Identification of novel genes and networks governing hematopoietic stem cell development

Hematopoietic stem cells (HSCs) are capable of giving rise to all blood cell lineages throughout adulthood, and the generation of engraftable HSCs from human pluripotent stem cells is a major goal for regenerative medicine. Here, we describe a functional genome‐wide RNAi screen to identify genes required for the differentiation of embryonic stem cell (ESC) into hematopoietic stem/progenitor cells (HSPCs) in vitro. We report the discovery of novel genes important for the endothelial‐to‐hematopoietic transition and subsequently for HSPC specification. High‐throughput sequencing and bioinformatic analyses identified twelve groups of genes, including a set of 351 novel genes required for HSPC specification. As in vivo proof of concept, four of these genes, Ap2a1, Mettl22, Lrsam1, and Hal, are selected for validation, confirmed to be essential for HSPC development in zebrafish and for maintenance of human HSCs. Taken together, our results not only identify a number of novel regulatory genes and pathways essential for HSPC development but also serve as valuable resource for directed differentiation of therapy grade HSPCs using human pluripotent stem cells.     

Epstein–Barr virus and thyroid cancer: The role of viral expressed proteins

Background : Thyroid cancer is a common endocrine malignancy whose incidence has increased in recent years. Several internal and external risk factors are involved in the development of this cancer, such as infectious agents. Evidence supporting the role of viral infection as an etiology for the invasiveness of thyroid cancer is increasing. The aim of this study was to determine the presence of the Epstein–Barr virus (EBV) and the association between viral gene products and thyroid tumor development. Methods : Fifty-seven thyroid cancer specimens were collected from the same number of patients as well as 18 samples from healthy controls. The presence of the EBV genome and the genotyping was examined by polymerase chain reaction (PCR). Also, an enzyme-linked immunosorbent assay and real-time PCR were used to measure the expression levels of viral and cellular genes. Results : The EBV DNA was detected in 71.9% of the samples, and it was also found that the presence of the EBV was associated with increasing development of thyroid tumor. Conclusion : Our results demonstrated that EBV infection may play a role in the development of thyroid tumor.     

Roller Compaction of Hydrophilic Extended Release Tablets—Combined Effects of Processing Variables and Drug/Matrix Former Particle Size

The present study shows that roller compaction (RC) can successfully be used as a granulation method to prepare hydroxypropyl methylcellulose (HPMC)-based extended release matrix tablets containing a high drug load, both for materials deforming mainly by fragmentation (paracetamol) as for those having mainly plastic deformation (ibuprofen). The combined effect of RC process variables and composition on the manufacturability of HPMC tablets was investigated. Standard wet granulation grade HPMC was compared with a larger particle size direct compressible HPMC grade. Higher roll pressure was found to result in larger paracetamol granules and narrower granule particle size distributions, especially for formulations containing smaller size HPMC. However, for ibuprofen, no clear effect of roll pressure was observed. High roll pressure also resulted in denser ribbon and less bypass fines during RC. Loss of compactibility was observed for granules compared to powder blends, which was found to be related to differences in granule porosity and morphology. Using the large-sized HPMC grade did in some cases result in lower tensile strength tablets but had the advantage to improve the powder flow into the roller compactor. This work also indicates that when the HPMC level lies near the percolation threshold, significant changes can occur in the drug release rate due to changes in other factors (raw material characteristics and processing).

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0219-3) contains supplementary material, which is available to authorized users.KEY WORDS: dry granulation, extended release, hydroxypropyl methylcellulose, roller compaction, work hardening     

Heterogeneity of yield strain in low-density versus high-density human trabecular bone

Understanding the off-axis behavior of trabecular yield strains may lend unique insight into the etiology of fractures since yield strains provide measures of failure independent of elastic behavior. We sought to address anisotropy of trabecular yield strains while accounting for variations in both density and anatomic site and to determine the mechanisms governing this behavior. Cylindrical specimens were cored from vertebral bodies (n=22, BV/TV=0.11±0.02) and femoral necks (n=28, BV/TV=0.22±0.06) with the principal trabecular orientation either aligned along the cylinder axis (on-axis, n=22) or at an oblique angle of 15° or 45° (off-axis, n=28). Each specimen was scanned with micro-CT, mechanically compressed to failure, and analysed with nonlinear micro-CT-based finite element analysis. Yield strains depended on anatomic site (p=0.03, ANOVA), and the effect of off-axis loading was different for the two sites (p=0.04)—yield strains increased for off-axis loading of the vertebral bone (p=0.04), but were isotropic for the femoral bone (p=0.66). With sites pooled together, yield strains were positively correlated with BV/TV for on-axis loading (R²=58%, p<0.0001), but no such correlation existed for off-axis loading (p=0.79). Analysis of the modulus-BV/TV and strength-BV/TV relationships indicated that, for the femoral bone, the reduction in strength associated with off-axis loading was greater than that for modulus, while the opposite trend occurred for the vertebral bone. The micro-FE analyses indicated that these trends were due to different failure mechanisms for the two types of bone and the different loading modes. Taken together, these results provide unique insight into the failure behavior of human trabecular bone and highlight the need for a multiaxial failure criterion that accounts for anatomic site and bone volume fraction.     

Efficacy of Setarud (IMod), a novel drug with potent anti-toxic stress potential in rat inflammatory bowel disease and comparison with dexamethasone and infliximab

The inflammatory bowel disease (IBD) is an idiopathic, immune-mediated and chronic intestinal condition. In the present study, the effect of Setarud (IMOD), a novel natural drug with known immunomodulatory, anti-inflammatory and antioxidant properties was investigated in experimental colitis in rats and compared with the dexamethasone and infliximab. Immunologic colitis was induced by intracolonic administration of a mixture of trinitrobenzene sulfonic acid (TNBS) and absolute ethanol in male Wistar rats. Animals were divided into 6 groups of sham (normal group), control (vehicle-treated), positive control (dexamethasone 1 mg/kg/day given orally and infliximab 5 mg/kg/day given subcutaneously) and 3 Setarud-treated groups (13.3, 20, 30 mg/kg/day given intraperitoneally). The treatment continued for 14 consecutive days and then animals were decapitated on the day 15 and distal colons were removed for macroscopic, microscopic, and biochemical assays. Biochemical markers, including TNF-alpha, IL-1beta, ferric reducing/antioxidant power (FRAP), myeloperoxidase (MPO) activity and thiobarbitoric acid-reactive substance (TBARS) were measured in the homogenate of colonic tissue. A remarkable reduction in macroscopic and histological damage scores was observed in the animals treated with Setarud. These findings were confirmed by decreased levels of TNF-alpha, interleukin-1beta, MPO activity and TBARS, and raised levels of FRAP in the colon tissue. These observations confirmed the immunomodulatory, anti-inflammatory and antioxidant properties of Setarud in experimental colitis, which was comparable to those of dexamethasone and infliximab.     

Dissociation of the Octameric Enolase from S. Pyogenes - One Interface Stabilizes Another

Most enolases are homodimers. There are a few that are octamers, with the eight subunits arranged as a tetramer of dimers. These dimers have the same basic fold and same subunit interactions as are found in the dimeric enolases. The dissociation of the octameric enolase from S. pyogenes was examined, using NaClO₄, a weak chaotrope, to perturb the quaternary structure. Dissociation was monitored by sedimentation velocity. NaClO₄ dissociated the octamer into inactive monomers. There was no indication that dissociation of the octamer into monomers proceeded via formation of significant amounts of dimer or any other intermediate species. Two mutations at the dimer-dimer interface, F137L and E363G, were introduced in order to destabilize the octameric structure. The double mutant was more easily dissociated than was the wild type. Dissociation could also be produced by other salts, including tetramethylammonium chloride (TMACl) or by increasing pH. In all cases, no significant amounts of dimers or other intermediates were formed. Weakening one interface in this protein weakened the other interface as well. Although enolases from most organisms are dimers, the dimeric form of the S. pyogenes enzyme appears to be unstable.     

Abdominal pain as presentation of Takayasu's arteritis in an adolescent male patient

We describe a boy with chronic abdominal pain, nausea and vomiting, and weight loss. The imaging was compatible with Takayasu's arteritis. Chronic mesenteric ischemia was the etiology of the patient's symptoms.     

Role of the p38 mitogen-activated protein kinase pathway in the generation of the effects of imatinib mesylate (STI571) in BCR-ABL-expressing cells

Imatinib mesylate (STI571), a specific inhibitor of the BCR-ABL tyrosine kinase, exhibits potent antileukemic effects in vitro and in vivo. Despite the well established role of STI571 in the treatment of chronic myelogenous leukemia, the precise mechanisms by which inhibition of BCR-ABL tyrosine kinase activity results in generation of antileukemic responses remain unknown. In the present study we provide evidence that treatment of CML-derived BCR-ABL-expressing leukemia cells with STI571 results in activation of the p38 mitogen-activated protein (MAP) kinase signaling pathway. Our data indicate that STI571 induces phosphorylation of the p38 and activation of its kinase domain, in KT-1 cells and other BCR-ABL-expressing cell lines. We also identify the kinases MAP kinase-activated protein kinase-2 and Msk1 as two downstream effectors of p38, activated during inhibition of BCR-ABL activity by STI571. Importantly, pharmacological inhibition of p38 reverses the growth inhibitory effects of STI571 on primary leukemic colony-forming unit granulocyte/macrophage progenitors from patients with CML. Altogether, our data establish that activation of the p38 MAP kinase signaling cascade plays an important role in the generation of the effects of STI571 on BCR-ABL-expressing cells. They also suggest that, in addition to activation of mitogenic pathways, BCR-ABL promotes leukemogenesis by suppressing the function of growth inhibitory signaling cascades.