The cerebellum (Cb) controls movement related physiology using a diverse array of morphologically and biochemically distinct neurons. During development, the Cb is derived from rhombomere 1 (r1), an embryonic compartment patterned by a signaling center referred to as the isthmus organizer. The secreted glycoprotein WNT1 is expressed in the midbrain primordia (mesencephalon, mes) and at the posterior limit of the mes. WNT1 plays a pivotal role in maintaining the isthmus organizer and mutations in Wnt1 produce severe Cb defects that are generally attributed to aberrant organizer activity. Interestingly, Wnt1 is also expressed at the most posterior limit of dorsal r1, in a region known as the upper rhombic lip (URL). However, the distribution and molecular identity of Wnt1 expressing progenitors have not been carefully described in r1. We used Wnt1-Venus transgenic mice to generate a molecular map of Wnt1 expressing progenitors in relation to other well characterized Cb biomarkers such as MATH1 (ATOH1), LMX1a and OTX2. Our analysis validated Wnt1 expression in the URL and revealed molecularly-defined developmental zones in r1. We then used genetic inducible fate mapping (GIFM) to link transient Wnt1 expression in r1 to terminal cell fates in the mature Cb. Wnt1 expressing progenitors primarily contributed to neurons in deep cerebellar nuclei, granule cells, and unipolar brush cells in distinct but overlapping temporal windows and sparsely contributed to inhibitory neurons and Bergmann glia. We further demonstrate that the Wnt1 lineage does not follow a competency model of progressive lineage restriction to generate the Cb or the functionally related precerebellar system. Instead, progenitors initiate Wnt1 expression de novo to give rise to each Cb cell type and precerebellar nuclei. We also used GIFM to determine how the temporal control of Wnt1 expression is related to molecular identity and cell migration in Cb development. Our findings provide new insight into how lineage and timing establish cell diversity within the Cb system.

A liquid chromatography-tandem mass spectrometry method for the analysis of seven antiviral drugs, zanamivir, ribavirin, oseltamivir, oseltamivir carboxylate, amantadine, rimantadine and arbidol, in poultry muscle is reported. The antiviral drugs were extracted from the homogenized poultry muscle sample using methanol. The extract was purified using tandem solid-phase extraction combining a cation exchange cartridge and a phenylboronic acid cartridge. To prevent excessive matrix effects, the analytes were separated from the matrix constituents using a column-switch liquid chromatography system combining a reversed-phase and a Hypercarb analytical column. Detection was carried out using tandem mass spectrometry. The method was fully validated according to 2002/657/EC [1] and proved to be adequate for quantification and confirmation of zanamivir and ribavirin at 10 μg kg(-1), oseltamivir, oseltamivir carboxylate, amantadine and rimantadine at levels below 1.0 μg kg(-1) and for qualitative confirmatory analysis of arbidol at levels below 1 μg kg(-1).

Animals that hunt and scavenge are often exposed to a broad array of pathogens. Theory predicts the immune systems of animals specialized for scavenging should have been molded by selective pressures associated with surviving microbial assaults from their food. Spotted hyenas (Crocuta crocuta) are capable hunters that have recently descended from carrion feeding ancestors. Hyenas have been documented to survive anthrax and rabies infections, and outbreaks of several other viral diseases that decimated populations of sympatric carnivores. In light of the extreme disease resistance manifested by spotted hyenas, our objective was to identify tools available for studying immune function in spotted hyenas and use these tools to document the hyena antibody response to immunization. Domestic cats (Felis catus) are the closest phylogenetic relatives of hyenas that have been studied in detail immunologically, and we hypothesized that anti-cat isotype-specific antibodies would cross react with hyena immunoglobulin epitopes. We used ELISA and Western blots to test isotype-specific anti-feline antibodies for specific cross-reaction to hyena Ig epitopes. Molecular weights of heavy (IgA, IgG, IgM) and light chains of hyena immunoglobulins were determined by protein electrophoresis, and as expected, they were found to be similar to feline immunoglobulins. In order to further validate the cross-reactivity of the anti-feline antibodies and document the hyena humoral response, eight spotted hyenas were immunized with dinitrophenol conjugated keyhole limpet hemocyanin (DNP-KLH) and serum anti-DNP responses were monitored by enzyme-linked immunosorbent assay (ELISA) for one year. The full array of isotype-specific antibodies identified here will allow veterinarians and other researchers to thoroughly investigate the hyena antibody response, and can be used in future studies to test hypotheses about pathogen exposure and immune function in this species.

MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally silence gene expression by binding to target mRNAs. Previous studies have identified the miRNA miR-8 as a pleiotropic regulator of Drosophila development, controlling body size and neuronal survival by targeting multiple mRNAs. In this study we demonstrate that miR-8 is also required for proper spatial patterning of pigment on the adult abdominal cuticle in females but not males.

Fibrodysplasia Ossificans Progressiva (FOP) is an autosomal dominant disorder characterized by episodic deposition of heterotopic bone in place of soft connective tissue. All FOP-associated mutations map to the BMP type I receptor, ALK2, with the ALK2(R206H) mutant form found in the vast majority of patients. The mechanism(s) regulating the expressivity of hyperactive ALK2(R206H) signaling throughout a patient's life is not well understood.

Large volumes of treated process water are required for protein extraction. Evaporation of this water contributes greatly to the energy consumed in enriching protein products. Thin stillage remaining from ethanol production is available in large volumes and may be suitable for extracting protein rich materials. In this work protein was extracted from ground defatted oriental mustard (Brassica juncea (L.) Czern.) meal using thin stillage. Protein extraction efficiency was studied at pHs between 7.6 and 10.4 and salt concentrations between 3.4 × 10-2 and 1.2 M. The optimum extraction efficiency was pH 10.0 and 1.0 M NaCl. Napin and cruciferin were the most prevalent proteins in the isolate. The isolate exhibited high in vitro digestibility (74.9 ± 0.80%) and lysine content (5.2 ± 0.2 g/100 g of protein). No differences in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability, or amino acid composition were observed between protein extracted with thin stillage and that extracted with NaCl solution. The use of thin stillage, in lieu of water, for protein extraction would decrease the energy requirements and waste disposal costs of the protein isolation and biofuel production processes.

The current study explored the effects of talking on respiratory sinus arrhythmia (RSA) during a semi-structured emotional interview (Adult Attachment Interview) using 76 female undergraduates. The effectiveness of 2 different methodological approaches (i.e. talking baseline or transfer function) was explored as respiratory control during talking tasks. RSA was collected during resting baseline, talking baseline, and interview conditions. Subjective reports of distress were higher in the interview than in the other 2 conditions. Mean RSA levels were significantly lower in the 2 talking tasks than in the resting baseline. After applying a transfer function for respiratory control, there were no significant differences between the 3 conditions. Moderator analyses yielded lower RSA values in the talking baseline and interview conditions for participants who reported greater distress during the interview. It was concluded that respiratory controls are likely necessary when using RSA in talking paradigms and that both approaches appeared to be adequate.

High-throughput tag-sequencing (Tag-seq) analysis based on the Solexa Genome Analyzer platform was applied to analyze the gene expression profiling of cucumber plant at 5 time points over a 24h period of waterlogging treatment. Approximately 5.8 million total clean sequence tags per library were obtained with 143013 distinct clean tag sequences. Approximately 23.69%-29.61% of the distinct clean tags were mapped unambiguously to the unigene database, and 53.78%-60.66% of the distinct clean tags were mapped to the cucumber genome database. Analysis of the differentially expressed genes revealed that most of the genes were down-regulated in the waterlogging stages, and the differentially expressed genes mainly linked to carbon metabolism, photosynthesis, reactive oxygen species generation/scavenging, and hormone synthesis/signaling. Finally, quantitative real-time polymerase chain reaction using nine genes independently verified the tag-mapped results. This present study reveals the comprehensive mechanisms of waterlogging-responsive transcription in cucumber.

Gap junctions play a central role in coordinating intercellular signal-transduction pathways to control tissue homeostasis. Deregulation of gap junctional intercellular communication is a common phenotype of cancer cells and supports its involvement in the carcinogenesis process. Many carcinogens, like environmental heavy-metal chemical pollutants, are known to activate various signal transduction mechanisms and modulate GJIC. They act as tumor promoters on preexisting "initiated" cells, rather than as genotoxic initiators, albeit their mode of action is often unknown. In this study we investigated the effect of Hg(II) (HgCl(2)) on GJIC in cultured human keratinocytes. It is shown that subcytotoxic concentrations of HgCl(2) as low as 10 nM cause inhibition of the GJIC, assessed by dye transfer assay, despite enhanced expression of connexins. In addition, HgCl(2)-treated keratinocytes exhibited a decrease of free thiols and accumulation of mitochondria-derived reactive oxygen species, albeit no effect on the respiratory chain activity was observed. Treatment of HgCl(2)-exposed keratinocytes with the PKC inhibitor calphostin C and with all-trans retinoic acid resulted in rescue of the mitochondrial ROS overproduction and full recovery of the GJIC. Similar results were obtained with the PKA activator db-cAMP. Overall, the presented results support a cross-talk between the altered intracellular redox tone and PKA- and PKC-mediated signaling in HgCl(2)-challenged keratinocytes. These events, although not cytotoxic, lead to inhibition of GJIC and possibly to carcinogenic priming.

Activation of microglia causes the production of proinflammatory factors and upregulation of NADPH oxidase (NOX) that form reactive oxygen species (ROS) that lead to neurodegeneration. Previously, we reported that 10 daily doses of ethanol treatment induced innate immune genes in brain. In the present study, we investigate the effects of chronic ethanol on activation of NOX and release of ROS, and their contribution to ethanol neurotoxicity.

Galectins, a family of small carbohydrate binding proteins, have been implicated in regulation of inflammatory reactions, including asthma and fibrosis in the lungs. Galectins are found in cells of the airways and in airway secretions, but their glycoprotein ligands there have only been studied to a very limited extent.

Status epilepticus (SE) induces severe vasogenic edema in the piriform cortex (PC) accompanied by neuronal and astroglial damages. To elucidate the mechanism of SE-induced vasogenic edema, we investigated the roles of tumor necrosis factor (TNF)-α in blood-brain barrier (BBB) disruption during vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced SE.

Mobile technology is increasingly being used by clinicians to access up-to-date information for patient care. These offer learning opportunities in the clinical setting for medical students but the underlying pedagogic theories are not clear. A conceptual framework is needed to understand these further. Our initial questions were how the medical students used the technology, how it enabled them to learn and what theoretical underpinning supported the learning.

The tumor suppressor menin (MEN1) is mutated in the inherited disease multiple endocrine neoplasia type I, and has several documented cellular roles, including the activation and repression of transcription effected by several transcription factors. As an activator, MEN1 is a component of the Set1-like mixed lineage leukemia (MLL) MLL1/MLL2 methyltransferase complex that methylates histone H3 lysine 4 (H3K4). MEN1 is localized to the signal transducer and activator of transcription 1 (STAT1)-dependent gene, interferon regulatory factor 1 (IRF1), and is further recruited when IRF1 transcription is triggered by interferon-γ signaling.

To explore the feasibility of constructing engineered myocardial tissues (EMTs) in vivo, using polylactic acid -co-glycolic acid (PLGA) for scaffold and cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells (BMMSCs) for seeded cells.

Increased Aurora kinase A gene copy number (AURKA-CN) has been reported in metastatic colorectal cancer (mCRC), with unknown relationship to clinical outcome. We correlated increased AURKA-CN in mCRC tumours with KRAS mutation status, overall and progression-free survival (OS, PFS).

A generally applicable, easy-to-use method of focusing a patient's immune system to eradicate or prevent cancer has been elusive. We are attempting to develop a targeted virus to accomplish these aims. We previously created a recombinant replicating vesicular stomatitis virus (VSV) that preferentially infected Her2/neu expressing breast cancer cells and showed therapeutic efficacy in an implanted Balb/c mouse tumor model. The current work shows that this therapy generated therapeutic anti-tumor CD4 T cells against multiple tumor antigens. CD4 T cells transferred directly from cured donor mice could eradicate established tumors in host mice. T cells were transferred directly from donor mice and were not stimulated ex vivo. Both tumors that expressed Her2/neu and those that did not were cured by transferred T cells. Analysis of cytokines secreted by anti-tumor memory CD4 T cells displayed a multifunctional pattern with high levels of interferon-γ, interleukin (IL)-4 and IL-17. Anti-tumor memory CD4 T cells traveled to the mesenteric lymph nodes and were activated there. Treatment with targeted recombinant replicating VSV is a potent immune adjuvant that generates therapeutic, multifunctional anti-tumor memory CD4 T cells that recognize multiple tumor antigens. Immunity elicited by viral therapy is independent of host major histocompatibility complex or knowledge of tumor antigens. Virus-induced tumor immunity could have great benefit in the prevention and treatment of tumor metastases.

Special AT-rich sequence-binding protein 1 (SATB1) is a global chromatin organizer and gene expression regulator essential for T-cell development and breast cancer tumor growth and metastasis. The oligomerization of the N-terminal domain of SATB1 is critical for its biological function. We determined the crystal structure of the N-terminal domain of SATB1. Surprisingly, this domain resembles a ubiquitin domain instead of the previously proposed PDZ domain. Our results also reveal that SATB1 can form a tetramer through its N-terminal domain. The tetramerization of SATB1 plays an essential role in its binding to highly specialized DNA sequences. Furthermore, isothermal titration calorimetry results indicate that the SATB1 tetramer can bind simultaneously to two DNA targets. Based on these results, we propose a molecular model whereby SATB1 regulates the expression of multiple genes both locally and at a distance.

Exome sequencing strategy is promising for finding novel mutations of human monogenic disorders. However, pinpointing the casual mutation in a small number of samples is still a big challenge. Here, we propose a three-level filtration and prioritization framework to identify the casual mutation(s) in exome sequencing studies. This efficient and comprehensive framework successfully narrowed down whole exome variants to very small numbers of candidate variants in the proof-of-concept examples. The proposed framework, implemented in a user-friendly software package, named KGGSeq (http://statgenpro.psychiatry.hku.hk/kggseq), will play a very useful role in exome sequencing-based discovery of human Mendelian disease genes.

How stable synchrony in neuronal networks is sustained in the presence of conduction delays is an open question. The Dynamic Clamp was used to measure phase resetting curves (PRCs) for entorhinal cortical cells, and then to construct networks of two such neurons. PRCs were in general Type I (all advances or all delays) or weakly type II with a small region at early phases with the opposite type of resetting. We used previously developed theoretical methods based on PRCs under the assumption of pulsatile coupling to predict the delays that synchronize these hybrid circuits. For excitatory coupling, synchrony was predicted and observed only with no delay and for delays greater than half a network period that cause each neuron to receive an input late in its firing cycle and almost immediately fire an action potential. Synchronization for these long delays was surprisingly tight and robust to the noise and heterogeneity inherent in a biological system. In contrast to excitatory coupling, inhibitory coupling led to antiphase for no delay, very short delays and delays close to a network period, but to near-synchrony for a wide range of relatively short delays. PRC-based methods show that conduction delays can stabilize synchrony in several ways, including neutralizing a discontinuity introduced by strong inhibition, favoring synchrony in the case of noisy bistability, and avoiding an initial destabilizing region of a weakly type II PRC. PRCs can identify optimal conduction delays favoring synchronization at a given frequency, and also predict robustness to noise and heterogeneity.