Prospective epidemiologic studies have consistently shown that levels of circulating androgens in postmenopausal women are positively associated with breast cancer risk. However, data in premenopausal women are limited.

Two-component signal transduction (TCST) is the predominant signaling scheme used in bacteria to sense and respond to environmental changes in order to survive and thrive. A typical TCST system consists of a sensor histidine kinase to detect external signals and an effector response regulator to respond to external changes. In the signaling scheme, the histidine kinase phosphorylates and activates the response regulator, which functions as a transcription factor to modulate gene expression. One promising strategy toward antibacterial development is to target TCST regulatory systems, specifically the response regulators to disrupt the expression of genes important for virulence. In Salmonella enterica, the PhoQ/PhoP signal transduction system is used to sense and respond to low magnesium levels and regulates the expression for over 40 genes necessary for growth under these conditions, and more interestingly, genes that are important for virulence. In this study, a hybrid approach coupling computational and experimental methods was applied to identify drug-like compounds to target the PhoP response regulator. A computational approach of structure-based virtual screening combined with a series of biochemical and biophysical assays was used to test the predictability of the computational strategy and to characterize the mode of action of the compounds. Eight compounds from virtual screening inhibit the formation of the PhoP-DNA complex necessary for virulence gene regulation. This investigation served as an initial case study for targeting TCST response regulators to modulate the gene expression of a signal transduction pathway important for bacterial virulence. With the increasing resistance of pathogenic bacteria to current antibiotics, targeting TCST response regulators that control virulence is a viable strategy for the development of antimicrobial therapeutics with novel modes of action.

Newcastle disease virus (NDV) causes severe and economically important disease in poultry around the globe. None of NDV strains in Pakistan have been completely characterized and the role of rural poultry in harbouring NDV is unclear. Since they have a very important role for long-term circulation of the virus, samples were collected from apparently healthy backyard poultry (BYP) flocks. These samples were biologically analyzed using mean death time (MDT) and intracerebral pathogenicity index (ICPI), whereas genotypically characterized by the real-time PCRs coupled with sequencing of the complete genome.

Diabetes risk assessment has been proposed as part of the National Health Service Health Checks programme, and HbA(1c) has recently been recommended as a diagnostic test for diabetes at a threshold of 48 mmol/mol (6.5%). We estimated the potential population impact of different stepwise screening strategies to identify individuals at high risk who might be offered preventive interventions.

The clinical picture of the pandemic influenza A (H1N1)v ranges from a self-limiting afebrile infection to a rapidly progressive pneumonia. Prompt diagnosis and well-timed treatment are recommended. Chest radiography (CRx) often fails to detect the early interstitial stage. The aim of this study was to evaluate the role of bedside chest ultrasonography (US) in the early management of the 2009 influenza A (H1N1)v infection.

An increasing number of publications are suggesting that galectin-3 (Gal-3) and soluble cadherin fragments, such as E-cadherin (sE-CAD) and N-cadherin (sN-CAD), may be considered as cancer markers. Despite the promising results of the studies, there are no data concerning their levels in the plasma of echinococcosis patients. In most cases, echinoccocosis affects the liver, and its symptoms and disease course are very similar to those of liver cancer. The aim of the present study was to observe whether echinococcosis affects the concentration of soluble sN-CAD, sE-CAD fragments and Gal-3 in plasma and to determine which of them could be considered reliable liver cancer markers for further research.

Sonic hedgehog (SHH) is a regulator of forebrain development that acts through its receptor, patched 1. However, little is known about cellular mechanisms at neurulation, whereby SHH from the prechordal plate governs specification of the rostral diencephalon ventral midline (RDVM), a major forebrain organizer. We identified LRP2, a member of the LDL receptor gene family, as a component of the SHH signaling machinery in the RDVM. LRP2 acts as an apical SHH-binding protein that sequesters SHH in its target field and controls internalization and cellular trafficking of SHH/patched 1 complexes. Lack of LRP2 in mice and in cephalic explants results in failure to respond to SHH, despite functional expression of patched 1 and smoothened, whereas overexpression of LRP2 variants in cells increases SHH signaling capacity. Our data identify a critical role for LRP2 in SHH signaling and reveal the molecular mechanism underlying forebrain anomalies in mice and patients with Lrp2 defects.

Signaling events that regulate central nervous system (CNS) angiogenesis and blood-brain barrier (BBB) formation are only beginning to be elucidated. By evaluating the gene expression profile of mouse vasculature, we identified DR6/TNFRSF21 and TROY/TNFRSF19 as regulators of CNS-specific angiogenesis in both zebrafish and mice. Furthermore, these two death receptors interact both genetically and physically and are required for vascular endothelial growth factor (VEGF)-mediated JNK activation and subsequent human brain endothelial sprouting in vitro. Increasing beta-catenin levels in brain endothelium upregulate DR6 and TROY, indicating that these death receptors are downstream target genes of Wnt/beta-catenin signaling, which has been shown to be required for BBB development. These findings define a role for death receptors DR6 and TROY in CNS-specific vascular development.

Angiogenesis requires coordination of distinct cell behaviors between tip and stalk cells. Although this process is governed by regulatory interactions between the vascular endothelial growth factor (Vegf) and Notch signaling pathways, little is known about the potential role of microRNAs. Through deep sequencing and functional screening in zebrafish, we find that miR-221 is essential for angiogenesis. miR-221 knockdown phenocopied defects associated with loss of the tip cell-expressed Flt4 receptor. Furthermore, miR-221 was required for tip cell proliferation and migration, as well as tip cell potential in mosaic blood vessels. miR-221 knockdown also prevented "hyper-angiogenesis" defects associated with Notch deficiency and miR-221 expression was inhibited by Notch signaling. Finally, miR-221 promoted tip cell behavior through repression of two targets: cyclin dependent kinase inhibitor 1b (cdkn1b) and phosphoinositide-3-kinase regulatory subunit 1 (pik3r1). These results identify miR-221 as an important regulatory node through which tip cell migration and proliferation are controlled during angiogenesis.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit overexpression or amplification of the MYC oncogene (c-MYC) usually have an extremely poor prognosis, but there are no animal models of this subtype of the disease. Here, we show that cerebellar stem cells expressing Myc and mutant Trp53 (p53) generate aggressive tumors following orthotopic transplantation. These tumors consist of large, pleiomorphic cells and resemble human MYC-driven MB at a molecular level. Notably, antagonists of PI3K/mTOR signaling, but not Hedgehog signaling, inhibit growth of tumor cells. These findings suggest that cerebellar stem cells can give rise to MYC-driven MB and identify a novel model that can be used to test therapies for this devastating disease.

Elevated Aurora kinase-A expression is correlated with abrogation of DNA damage-induced apoptotic response and mitotic spindle assembly checkpoint (SAC) override in human tumor cells. We report that Aurora-A phosphorylation of p73 at serine235 abrogates its transactivation function and causes cytoplasmic sequestration in a complex with the chaperon protein mortalin. Aurora-A phosphorylated p73 also facilitates inactivation of SAC through dissociation of the MAD2-CDC20 complex in cells undergoing mitosis. Cells expressing phosphor-mimetic mutant (S235D) of p73 manifest altered growth properties, resistance to cisplatin- induced apoptosis, as well as premature dissociation of the MAD2-CDC20 complex, and accelerated mitotic exit with SAC override in the presence of spindle damage. Elevated cytoplasmic p73 in Aurora-A overexpressing primary human tumors corroborates the experimental findings.

Lupulin glands of hop produce a specific metabolome including hop bitter acids valuable for the brewing process and prenylflavonoids with promising health-beneficial activities. The detailed analysis of the transcription factor (TF)-mediated regulation of the oligofamily of one of the key enzymes, i.e., chalcone synthase CHS_H1 that efficiently catalyzes the production of naringenin chalcone, a direct precursor of prenylflavonoids in hop, constitutes an important part of the dissection of the biosynthetic pathways leading to the accumulation of these compounds.

Small molecules with potent biological effects on the fate of normal and cancer-derived stem cells represent both useful research tools and new drug leads for regenerative medicine and oncology. Long-term expansion of mouse and human neural stem cells is possible using adherent monolayer culture. These cultures represent a useful cellular resource to carry out image-based high content screening of small chemical libraries. Improvements in automated microscopy, desktop computational power, and freely available image processing tools, now means that such chemical screens are realistic to undertake in individual academic laboratories. Here we outline a cost effective and versatile time lapse imaging strategy suitable for chemical screening. Protocols are described for the handling and screening of human fetal Neural Stem (NS) cell lines and their malignant counterparts, Glioblastoma-derived neural stem cells (GNS). We focus on identification of cytostatic and cytotoxic "hits" and discuss future possibilities and challenges for extending this approach to assay lineage commitment and differentiation.

We recently reported that the α(2)-adrenoreceptor (AR) ligand allyphenyline (9) significantly enhanced morphine analgesia (due to its α(2C)-AR agonism), was devoid of sedative side effects (due to its α(2A)-AR antagonism), prevented and reversed morphine tolerance and dependence. To highlight the molecular characteristics compatible with this behaviour and to obtain novel agents potentially useful in chronic pain and opioid addiction management, the allyl group of 9 was replaced by substituents of moderate steric bulk (MR) and positive or negative lipophilic (π) and electronic (σ) contributions in all the possible combinations. Effective novel α(2C)-agonists/α(2A)-antagonists (2, 3, 10, 12, and 17) were obtained. This study also demonstrated that contradictory combinations of the physicochemical parameters were similarly able to induce the α(2A)-activation. Since we had previously observed that the absolute configuration affected only the potency, but not the functional profile of the ligands, we hypothesized that the α(2A)-activation was governed by a ligand preferred conformation. From a structural overlay investigation it emerged that an extended conformation appeared to be associated with dual α(2C)-agonism/α(2A)-antagonism, whereas a folded conformation associated with α(2C)-/α(2A)-agonism.

Genetically encoded calcium indicators (GECIs), together with modern microscopy, allow repeated activity measurement, in real time and with cellular resolution, of defined cellular populations. Recent efforts in protein engineering have yielded several high-quality GECIs that facilitate new applications in neuroscience. Here, we summarize recent progress in GECI design, optimization, and characterization, and provide guidelines for selecting the appropriate GECI for a given biological application. We focus on the unique challenges associated with imaging in behaving animals.

To assess telomerase as a cancer therapeutic target and determine adaptive mechanisms to telomerase inhibition, we modeled telomerase reactivation and subsequent extinction in T cell lymphomas arising in Atm(-/-) mice engineered with an inducible telomerase reverse transcriptase allele. Telomerase reactivation in the setting of telomere dysfunction enabled full malignant progression with alleviation of telomere dysfunction-induced checkpoints. These cancers possessed copy number alterations targeting key loci in human T cell lymphomagenesis. Upon telomerase extinction, tumor growth eventually slowed with reinstatement of telomere dysfunction-induced checkpoints, yet growth subsequently resumed as tumors acquired alternative lengthening of telomeres (ALT) and aberrant transcriptional networks centering on mitochondrial biology and oxidative defense. ALT+ tumors acquired amplification/overexpression of PGC-1β, a master regulator of mitochondrial biogenesis and function, and they showed marked sensitivity to PGC-1β or SOD2 knockdown. Genetic modeling of telomerase extinction reveals vulnerabilities that motivate coincidental inhibition of mitochondrial maintenance and oxidative defense mechanisms to enhance antitelomerase cancer therapy.

Inflammatory pain impacts adversely on the quality of life of patients, often resulting in motor disabilities. Therefore, we studied the effect of peripheral inflammation induced by intraplantar administration of complete Freund's adjuvant (CFA) in mice on a particular form of voluntary locomotion, wheel running, as an index of mobility impairment produced by pain. The distance traveled over 1 hour of free access to activity wheels decreased significantly in response to hind paw inflammation, peaking 24 hours after CFA administration. Recovery of voluntary wheel running by day 3 correlated with the ability to support weight on the inflamed limb. Inflammation-induced mechanical hypersensitivity, measured with von Frey hairs, lasted considerably longer than the impaired voluntary wheel running and is not driving; therefore, the change in voluntary behavior. The CFA-induced decrease in voluntary wheel running was dose-dependently reversed by subcutaneous administration of antiinflammatory and analgesic drugs, including naproxen (10-80 mg/kg), ibuprofen (2.5-20mg/kg), diclofenac (1.25-10mg/kg), celecoxib (2.5-20mg/kg), prednisolone (0.62-5mg/kg), and morphine (0.06-0.5mg/kg), all at much lower doses than reported in most rodent models. Furthermore, the doses that induced recovery in voluntary wheel running did not reduce CFA-induced mechanical allodynia, indicating a greater sensitivity of the former as a surrogate measure of inflammatory pain. We conclude that monitoring changes in voluntary wheel running in mice during peripheral inflammation is a simple, observer-independent objective measure of functional changes produced by inflammation, likely more aligned to the global level of pain than reflexive measures, and much more sensitive to analgesic drug effects.

Testosterone is a steroid hormone thought to influence both emotional and cognitive functions. It is unknown, however, if testosterone also affects the interaction between these two domains, such as the emotional arousal-induced enhancement of memory. Healthy subjects (N=234) encoded pictures taken from the International Affective Picture System (IAPS) during functional magnetic resonance imaging (fMRI) and underwent a free recall test 10 min after memory encoding. We show that higher endogenous testosterone levels at encoding were associated with higher arousal ratings of neutral pictures in men. fMRI analysis revealed that higher testosterone levels were related to increased brain activation in the amygdala during encoding of neutral pictures. Moreover, endogenous testosterone levels were positively correlated with the number of freely recalled neutral pictures. No such relations were found in women. These findings point to a male-specific role for testosterone in enhancing memory by increasing the biological salience of incoming information.

The neural mechanisms used in tone rises and falls in Mandarin were investigated. Nine participants were scanned while they named one-character pictures that required rising or falling tone responses in Mandarin: the left insula and right putamen showed stronger activation between rising and falling tones; the left brainstem showed weaker activation between rising and falling tones. Connectivity analysis showed that the significant projection from the laryngeal motor cortex to the brainstem which was present in rising tones was absent in falling tones. Additionally, there was a significant difference between the connection from the insula to the laryngeal motor cortex which was negative in rising tones but positive in falling tones. These results suggest that the significant projection from the laryngeal motor cortex to the brainstem used in rising tones was not active in falling tones. The connection from the left insula to the laryngeal motor cortex that differs between rising and falling tones may control whether the rise mechanism is active or not.

Revertant mosaicism is an infrequently observed phenomenon caused by spontaneous correction of a pathogenic allele. We have observed such reversions caused by mitotic recombination of mutant TERC (telomerase RNA component) alleles in six patients from four families affected by dyskeratosis congenita (DC). DC is a multisystem disorder characterized by mucocutaneous abnormalities, dystrophic nails, bone-marrow failure, lung fibrosis, liver cirrhosis, and cancer. We identified a 4 nt deletion in TERC in a family with an autosomal-dominant form of DC. In two affected brothers without bone-marrow failure, sequence analysis revealed pronounced overrepresentation of the wild-type allele in blood cells, whereas no such skewing was observed in the other tissues tested. These observations suggest that this mosaic pattern might have resulted from somatic reversion of the mutated allele to the normal allele in blood-forming cells. SNP-microarray analysis on blood DNA from the two brothers indeed showed independent events of acquired segmental isodisomy of chromosome 3q, including TERC, indicating that the reversions must have resulted from mitotic recombination events. Subsequently, after developing a highly sensitive method of detecting mosaic homozygosity, we have found four additional cases with a mosaic-reversion pattern in blood cells; these four cases are part of a cohort of 17 individuals with germline TERC mutations. This shows that revertant mosaicism is a recurrent event in DC. This finding has important implications for improving diagnostic testing and understanding the variable phenotype of DC.