Molecular mechanisms of action of herbal antifungal alkaloid berberine, in Candida albicans.

Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

Early dyadic patterns of mother-infant interactions and outcomes of prematurity at 18 months.

OBJECTIVE: With the increased survival of very preterm infants, there is a growing concern for their developmental and socioemotional outcomes. The quality of the early mother-infant relationship has been noted as 1 of the factors that may exacerbate or soften the potentially adverse impact of preterm birth, particularly concerning the infant's later competencies and development. The first purpose of the study was to identify at 6 months of corrected age whether there were specific dyadic mother-infant patterns of interaction in preterm as compared with term mother-infant dyads. The second purpose was to examine the potential impact of these dyadic patterns on the infant's behavioral and developmental outcomes at 18 months of corrected age. METHODS: During a 12-month period (January-December 1998), all preterm infants who were <34 weeks of gestational age and hospitalized at the NICU of the Lausanne University Hospital were considered for inclusion in this longitudinal prospective follow-up study. Control healthy term infants were recruited during the same period from the maternity ward of our hospital. Mother-infant dyads with preterm infants (n = 47) and term infants (n = 25) were assessed at 6 months of corrected age during a mother-infant play interaction and coded according to the Care Index. This instrument evaluates the mother's interactional behavior according to 3 scales (sensitivity, control, and unresponsiveness) and the child's interactional behavior according to 4 scales (cooperation, compliance, difficult, and passivity). At 18 months, behavioral outcomes of the children were assessed on the basis of a semistructured interview of the mother, the Symptom Check List. The Symptom Check List explores 4 groups of behavioral symptoms: sleeping problems, eating problems, psychosomatic symptoms, and behavioral and emotional disorders. At the same age, developmental outcomes were evaluated using the Griffiths Developmental Scales. Five areas were evaluated: locomotor, personal-social, hearing and speech, eye-hand coordination, and performance. RESULTS: Among the possible dyadic patterns of interaction, 2 patterns emerge recurrently in mother-infant preterm dyads: a "cooperative pattern" with a sensitive mother and a cooperative-responsive infant (28%) and a "controlling pattern" with a controlling mother and a compulsive-compliant infant (28%). The remaining 44% form a heterogeneous group that gathers all of the other preterm dyads and is composed of 1 sensitive mother-passive infant; 10 controlling mothers with a cooperative, difficult, or passive infant; and 10 unresponsive mothers with a cooperative, difficult, or passive infant. Among the term control subjects, 68% of the dyads are categorized as cooperative pattern dyads, 12% as controlling pattern dyads, and the 20% remaining as heterogeneous dyads. At 18 months, preterm infants of cooperative pattern dyads have similar outcomes as the term control infants. Preterm infants of controlling pattern dyads have significantly fewer positive outcomes as compared with preterm infants of cooperative pattern dyads, as well as compared with term control infants. They display significantly more behavioral symptoms than term infants, including more eating problems than term infants as well as infants from cooperative preterm dyads. Infants of the controlling preterm dyads do not differ significantly for the total development quotient but have worse personal-social development than term infants and worse hearing-speech development than infants from cooperative preterm dyads. The preterm infants of the heterogeneous group have outcomes that can be considered as intermediate with no significant differences compared with preterm infants from the cooperative pattern or the controlling pattern dyads. CONCLUSION: Among mother-preterm infant dyads, we identified 2 specific patterns of interaction that could play either a protective (cooperative pattern) or a risk-precipitating (controlling pattern) role on developmental and behavioral outcome, independent of perinatal risk factors and of the family's socioeconomic background. The controlling pattern is much more prevalent among preterm than term dyads and is related to a less favorable infant outcome. However, the cooperative pattern still represents almost 30% of the preterm dyads, with infants' outcome comparable to the ones of term infants. These results point out the impact of the quality of mother-infant relationship on the infant's outcome. The most important clinical implication should be to support a healthy parent-infant relationship already in the NICU but also in the first months of the infant's life. Early individualized family-based interventions during neonatal hospitalization and transition to home have been shown to reduce maternal stress and depression and increase maternal self-esteem and to improve positive early parent-preterm infant interactions.

Plasticity of protein expression during culture of fetal skin cells.

In order to gain insight into the biology of fetal skin during culture, cellular proteins were studied during four culture passages (P00, P01, P04 as well as P10) using high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS). Bioinformatic analyses were focused on a region of each gel corresponding to pI between 4 and 8 and M(r) from 8000 to 35 000. In this area, 373 +/- 42 spots were detected (N = 18). Twenty-six spots presented an integrated intensity that increased in the higher passages, whereas five spots showed a progressively lower intensity in subsequent passaging. MS analysis was performed on spots that were unambiguously identified on preparative 2-D gels. Among the 26 spots showing an increased size between P00 and P10, 9 were identified, and corresponded to 3 proteins: (i) peptidyl-prolyl cis-trans isomerase A (P05092; cyclophilin A or cyclosporin A-binding protein), (ii) triosephosphate isomerase (P00938), and (iii) enoyl-CoA hydratase (P30084). Among these nine identified spots, three were absent at P00, but were present at P10. They corresponded to isoforms of peptidyl-prolyl cis-trans isomerase and triosephosphate isomerase, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the acidic isoforms of triosephosphate isomerase showed modifications of cysteine residues to cysteic acid. All these isoforms were clearly present in the skin cells of a 4-year-old child, as well as in skin cells from a 80-year-old man, at P00. These observations probably reflect either an oxidative stress related to cell culture, or, alternatively, maturation, differentiation and the aging of the cells.

Optimization of preservation conditions of As (III) bioreporter bacteria.

Long-term preservation of bioreporter bacteria is essential for the functioning of cell-based detection devices, particularly when field application, e.g., in developing countries, is intended. We varied the culture conditions (i.e., the NaCl content of the medium), storage protection media, and preservation methods (vacuum drying vs. encapsulation gels remaining hydrated) in order to achieve optimal preservation of the activity of As (III) bioreporter bacteria during up to 12 weeks of storage at 4 degrees C. The presence of 2% sodium chloride during the cultivation improved the response intensity of some bioreporters upon reconstitution, particularly of those that had been dried and stored in the presence of sucrose or trehalose and 10% gelatin. The most satisfying, stable response to arsenite after 12 weeks storage was obtained with cells that had been dried in the presence of 34% trehalose and 1.5% polyvinylpyrrolidone. Amendments of peptone, meat extract, sodium ascorbate, and sodium glutamate preserved the bioreporter activity only for the first 2 weeks, but not during long-term storage. Only short-term stability was also achieved when bioreporter bacteria were encapsulated in gels remaining hydrated during storage.

Moving On Moves Recidivism Rates Lower, Data Download, Iowa Department of Corrections, October 2009

The results of a recently conducted evaluation show that a gender-responsive program for women probationers holds promise in significantly reducing recidivism rates. The University of Cincinnati conducted the evaluation of the cognitive-behavioral program, Moving On, in which it compared recidivism outcomes for moderate to high-risk Iowa offenders completing the program with a similar group of offenders not having attended any cognitive program.

Iowa Criminal and Juvenile Justice Plan, 1998

Pursuant to Iowa Code 216A, subchapter 9, CJJP is required to issue an annual report containing long-range systems goals, special issue planning recommendations and research findings. CJJP’s 1998 response to its reporting requirement is replicated in the manner of the distribution of the 1997 Update. Again this year, CJJP is issuing one large document which contains many separate reports. Single-issue 1998 Update reports will be made available based on reader interest and need. Having utilized this disseminating approach of CJJP research and reports in 1997, it proved to be cost effective and responsive to the planning activities and information needs of Iowa’s policy makers, justice system officials and others.

Induction of the acyl-coenzyme A synthetase gene by fibrates and fatty acids is mediated by a peroxisome proliferator response element in the C promoter.

The long-chain acyl-coenzyme A synthetase (ACS) gene gives rise to three transcripts containing different first exons preceded by specific regulatory regions A, B, and C. Exon-specific oligonucleotide hybridization indicated that only A-ACS mRNA is expressed in rat liver. Fibrate administration induced liver C-ACS strongly and A-ACS mRNA to a lesser extent. B-ACS mRNA remained undetectable. In primary rat hepatocytes and Fa-32 hepatoma cells C-ACS mRNA increased after treatment with fenofibric acid, alpha-bromopalmitate, tetradecylthioacetic acid, or alpha-linolenic acid. Nuclear run-on experiments indicated that fenofibric acid and alpha-bromopalmitate act at the transcriptional level. Transient transfections showed a 3.4-, 2.3-, and 2.2-fold induction of C-ACS promoter activity after fenofibric acid, alpha-bromopalmitate, and tetradecylthioacetic acid, respectively. Unilateral deletion and site-directed mutagenesis identified a peroxisome proliferator activator receptor (PPAR)-responsive element (PPRE) mediating the responsiveness to fibrates and fatty acids. This ACS PPRE contains three imperfect half sites spaced by 1 and 3 oligonucleotides and binds PPAR.retinoid X receptor heterodimers in gel retardation assays. In conclusion, the regulation of C-ACS mRNA expression by fibrates and fatty acids is mediated by PPAR.retinoid X receptor heterodimers interacting through a PPRE in the C-ACS promoters. PPAR therefore occupies a key position in the transcriptional control of a pivotal enzyme controlling the channeling of fatty acids into various metabolic pathways.

Iowa Criminal and Juvenile Justice Plan 1997

Pursuant to Iowa Code 216A, subchapter 9, CJJP is required to issue an annual report containing long-range system goals, special issue planning recommendations and research findings. CJJP’s 1997 response to its reporting requirement is different from past years. Rather than issuing one large document containing many separate reports, single-issue 1997 Update reports now are being made available based on reader interest and need. It is hoped this approach to disseminating CJJP research and planning reports will be more cost effective and more responsive to the planning activities and information needs of Iowa’s policy makers, justice system officials and others.

Integrated methodologies to study human transcriptional regulation from functional genomics data

Abstract : The human body is composed of a huge number of cells acting together in a concerted manner. The current understanding is that proteins perform most of the necessary activities in keeping a cell alive. The DNA, on the other hand, stores the information on how to produce the different proteins in the genome. Regulating gene transcription is the first important step that can thus affect the life of a cell, modify its functions and its responses to the environment. Regulation is a complex operation that involves specialized proteins, the transcription factors. Transcription factors (TFs) can bind to DNA and activate the processes leading to the expression of genes into new proteins. Errors in this process may lead to diseases. In particular, some transcription factors have been associated with a lethal pathological state, commonly known as cancer, associated with uncontrolled cellular proliferation, invasiveness of healthy tissues and abnormal responses to stimuli. Understanding cancer-related regulatory programs is a difficult task, often involving several TFs interacting together and influencing each other's activity. This Thesis presents new computational methodologies to study gene regulation. In addition we present applications of our methods to the understanding of cancer-related regulatory programs. The understanding of transcriptional regulation is a major challenge. We address this difficult question combining computational approaches with large collections of heterogeneous experimental data. In detail, we design signal processing tools to recover transcription factors binding sites on the DNA from genome-wide surveys like chromatin immunoprecipitation assays on tiling arrays (ChIP-chip). We then use the localization about the binding of TFs to explain expression levels of regulated genes. In this way we identify a regulatory synergy between two TFs, the oncogene C-MYC and SP1. C-MYC and SP1 bind preferentially at promoters and when SP1 binds next to C-NIYC on the DNA, the nearby gene is strongly expressed. The association between the two TFs at promoters is reflected by the binding sites conservation across mammals, by the permissive underlying chromatin states 'it represents an important control mechanism involved in cellular proliferation, thereby involved in cancer. Secondly, we identify the characteristics of TF estrogen receptor alpha (hERa) target genes and we study the influence of hERa in regulating transcription. hERa, upon hormone estrogen signaling, binds to DNA to regulate transcription of its targets in concert with its co-factors. To overcome the scarce experimental data about the binding sites of other TFs that may interact with hERa, we conduct in silico analysis of the sequences underlying the ChIP sites using the collection of position weight matrices (PWMs) of hERa partners, TFs FOXA1 and SP1. We combine ChIP-chip and ChIP-paired-end-diTags (ChIP-pet) data about hERa binding on DNA with the sequence information to explain gene expression levels in a large collection of cancer tissue samples and also on studies about the response of cells to estrogen. We confirm that hERa binding sites are distributed anywhere on the genome. However, we distinguish between binding sites near promoters and binding sites along the transcripts. The first group shows weak binding of hERa and high occurrence of SP1 motifs, in particular near estrogen responsive genes. The second group shows strong binding of hERa and significant correlation between the number of binding sites along a gene and the strength of gene induction in presence of estrogen. Some binding sites of the second group also show presence of FOXA1, but the role of this TF still needs to be investigated. Different mechanisms have been proposed to explain hERa-mediated induction of gene expression. Our work supports the model of hERa activating gene expression from distal binding sites by interacting with promoter bound TFs, like SP1. hERa has been associated with survival rates of breast cancer patients, though explanatory models are still incomplete: this result is important to better understand how hERa can control gene expression. Thirdly, we address the difficult question of regulatory network inference. We tackle this problem analyzing time-series of biological measurements such as quantification of mRNA levels or protein concentrations. Our approach uses the well-established penalized linear regression models where we impose sparseness on the connectivity of the regulatory network. We extend this method enforcing the coherence of the regulatory dependencies: a TF must coherently behave as an activator, or a repressor on all its targets. This requirement is implemented as constraints on the signs of the regressed coefficients in the penalized linear regression model. Our approach is better at reconstructing meaningful biological networks than previous methods based on penalized regression. The method is tested on the DREAM2 challenge of reconstructing a five-genes/TFs regulatory network obtaining the best performance in the "undirected signed excitatory" category. Thus, these bioinformatics methods, which are reliable, interpretable and fast enough to cover large biological dataset, have enabled us to better understand gene regulation in humans.

The role of actions in auditory object discrimination.

Action representations can interact with object recognition processes. For example, so-called mirror neurons respond both when performing an action and when seeing or hearing such actions. Investigations of auditory object processing have largely focused on categorical discrimination, which begins within the initial 100 ms post-stimulus onset and subsequently engages distinct cortical networks. Whether action representations themselves contribute to auditory object recognition and the precise kinds of actions recruiting the auditory-visual mirror neuron system remain poorly understood. We applied electrical neuroimaging analyses to auditory evoked potentials (AEPs) in response to sounds of man-made objects that were further subdivided between sounds conveying a socio-functional context and typically cuing a responsive action by the listener (e.g. a ringing telephone) and those that are not linked to such a context and do not typically elicit responsive actions (e.g. notes on a piano). This distinction was validated psychophysically by a separate cohort of listeners. Beginning approximately 300 ms, responses to such context-related sounds significantly differed from context-free sounds both in the strength and topography of the electric field. This latency is >200 ms subsequent to general categorical discrimination. Additionally, such topographic differences indicate that sounds of different action sub-types engage distinct configurations of intracranial generators. Statistical analysis of source estimations identified differential activity within premotor and inferior (pre)frontal regions (Brodmann's areas (BA) 6, BA8, and BA45/46/47) in response to sounds of actions typically cuing a responsive action. We discuss our results in terms of a spatio-temporal model of auditory object processing and the interplay between semantic and action representations.

An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Biological activity of ectodysplasin A is conditioned by its collagen and heparan sulfate proteoglycan-binding domains.

Mutations in the TNF family ligand EDA1 cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition characterized by defective development of skin appendages. The EDA1 protein displays a proteolytic processing site responsible for its conversion to a soluble form, a collagen domain, and a trimeric TNF homology domain (THD) that binds the receptor EDAR. In-frame deletions in the collagen domain reduced the thermal stability of EDA1. Removal of the collagen domain decreased its activity about 100-fold, as measured with natural and engineered EDA1-responsive cell lines. The collagen domain could be functionally replaced by multimerization domains or by cross-linking antibodies, suggesting that it functions as an oligomerization unit. Surprisingly, mature soluble EDA1 containing the collagen domain was poorly active when administered in newborn, EDA-deficient (Tabby) mice. This was due to a short stretch of basic amino acids located at the N terminus of the collagen domain that confers EDA1 with proteoglycan binding ability. In contrast to wild-type EDA1, EDA1 with mutations in this basic sequence was a potent inducer of tail hair development in vivo. Thus, the collagen domain activates EDA1 by multimerization, whereas the proteoglycan-binding domain may restrict the distribution of endogeneous EDA1 in vivo.

9-cis-retinoic acid enhances fatty acid-induced expression of the liver fatty acid-binding protein gene.

The role of retinoic acids (RA) on liver fatty acid-binding protein (L-FABP) expression was investigated in the well differentiated FAO rat hepatoma cell line. 9-cis-Retinoic acid (9-cis-RA) specifically enhanced L-FABP mRNA levels in a time- and dose-dependent manner. The higher induction was found 6 h after addition of 10(-6) M 9-cis-RA in the medium. RA also enhanced further both L-FABP mRNA levels and cytosolic L-FABP protein content induced by oleic acid. The retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR), which are known to be activated, respectively, by 9-cis-RA and long chain fatty acid (LCFA), co-operated to bind specifically the peroxisome proliferator-responsive element (PPRE) found upstream of the L-FABP gene. Our result suggest that the PPAR-RXR complex is the molecular target by which 9-cis-RA and LCFA regulate the L-FABP gene.

Differential role of naïve and memory CD4 T-cell subsets in primary alloresponses.

The T cell response to major histocompatibility complex (MHC) alloantigens occurs via two main pathways. The direct pathway involves the recognition of intact allogeneic MHC:peptide complexes on donor cells and provokes uniquely high frequencies of responsive T cells. The indirect response results from alloantigens being processed like any other protein antigen and presented as peptide by autologous antigen-presenting cells. The frequencies of T cells with indirect allospecificity are orders of magnitude lower and comparable to other peptide-specific responses. In this study, we explored the contributions of naïve and memory CD4(+) T cells to these two pathways. Using an adoptive transfer and skin transplantation model we found that naive and memory CD4(+) T cells, both naturally occurring and induced by sensitization with multiple third-party alloantigens, contributed equally to graft rejection when only the direct pathway was operative. In contrast, the indirect response was predominantly mediated by the naïve subset. Elimination of regulatory CD4(+)CD25(+) T cells enabled memory cells to reject grafts through the indirect pathway, but at a much slower tempo than for naïve cells. These findings have implications for better targeting of immunosuppression to inhibit immediate and later forms of alloimmunity.

The major transcription initiation site of the SV40 late promoter is a potent thyroid hormone response element.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily, which act as transcription factors upon binding to specific DNA sequences called thyroid hormone (T3) response elements (TREs). Such elements are found in the upstream regulatory region of promoters as well as in intragenic sequences of T3-responsive genes. In this report, we demonstrate that SV40 late (SVL) promoter activity is strongly down-regulated by TR in the absence of ligand. Addition of T3 releases this repression, but does not further induce SVL promoter activity. Electrophoretic mobility shift analyses reveal a TR binding element that overlaps with the SV40 major late transcription initiation site. This element closely fits the consensus TRE, formed of two hexanucleotides organized in a tandem repeat separated by 4 nt, and is able to confer T3 responsiveness on a heterologous promoter. We further show that, although the presence of TR leads to quantitatively modified expression of an SVL-driven reporter gene, neither displacement of the site of transcription initiation nor modification of the splicing pattern of the primary transcripts occur.