Khoa Tran

E2-2 protein and Fuchs's corneal dystrophy

BACKGROUND Fuchss corneal dystrophy (FCD) is a leading cause of corneal transplantation and affects 5% of persons in the United States who are over the age of 40 years. Clinically visible deposits called guttae develop under the corneal endothelium in patients with FCD. A loss of endothelial cells and deposition of an abnormal extracellular matrix are observed microscopically. In advanced disease, the cornea swells and becomes cloudy because the remaining endothelial cells are not sufficient to keep the cornea dehydrated and clear. Although rare genetic variation that contributes to both early-onset and typical late-onset forms of FCD has been identified, to our knowledge, no common variants have been reported. METHODS We performed a genomewide association study and replicated the most significant observations in a second, independent group of subjects. RESULTS Alleles in the transcription factor 4 gene (TCF4), encoding a member of the E-protein family (E2-2), were associated with typical FCD (P=2.3x10(-26)). The association increased the odds of having FCD by a factor of 30 for persons with two copies of the disease variants (homozygotes) and discriminated between case subjects and control subjects with about 76% accuracy. At least two regions of the TCF4 locus were associated independently with FCD. Alleles in the gene encoding protein tyrosine phosphatase receptor type G (PTPRG) were associated with FCD (P=4.0x10(-7)), but the association did not reach genomewide significance. CONCLUSIONS Genetic variation in TCF4 contributes to the development of FCD. (Funded by the National Eye Institute and others.)

Malignant precursor cells pre-exist in human breast DCIS and require autophagy for survival.

Background While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment. Methodology and Principal Findings We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patients breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patients original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation. Conclusions Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.

A randomised controlled comparison of early post-pyloric versus early gastric feeding to meet nutritional targets in ventilated intensive care patients

IntroductionTo compare outcomes from early post-pyloric to gastric feeding in ventilated, critically ill patients in a medical intensive care unit (ICU).MethodsProspective randomized study. Ventilated patients were randomly assigned to receive enteral feed via a nasogastric or a post-pyloric tube. Post-pyloric tubes were inserted by the bedside nurse and placement was confirmed radiographically.ResultsA total of 104 patients were enrolled, 54 in the gastric group and 50 in the post-pyloric group. Bedside post-pyloric tube insertion was successful in 80% of patients. Patients who failed post-pyloric insertion were fed via the nasogastric route, but were analysed on an intent-to treat basis. A per protocol analysis was also performed. Baseline characteristics were similar for all except Acute Physiology and Chronic Health Evaluation II (APACHE II) score, which was higher in the post-pyloric group. There was no difference in length of stay or ventilator days. The gastric group was quicker to initiate feed 4.3 hours (2.9 - 6.5 hours) as compared to post-pyloric group 6.6 hours (4.5 - 13.0 hours) (P = 0.0002). The time to reach target feeds from admission was also faster in gastric group: 8.7 hours (7.6 - 13.0 hours) compared to 12.3 hours (8.9 - 17.5 hours). The average daily energy and protein deficit were lower in gastric group 73 Kcal (2 - 288 Kcal) and 3.5 g (0 - 15 g) compared to 167 Kcal (70 - 411 Kcal) and 6.5 g (2.8 - 17.3 g) respectively but was only statistically significant for the average energy deficit (P = 0.035). This difference disappeared in the per protocol analysis. Complication rates were similar.ConclusionsEarly post-pyloric feeding offers no advantage over early gastric feeding in terms of overall nutrition received and complicationsTrial RegistrationClinical Trial: anzctr.org.au:ACTRN12606000367549

Knockdown of Parhyale Ultrabithorax recapitulates evolutionary changes in crustacean appendage morphology

Crustaceans possess remarkably diverse appendages, both between segments of a single individual as well as between species. Previous studies in a wide range of crustaceans have demonstrated a correlation between the anterior expression boundary of the homeotic (Hox) gene Ultrabithorax (Ubx) and the location and number of specialized thoracic feeding appendages, called maxillipeds. Given that Hox genes regulate regional identity in organisms as diverse as mice and flies, these observations in crustaceans led to the hypothesis that Ubx expression regulates the number of maxillipeds and that evolutionary changes in Ubx expression have generated various aspects of crustacean appendage diversity. Specifically, evolutionary changes in the expression boundary of Ubx have resulted in crustacean species with either 0, 1, 2, or 3 pairs of thoracic maxillipeds. Here we test this hypothesis by altering the expression of Ubx in Parhyale hawaiensis, a crustacean that normally possesses a single pair of maxillipeds. By reducing Ubx expression, we can generate Parhyale with additional maxillipeds in a pattern reminiscent of that seen in other crustacean species, and these morphological alterations are maintained as the animals molt and mature. These results provide critical evidence supporting the proposition that changes in Ubx expression have played a role in generating crustacean appendage diversity and lend general insights into the mechanisms of morphological evolution.

Pdm and Castor close successive temporal identity windows in the NB3-1 lineage

Neurogenesis in Drosophila and mammals requires the precise integration of spatial and temporal cues. In Drosophila, embryonic neural progenitors (neuroblasts) sequentially express the transcription factors Hunchback, Kruppel, Pdm1/Pdm2 (Pdm) and Castor as they generate a stereotyped sequence of neuronal and glial progeny. Hunchback and Kruppel specify early temporal identity in two posterior neuroblast lineages (NB7-1 and NB7-3), whereas Pdm and Castor specify late neuronal identity in the NB7-1 lineage. Because Pdm and Castor have only been assayed in one lineage, it is unknown whether their function is restricted to neuronal identity in the NB7-1 lineage, or whether they function more broadly as late temporal identity genes in all neuroblast lineages. Here, we identify neuronal birth-order and molecular markers within the NB3-1 cell lineage, and then use this lineage to assay Pdm and Castor function. We show that Hunchback and Kruppel specify first and second temporal identities, respectively. Surprisingly, Pdm does not specify the third temporal identity, but instead acts as a timing factor to close the second temporal identity window. Similarly, Castor closes the third temporal identity window. We conclude that Hunchback and Kruppel specify the first and second temporal identities, an unknown factor specifies the third temporal identity, and Pdm and Castor are timing factors that close the second and third temporal identity windows in the NB3-1 lineage. Our results provide a new neuroblast lineage for investigating temporal identity and reveal the importance of Pdm and Cas as timing factors that close temporal identity windows.

Evaluation of regional and whole gut motility using the wireless motility capsule: relevance in clinical practice:

The wireless motility capsule (WMC) is an ambulatory noninvasive and nonradioactive diagnostic sensor that continuously samples intraluminal pH, temperature, and pressure as it moves through the gastrointestinal (GI) tract. This review summarizes the data obtained in clinical trials with the WMC and discusses its role in clinical practice. The United States Food and Drug Administration has approved the SmartPill GI monitoring system for the evaluation of gastric emptying time in patients with suspected gastroparesis, the evaluation of colonic transit time in patients with suspected chronic constipation, and for the characterization of pressure profiles from the antrum and duodenum. Clinical studies have shown that WMC-measured GI transit times can distinguish patients with motility abnormalities similarly to conventional testing. However, the WMC offers the advantage of providing a full GI-tract profile, enabling the detection of multiregional GI transit abnormalities in patients with suspected upper or lower GI dysmotility. The WMC also characterizes pressure profiles of the GI tract and impaired pressure profile limits are reported for the antrum and duodenum. In comparison with manometry, interpretations of pressure measurements obtained by the WMC are limited by an inability to detect a peristaltic pressure wave front, and further investigation is required to develop clinical applications. Clinical studies with the WMC indicated that it should be considered for the evaluation of regional and whole gut transit time in patients with suspected upper or lower dysmotility, particularly if there are concerns about multiregional dysmotility.

Selective modulation of autophagy, innate immunity, and adaptive immunity by small molecules

Autophagy is an evolutionarily conserved catabolic process that directs cytoplasmic proteins, organelles and microbes to lysosomes for degradation. Autophagy acts at the intersection of pathways involved in cellular stress, host defense, and modulation of inflammatory and immune responses; however, the details of how the autophagy network intersects with these processes remain largely undefined. Given the role of autophagy in several human diseases, it is important to determine the extent to which modulators of autophagy also modify inflammatory or immune pathways and whether it is possible to modulate a subset of these pathways selectively. Here, we identify small-molecule inducers of basal autophagy (including several FDA-approved drugs) and characterize their effects on IL-1β production, autophagic engulfment and killing of intracellular bacteria, and development of Treg, TH17, and TH1 subsets from naïve T cells. Autophagy inducers with distinct, selective activity profiles were identified that reveal the functional architecture of connections between autophagy, and innate and adaptive immunity. In macrophages from mice bearing a conditional deletion of the essential autophagy gene Atg16L1, the small molecules inhibit IL-1β production to varying degrees suggesting that individual compounds may possess both autophagy-dependent and autophagy-independent activity on immune pathways. The small molecule autophagy inducers constitute useful probes to test the contributions of autophagy-related pathways in diseases marked by impaired autophagy or elevated IL-1β and to test novel therapeutic hypotheses.

The kinase DYRK1A reciprocally regulates the differentiation of Th17 and regulatory T cells

The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity. DOI: http://dx.doi.org/10.7554/eLife.05920.001

Sleep-disordered breathing in spinal cord injured patients: A short-term longitudinal study

Background and objective:  Previous studies have demonstrated an increased incidence of sleep apnoea in spinal cord‐injured patients. Many of these studies were performed in long‐term, stable spinal cord injury (SCI). The aims of this study were: (i) to determine the prevalence of sleep‐disordered breathing (SDB) in acute SCI; (ii) to document the change in SDB over time during the rehabilitation period; and (iii) to correlate the degree of SDB with ventilatory parameters.

Recombineering Hunchback identifies two conserved domains required to maintain neuroblast competence and specify early-born neuronal identity.

The Hunchback/Ikaros family of zinc-finger transcription factors is essential for specifying the anterior/posterior body axis in insects, the fate of early-born pioneer neurons in Drosophila, and for retinal and immune development in mammals. Hunchback/Ikaros proteins can directly activate or repress target gene transcription during early insect development, but their mode of action during neural development is unknown. Here, we use recombineering to generate a series of Hunchback domain deletion variants and assay their function during neurogenesis in the absence of endogenous Hunchback. Previous studies have shown that Hunchback can specify early-born neuronal identity and maintain ‘young’ neural progenitor (neuroblast) competence. We identify two conserved domains required for Hunchback-mediated transcriptional repression, and show that transcriptional repression is necessary and sufficient to induce early-born neuronal identity and maintain neuroblast competence. We identify pdm2 as a direct target gene that must be repressed to maintain competence, but show that additional genes must also be repressed. We propose that Hunchback maintains early neuroblast competence by silencing a suite of late-expressed genes.