Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment.     

Chondroblastoma in a metatarsal treated with autogenous fibular graft: a case report

Chondroblastoma is a relatively rare tumor that mimics giant cell tumor and displays a predilection for long bones. In the present report, we describe the case of a benign chondroblastoma localized to the second metatarsal in a 20-year-old male who presented with a 2-year history of painless left foot swelling. Treatment of the tumor involved excision of the second metatarsal with use of an autologous structural fibular bone graft to stabilize the metatarsus and second toe. After 27 months of follow-up, the patient was ambulating well in regular shoes, with no clinical or radiographic evidence of tumor recurrence.     

Bilateral inferior dislocation of the hip---a case report

Inferior dislocation of the hip is the rarest type in hip dislocation.Very few cases have been reported in the anglophonic literature,most of which involved the pediatric age group.Surprisingly,we came...     

Eyelash alopecia areata: case series and literature review

ObjectivesTo characterize the clinical presentations, features, and outcomes of eyelash alopecia areata.DesignRetrospective chart review of patients evaluated for eyelash loss and found to have eyelash alopecia areata. A 3-year follow-up was required.ParticipantsThe study involved 15 patients.MethodsPatients who presented at a tertiary care eye clinic for evaluation of eyelash loss and were found to have eyelash alopecia areata were reviewed. Demographic considerations were categorized; they included age at presentation, areas of other hair loss, other relevant history, and treatment responses.ResultsPatients were young (mean age 18 years) and had a large female predominance (female-to-male ratio, 14:1). Concurrent scalp and brow involvement, either in isolation (20% and 13%, respectively) or together (13%), were seen in half of the patients (46%). Upper eyelid involvement was more common and severe than lower eyelid involvement, and 40% of patients experienced complete regrowth of lashes, with a mean time to regrowth of 28 months (range, 6 to 60 months). Of these, 13% experienced partial regrowth. Younger age at presentation was associated with regrowth, whereas presence or absence of other involved sites, personal or family histories of atopy, family history of alopecia, other autoimmune diseases, or the use of topical steroids did not appear to affect prognosis.ConclusionsEyelash alopecia areata is a unique entity, although it remains potentially underdiagnosed. The key differential diagnosis is trichotillomania, which is commonly associated with obsessive-compulsive disorders. The presence of exclamation-mark hairs in alopecia areata plays an important role in differentiating the 2 diagnoses.     

RPGR-Associated Retinal Degeneration in Human X-Linked RP and a Murine Model

Purpose. We investigated the retinal disease due to mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene in human patients and in an Rpgr conditional knockout (cko) mouse model. Methods. XLRP patients with RPGR-ORF15 mutations (n = 35, ages at first visit 5-72 years) had clinical examinations, and rod and cone perimetry. Rpgr-cko mice, in which the proximal promoter and first exon were deleted ubiquitously, were back-crossed onto a BALB/c background, and studied with optical coherence tomography and electroretinography (ERG). Retinal histopathology was performed on a subset. Results. Different patterns of rod and cone dysfunction were present in patients. Frequently, there were midperipheral losses with residual rod and cone function in central and peripheral retina. Longitudinal data indicated that central rod loss preceded peripheral rod losses. Central cone-only vision with no peripheral function was a late stage. Less commonly, patients had central rod and cone dysfunction, but preserved, albeit abnormal, midperipheral rod and cone vision. Rpgr-cko mice had progressive retinal degeneration detectable in the first months of life. ERGs indicated relatively equal rod and cone disease. At late stages, there was greater inferior versus superior retinal degeneration. Conclusions. RPGR mutations lead to progressive loss of rod and cone vision, but show different patterns of residual photoreceptor disease expression. Knowledge of the patterns should guide treatment strategies. Rpgr-cko mice had onset of degeneration at relatively young ages and progressive photoreceptor disease. The natural history in this model will permit preclinical proof-of-concept studies to be designed and such studies should advance progress toward human therapy.     

Development of a Genomic DNA Reference Material Panel for Rett Syndrome (MECP2-Related Disorders) Genetic Testing

Rett syndrome is a dominant X-linked disorder caused by point mutations (approximately 80%) or by deletions or insertions (approximately 15% to 18%) in the MECP2 gene. It is most common in females but lethal in males, with a distinctly different phenotype. Rett syndrome patients have severe neurological and behavioral problems. Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process; however, none are commercially available for MECP2 genetic testing. The Centers for Disease Control and Prevention’s Genetic Testing Reference Material Coordination Program, in collaboration with the genetic testing community and the Coriell Cell Repositories, established 27 new cell lines and characterized the MECP2 mutations in these and in 8 previously available cell lines. DNA samples from the 35 cell lines were tested by eight clinical genetic testing laboratories using DNA sequence analysis and methods to assess copy number (multiplex ligation-dependent probe amplification, semiquantitative PCR, or array-based comparative genomic hybridization). The eight common point mutations known to cause approximately 60% of Rett syndrome cases were identified, as were other MECP2 variants, including deletions, duplications, and frame shift and splice-site mutations. Two of the 35 samples were from males with MECP2 duplications. These MECP2 and other characterized genomic DNA samples are publicly available from the NIGMS Repository at the Coriell Cell Repositories.Rett syndrome is a dominant X-linked disorder usually caused by point mutations (approximately 80% in classical and 40% in atypical cases) and deletions or insertions (approximately 15% to 18% in classical and 3% in atypical cases) in the MECP2 gene, although patients with mutations in two other genes, CDKL5 and FOXG1, may also exhibit a Rett-like phenotype.^1,2 MECP2, located on Xq28 and comprising four exons, encodes methyl-CpG binding protein 2 (MeCP2). In males, Rett syndrome is usually lethal, because of abnormal MeCP2 function from the single X chromosome and severe neonatal encephalopathy, although Rett syndrome in males with an XXY karyotype has been reported.³ Duplications in MECP2 have also been observed, and these can cause severe neurodevelopmental disability in males.⁴ The prevalence of Rett syndrome in females is approximately 1:10,000.⁵Girls with classic Rett syndrome (OMIM #312750) exhibit a rapid decline in language and motor skills at approximately 1 year of age. These patients exhibit a loss of acquired purposeful hand use, loss of communication, gait ataxia and apraxia, and stereotypic hand movements. They may also exhibit additional symptoms, including bruxism, seizures, episodic apnea or hyperpnea, abnormal muscle tone, and often acquired microcephaly.^4,6,7Patients with Rett syndrome can present with various phenotypes. Those diagnosed with atypical Rett syndrome may have either more mild or more severe presentation than patients with the classical form. These patients share some of the same symptoms with classical Rett syndrome cases, but must also have some of the additional symptoms listed above.^4,6 Some patients present with only mild learning disabilities (females) or intellectual disability (males).^4,8Molecular diagnosis of Rett syndrome is performed by examination of the patient’s DNA for MECP2 mutations, using a variety of molecular diagnostic methods. Most MECP2 mutations are sporadic, and testing is performed on the proband, although predictive prenatal and preimplantation genetic testing may also be performed. DNA sequence analysis may detect point mutations and small insertions and deletions (indels). Larger deletions and duplications are detected using multiplex ligation-dependent probe amplification (MLPA), quantitative PCR, and array-based comparative genomic hybridization (CGH). To date, there are no US Food and Drug Administration (FDA)–approved assays for Rett syndrome. All testing is performed using laboratory-developed tests.Reference materials are needed by laboratories to comply with regulatory and accreditation requirements for assay development, assay validation, and quality control, and their use is recommended by professional guidelines for clinical laboratories [eg, http://www.acmg.net/Pages/ACMG_Activities/stds-2002/g.htm, last accessed October 16, 2013; Washington State Legislature, http://www.doh.wa.gov/hsqa/fsl/lqa_home.htm, last accessed January 11, 2013; College of American Pathologists http://www.cap.org/apps/cap.portal, last accessed January 11, 2013 (registration required); New York State Clinical Laboratory Evaluation Program, http://www.wadsworth.org/clep, last accessed January 11, 2013).^9–15 Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process. Ideally, these reference materials should closely resemble patient samples containing variants and types of variants common to the disorder and should be thoroughly characterized using methods different from those used in the user’s laboratory.¹⁵ For Rett syndrome, genomic DNA reference materials derived from females (and, if possible, males) containing common point mutations, indels, and larger deletions and duplications should be used. Careful use of a well-characterized and comprehensive set of reference materials helps to assure the proper design and function of a clinical assay. To date, there are no commercially available reference materials for Rett syndrome genetic testing.To address the need for characterized genomic DNA reference materials for Rett syndrome testing, the Centers for Disease Control and Prevention (CDC) based Genetic Testing Reference Material Coordination Program (GeT-RM), in collaboration with members of the genetic testing community and the National Institute of General Medical Sciences (NIGMS) Repository at the Coriell Cell Repositories, have characterized the MECP2 mutations in 35 publicly available cell lines. Twenty-seven of the 35 cell lines were generated as part of this project, using blood collected with informed consent from Rett syndrome patients with variants not previously represented in cell lines at the Coriell Repository. The availability of a renewable source of characterized reference materials for Rett syndrome helps to assure the accuracy of these genetic tests and facilitate research and test development.     

Mortality among homeless and unclaimed bodies in Mangalore city - An insight

Homelessness is a social as well as legal stigma on a Country's development index. In addition homeless people are exposed to increased incidence of diseases and accidents. Mangalore city, a bustling city located in Southern costal region of India, has seen tremendous growth in the past few years; with this the problem of migrants and homeless has also increased. This has invited a spectrum of problems relating to law and order including frequent incidences of unclaimed dead bodies, both due to natural and unnatural causes. This autopsy based study tries to highlight the situation of picture of homeless deaths in Mangalore and the problems faced by the Law enforcing authorities.     

Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds

Therapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC₅₀s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC₅₀s of <10 μM were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC₅₀s (compound concentrations at 50% cytotoxicity) of <30 μM from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.