significantly associated with a history of both intranasal and IDU (adjusted odds ratio (aOR) 35.4, 95% confidence interval (CI) 24.1-51.9), IDU alone (aOR 52.7, CI 25.3-109.9), intranasal drug use alone (aOR 2.6, CI 1.8-3.9) and incarceration (aOR 2.7, CI 2.0-3.8). By 4 October 2016, 574 persons with a reported history of drug use; 63 (11%) were interviewed further. Of 31 persons who used both intranasal and injection drugs, 26 (84%) reported previous intranasal drug use, occurring 1-18 years (median 5.5 years) before their first IDU. Our findings provide evidence that reported IDU, intranasal drug use and incarceration are independent indicators of risk for past or present HCV infection in the study population.

Heavy alcohol use as a risk factor for severe outcomes among adults hospitalized with laboratory-confirmed influenza, 2005-2012.

We examined heavy alcohol use as a risk factor for severe influenza (intensive care admission or death) among hospitalized adults. In <65- and ≥65-year-olds, heavy alcohol use increased disease severity [relative risk (RR) 1.34; 95 % confidence interval (CI): 1.04-1.74, and RR 2.47; 95 % CI: 1.69-3.60, respectively]. Influenza vaccination and early, empiric antiviral treatment should be emphasized in this population.

Clinical and laboratory characteristics of invasive infections due to methicillin-resistant Staphylococcus aureus isolates demonstrating a vancomycin MIC of 2 micrograms per milliliter: lack of effect of heteroresistant vancomycin-intermediate S. aureus phenotype.

We describe clinical and laboratory characteristics of invasive methicillin-resistant Staphylococcus aureus (MRSA) infections with vancomycin MICs of 2 µg/ml and compare heteroresistant-intermediate S. aureus (hVISA) to non-hVISA. Health care-associated community-onset infections were the most common and resulted in frequent complications and relapses. hVISA-infected patients were more likely to have been hospitalized in the year prior to MRSA culture.

Novel screening procedure for recombinant plasmids.

Lysed bacterial colonies containing potential recombinant plasmids were mixed with molten agar and sealed into slots of an agarose gel. After electrophoresis overnight, the gel was stained with ethidium bromide, which clearly reveals recombinant plasmids. Xenopus laevis ribosomal DNA and histone DNA of Psammechinus miliaris were ligated into pCRI plasmids and screened by this method.

Transcriptional activation modulated by homopolymeric glutamine and proline stretches.

Many transcription factors contain proline- or glutamine-rich activation domains. Here it is shown that simple homopolymeric stretches of these amino acids can activate transcription when fused to the DNA binding domain of GAL4 factor. In vitro, activity increased with polymer length, whereas in cell transfection assays maximal activity was achieved by 10 to 30 glutamines or about 10 prolines. Similar results were obtained when glutamine stretches were placed within a [GAL4]-VP16 chimeric protein. Because these stretches are encoded by rapidly evolving triplet repeats (microsatellites), they may be the main cause for modulation of transcription factor activity and thus result in subtle or overt genomic effects.

Molecular genetic analyses of the Tyrolean Ice Man.

An approximately 5000-year-old mummified human body was recently found in the Tyrolean Alps. The DNA from tissue samples of this Late Neolithic individual, the so-called "Ice Man," has been extracted and analyzed. The number of DNA molecules surviving in the tissue was on the order of 10 genome equivalents per gram of tissue, which meant the only multi-copy sequences could be analyzed. The degradation of the DNA made the enzymatic amplification of mitochondrial DNA fragments of more than 100 to 200 base pairs difficult. One DNA sequence of a hypervariable segment of the mitochondrial control region was determined independently in two different laboratories from internal samples of the body. This sequence showed that the mitochondrial type of the Ice Man fits into the genetic variation of contemporary Europeans and that it was most closely related to mitochondrial types determined from central and northern European populations.

Transcriptional enhancers can act in trans.

Enhancers have been defined operationally as cis-regulatory sequences that can stimulate transcription of RNA polymerase-II-transcribed genes over large distances and even when located downstream of the gene. Recently, it has become evident that enhancers can also stimulate transcription in trans if they are brought into close proximity to the promoter/gene. These reports provide clues to the mechanism of remote enhancer action. In addition, the findings, together with genetic studies in Drosophila, strongly suggest that enhancer action in trans could underlie phenomena such as 'transvection', where one chromosome affects gene expression in the paired homolog.

Conservation of glutamine-rich transactivation function between yeast and humans.

Several eukaryotic transcription factors such as Sp1 or Oct1 contain glutamine-rich domains that mediate transcriptional activation. In human cells, promoter-proximally bound glutamine-rich activation domains activate transcription poorly in the absence of acidic type activators bound at distal enhancers, but synergistically stimulate transcription with these remote activators. Glutamine-rich activation domains were previously reported to also function in the fission yeast Schizosaccharomyces pombe but not in the budding yeast Saccharomyces cerevisiae, suggesting that budding yeast lacks this pathway of transcriptional activation. The strong interaction of an Sp1 glutamine-rich domain with the general transcription factor TAF(II)110 (TAF(II)130), and the absence of any obvious TAF(II)110 homologue in the budding yeast genome, seemed to confirm this notion. We reinvestigated the phenomenon by reconstituting in the budding yeast an enhancer-promoter architecture that is prevalent in higher eukaryotes but less common in yeast. Under these conditions, we observed that glutamine-rich activation domains derived from both mammalian and yeast transcription factors activated only poorly on their own but strongly synergized with acidic activators bound at the remote enhancer position. The level of activation by the glutamine-rich activation domains of Sp1 and Oct1 in combination with a remote enhancer was similar in yeast and human cells. We also found that mutations in a glutamine-rich domain had similar phenotypes in budding yeast and human cells. Our results show that glutamine-rich activation domains behave very similarly in yeast and mammals and that their activity in budding yeast does not depend on the presence of a TAF(II)110 homologue.

The Drosophila homolog of mammalian zinc finger factor MTF-1 activates transcription in response to heavy metals.

Metallothioneins (MTs) are short, cysteine-rich proteins for heavy metal homeostasis and detoxification; they bind a variety of heavy metals and also act as radical scavengers. Transcription of mammalian MT genes is activated by heavy metal load via the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein whose elimination in mice leads to embryonic lethality due to liver decay. Here we characterize the Drosophila homolog of vertebrate MTF-1 (dMTF-1), a 791-amino-acid protein which is most similar to its mammalian counterpart in the DNA-binding zinc finger region. Like mammalian MTF-1, dMTF-1 binds to conserved metal-responsive promoter elements (MREs) and requires zinc for DNA binding, yet some aspects of heavy metal regulation have also been subject to divergent evolution between Drosophila and mammals. dMTF-1, unlike mammalian MTF-1, is resistant to low pH (6 to 6.5). Furthermore, mammalian MT genes are activated best by zinc and cadmium, whereas in Drosophila cells, cadmium and copper are more potent inducers than zinc. The latter species difference is most likely due to aspects of heavy metal metabolism other than MTF-1, since in transfected mammalian cells, dMTF-1 responds to zinc like mammalian MTF-1. Heavy metal induction of both Drosophila MTs is abolished by double-stranded RNA interference: small amounts of cotransfected double-stranded RNA of dMTF-1 but not of unrelated control RNA inhibit the response to both the endogenous dMTF-1 and transfected dMTF-1. These data underline an important role for dMTF-1 in MT gene regulation and thus heavy metal homeostasis.

Target gene search for the metal-responsive transcription factor MTF-1.

Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding alpha-fetoprotein, the liver-enriched transcription factor C/EBPalpha and tear lipocalin/von Ebner's gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns gamma-glutamylcysteine synthetase(hc) (gamma-GCS(hc)), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. gamma-GCS(hc) mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

Placenta growth factor gene expression is induced by hypoxia in fibroblasts: a central role for metal transcription factor-1.

Placenta growth factor (PlGF) is a mitogen for endothelial cells that can potentiate the growth and permeabilizing effects on endothelium of vascular endothelial growth factor. Here we report that hypoxia induces the expression of both PlGF mRNA and protein in immortalized/transformed mouse embryonic fibroblasts (mEFs) and in NIH 3T3 cells. Importantly, the magnitude of the induction of PlGF expression by hypoxia is enhanced by the presence of oncogenic Ras. To investigate the transcriptional component of hypoxia-inducible PlGF expression, we cloned and sequenced a 1350-bp fragment of the 5'-flanking region of the mouse gene. Analysis of the promoter region indicated the presence of putative consensus sequences for known hypoxia-responsive regulatory sites, including metal response elements and Sp1-like sites. In the present study, we show that the induction of PlGF expression by hypoxia is dependent on the presence of the metal response element-binding transcription factor 1 (MTF-1). Thus, in mEFs with targeted deletions of both MTF-1 alleles, hypoxia-induced increases of PIGF mRNA and protein levels were greatly attenuated compared with those in wild-type mEFs. Moreover, transient transfection of a PlGF promoter reporter gene into NIH 3T3 cells resulted in hypoxia-responsive transcriptional activation of the reporter. Finally, ectopic expression of MTF-1 resulted in increased basal transcriptional activity of a PlGF promoter reporter. Together, these findings demonstrate that the PlGF gene is responsive to hypoxia and that this response is mediated by MTF-1. It remains to be determined whether this activation is the result of direct and/or indirect transcriptional activation by MTF-1. The stimulatory effect of oncogenic Ras on the induction of PlGF expression in hypoxic cells suggests that PlGF could be an important proangiogenic factor in the tumor microenvironment.

Conserved cysteine residues of Oct-2 POU domain confer sensitivity to oxidation but are dispensable for sequence-specific DNA binding.

The POU family of proteins, including the Oct-2 transcription factor, is characterized by a highly conserved bipartite DNA binding domain containing a 'POU homeodomain', distantly related to homeodomains of other DNA binding proteins, and a 'POU specific' domain unique to this class of factors. Prompted by the finding that in vitro DNA binding by Oct-2 is reversibly inhibited by oxidation of the protein, we investigated the role of the cysteine residues in the POU domain. All POU homeodomains identified contain a cysteine in the helix 3 region presumed to contact DNA directly; many (including Oct-2) also contain a less-well conserved cysteine residue(s) in the POU specific domain. Replacement of these cysteines with serine residues rendered the DNA binding domain resistant to oxidation but did not appreciably change the binding to a canonical octamer sequence, suggesting that the conserved cysteine residues are not required for sequence-specific DNA contacts, but may be important for another function.

Synergistic activation of transcription by multiple binding sites for NF-kappa B even in absence of co-operative factor binding to DNA.

Regulation of eukaryotic genes is largely governed by multiple cis-acting DNA sequences recognized by specific transcription factors. The transcription factor NF-kappa B has been implicated as an important regulator of cellular and viral genes, including those of immunoglobulin kappa light chain, interleukin-2, beta-interferon, HIV-1 and cytomegalovirus. We have analyzed the effect of increasing the number of NF-kappa B sites, located directly upstream from the TATA box. Four copies of the sequence gave a more than 100-fold stimulation relative to a single copy, suggesting that NF-kappa B proteins act synergistically to bring about this dramatic increase in transcription. By DNase I footprinting we demonstrated factor binding to two adjacent NF-kappa B sites in vitro. However, we found no evidence for co-operative binding to these DNA sites. We propose that the high transcriptional activity results from another type of co-operation, based on multiple weak interactions of the NF-kappa B factors with another component of the transcription apparatus, perhaps RNA polymerase II itself.

Immediate early protein of pseudorabies virus is a general transactivator but stimulates only suboptimally utilized promoters. A clue to specificity?

Pseudorabies virus, a herpesvirus, encodes an immediate early (IE) protein that is known to be a general and strong transactivator of transcription. We have tested the activity of this IE protein with a set of well-defined promoters containing a TATA box and one type of upstream factor binding site (for Sp1, NF-kappa B, heavy metal responsive factors, octamer factors or glucocorticoid receptor). All promoters were strongly activated by IE protein, i.e. the IE protein did not preferentially activate transcription via a particular type of upstream element. Activation did not require a bona fide TATA box, since a promoter construct with three Sp1 sites but no TATA box was also activated. Our data are not compatible with a model in which IE protein would bypass the need for upstream factors. Rather, the properties of IE protein, especially a failure to induce strong transcription from a promoter with only a TATA box but no upstream sequences, mimic the action of a remotely placed, cis-active, enhancer DNA. The IE protein was found to have no effect on transcription units that are expressed to their maximal potential, irrespective of whether this was high or low. Such optimal transcription conditions are observed in the presence of a strong enhancer, or with multiple tandem copies of an upstream binding site and/or a high concentration of the corresponding factor. The property of stimulating only "suboptimally" utilized promoters may be exploited by pseudorabies virus to restrict the specificity of the IE protein to the viral early promoters and a subset of cellular promoters.

Redundancy of information in enhancers as a principle of mammalian transcription control.

In contrast to prokaryotes, in which strong transcriptional signals can be located within very short DNA segments, typical mammalian enhancers are about 200 base-pairs long. We reasoned that a minimal length of enhancer-active DNA is required for a high transcription rate in higher eukaryotes, and that segments from a single enhancer or from different enhancers might be multimerized or combined to satisfy such a requirement. To test this, enhancer fragments from different viruses were joined in a recombinant simian virus 40 (SV40) and screened for efficiency of viral growth. The 48 combinations tested show that the hypothesis is basically correct. For example, two subfunctional heterologous enhancer fragments can together form a functional enhancer. No enhancer shorter than 84 base-pairs could promote SV40 growth, i.e. in no case did we find a short "superstrong" enhancer segment. To test whether multimerization of a short fragment would result in a strong enhancer, we have synthesized a 50 base-pair enhancer segment derived from Herpesvirus saimiri. One to six copies of this oligonucleotide gave an incremental increase in enhancer activity. We propose, therefore, that mammalian gene regulation is based on a redundancy of information that can be provided either by a combination of different DNA sequence elements, or by multiple copies of the same element. Also, the finding of strong and weak enhancers suggests that in most cases an enhancer is permanently required for transcription of a gene, rather than acting in an all-or-none fashion to establish a transcription complex, after which it becomes dispensable.

Cimicifuga racemosa dried ethanolic extract in menopausal disorders: a double-blind placebo-controlled clinical trial.

OBJECTIVES: To compare the efficacy and safety of the black cohosh root extract Cr 99 with placebo in women with climacteric complaints. METHODS: A multicenter, randomized, placebo-controlled, double-blind, parallel group study was conducted in 122 menopausal women (intention-to-treat population) with > or =3 hot flashes a day, treated over 12 weeks. Two main efficacy measures - weekly weighted score of hot flashes and Kupperman Index - and secondary efficacy variables, e.g. Menopause Rating Scale, were defined. Routine safety laboratory parameters and adverse events were documented. RESULTS: The primary efficacy analysis showed no superiority of the tested black cohosh extract compared to placebo. However, in the subgroup of patients with a Kupperman Index> or =20 a significant superiority regarding this index could be demonstrated (P<0.018). A decrease of 47% and 21% was observed in the black cohosh and placebo group, respectively. The weekly weighted scores of hot flashes (P<0.052) and the Menopause Rating Scale (P<0.009) showed similar results. Prevalence and intensity of the adverse events did not differ in the two treatment groups. CONCLUSIONS: The results indicate a superiority of the tested Cimicifuga racemosa extract compared to placebo in patients with menopausal disorders of at least moderate intensity according to a Kupperman Index > or =20, but not in the intention-to-treat population as a whole.

Mumps transmission in hospitals.

Although sporadic transmission of mumps within hospitals to patients and staff is well documented, outbreaks of mumps within hospitals have only rarely been reported. The widespread mumps outbreaks that occurred in Tennessee in 1986-1987 provided an opportunity to assess the extent of the problems caused by mumps in hospitals. Information was obtained from 146 (95%) of 154 infection control practitioners in the state. Infection control problems caused by mumps were reported from 17 (12%) of 146 hospitals. The 17 hospitals in which these incidents occurred were located in counties that accounted for 67% of the reported mumps cases statewide during this period. Although most cases of mumps in health care workers were community-acquired, six health care workers in three different hospitals developed mumps following nosocomial exposure. In two institutions, nine patients contracted mumps while hospitalized. Both were long-term-care facilities housing adolescents, who had substantial contact with a community where mumps outbreaks were ongoing. This study suggests that mumps poses a small but real risk to both patients and staff in hospitals, particularly in long-term-care facilities caring for adolescents and young adults. In communities where mumps activity is ongoing, hospitals should consider identifying potentially susceptible staff members at risk for infection and offering vaccine. Likewise, susceptible patients in long-term-care facilities should be immunized.

Two versatile eukaryotic vectors permitting epitope tagging, radiolabelling and nuclear localisation of expressed proteins.

Two versatile eukaryotic expression vectors have been developed which permit the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells or by in vitro transcription-translation. The first vector, pCATCH, can be used to clone cDNA inserts in three different frames via eight unique restriction sites in a multiple cloning site (MCS) located downstream from both the FLAG epitope and the specific heart muscle kinase phosphorylation site, conferring the possibility of in vitro radiolabelling. A specific protease cleavage site enables the removal of the FLAG epitope, simplifying affinity purification of recombinant CATCH proteins. pCATCH possesses stop codons in all three reading frames at the 3' terminal end of the MCS. A derivate of this vector, pCATCH-NLS, was constructed by incorporating an SV40 nuclear localisation signal upstream from the MCS, for directed localisation of the tagged proteins.