Loss-of-function mutations in RAB18 cause Warburg micro syndrome

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.     

Rod Photoreceptors Express GPR55 in the Adult Vervet Monkey Retina

Cannabinoids exert their actions mainly through two receptors, the cannabinoid CB1 receptor (CB1R) and cannabinoid CB2 receptor (CB2R). In recent years, the G-protein coupled receptor 55 (GPR55) was suggested as a cannabinoid receptor based on its activation by anandamide and tetrahydrocannabinol. Yet, its formal classification is still a matter of debate. CB1R and CB2R expression patterns are well described for rodent and monkey retinas. In the monkey retina, CB1R has been localized in its neural (cone photoreceptor, horizontal, bipolar, amacrine and ganglion cells) and CB2R in glial components (Müller cells). The aim of this study was to determine the expression pattern of GPR55 in the monkey retina by using confocal microscopy. Our results show that GPR55 is strictly localized in the photoreceptor layer of the extrafoveal portion of the retina. Co-immunolabeling of GPR55 with rhodopsin, the photosensitive pigment in rods, revealed a clear overlap of expression throughout the rod structure with most prominent staining in the inner segments. Additionally, double-label of GPR55 with calbindin, a specific marker for cone photoreceptors in the primate retina, allowed us to exclude expression of GPR55 in cones. The labeling of GPR55 in rods was further assessed with a 3D visualization in the XZ and YZ planes thus confirming its exclusive expression in rods. These results provide data on the distribution of GPR55 in the monkey retina, different than CB1R and CB2R. The presence of GPR55 in rods suggests a function of this receptor in scotopic vision that needs to be demonstrated.     

Cardiomyocyte Protection by GATA-4 Gene Engineered Mesenchymal Stem Cells Is Partially Mediated by Translocation of miR-221 in Microvesicles

Introduction

microRNAs (miRs), a novel class of small non-coding RNAs, are involved in cell proliferation, differentiation, development, and death. In this study, we found that miR-221 translocation by microvesicles (MVs) plays an important role in cardioprotection mediated by GATA-4 overexpressed mesenchymal stem cells (MSC).

Methods and Results

Adult rat bone marrow MSC and neonatal rat ventricle cardiomyocytes (CM) were harvested as primary cultures. MSC were transduced with GATA-4 (MSC^GATA-4) using the murine stem cell virus (pMSCV) retroviral expression system. Empty vector transfection was used as a control (MSC^Null). The expression of miRs was assessed by real-time PCR and localized using in situ hybridization (ISH). MVs collected from MSC cultures were characterized by expression of CD9, CD63, and HSP70, and photographed with electron microscopy. Cardioprotection during hypoxia afforded by conditioned medium (CdM) from MSC cultures was evaluated by lactate dehydrogenase (LDH) release, MTS uptake by CM, and caspase 3/7 activity. Expression of miR-221/222 was significantly higher in MSC than in CM and miR-221 was upregulated in MSC^GATA-4. MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA). Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC. MVs derived from MSC expressed high levels of miR-221, and were internalized quickly by CM as documented in images obtained from a Time-Lapse Imaging System.

Conclusions

Our results demonstrate that cardioprotection by MSC^GATA-4 may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.     

Systematic Pharmacogenomics Analysis of a Malay Whole Genome: Proof of Concept for Personalized Medicine

Background

With a higher throughput and lower cost in sequencing, second generation sequencing technology has immense potential for translation into clinical practice and in the realization of pharmacogenomics based patient care. The systematic analysis of whole genome sequences to assess patient to patient variability in pharmacokinetics and pharmacodynamics responses towards drugs would be the next step in future medicine in line with the vision of personalizing medicine.

Methods

Genomic DNA obtained from a 55 years old, self-declared healthy, anonymous male of Malay descent was sequenced. The subject''s mother died of lung cancer and the father had a history of schizophrenia and deceased at the age of 65 years old. A systematic, intuitive computational workflow/pipeline integrating custom algorithm in tandem with large datasets of variant annotations and gene functions for genetic variations with pharmacogenomics impact was developed. A comprehensive pathway map of drug transport, metabolism and action was used as a template to map non-synonymous variations with potential functional consequences.

Principal Findings

Over 3 million known variations and 100,898 novel variations in the Malay genome were identified. Further in-depth pharmacogenetics analysis revealed a total of 607 unique variants in 563 proteins, with the eventual identification of 4 drug transport genes, 2 drug metabolizing enzyme genes and 33 target genes harboring deleterious SNVs involved in pharmacological pathways, which could have a potential role in clinical settings.

Conclusions

The current study successfully unravels the potential of personal genome sequencing in understanding the functionally relevant variations with potential influence on drug transport, metabolism and differential therapeutic outcomes. These will be essential for realizing personalized medicine through the use of comprehensive computational pipeline for systematic data mining and analysis.     

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.     

Molecular iodine catalyzed synthesis of aryl-14H-dibenzo[a, j]xanthenes under solvent-free condition

Molecular iodine efficiently catalyzes the reaction of beta-naphthol and araldehydes on a preheated hot plate at 90-95 degrees C to give biologically active aryl-14H-dibenzo[a, j]xanthenes under solvent-free condition. The yields are excellent and the reactions go to completion within 15-20 min.     

Estrogen receptor alpha (ERα) promoter methylation status in tumor and serum DNA in Egyptian breast cancer patients

Background

Status of DNA methylation is one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated the aberrant DNA methylation status of estrogen receptor alpha (ERα) in patient pretherapeutic sera and tissue.

Materials and methods

In this case control study the patient series consisted of 120 sporadic primary breast cancer cases and 100 patients with benign breast lesion. ER3, ER4, and ER5 primers were used for methylation-specific polymerase chain reaction (MSP) to analyze the CpG methylation of promoter region of ERα gene. Correlation between ER3, ER4, and ER5 methylation and clinicopathological characteristics of the patients was investigated.

Result

The methylation status of ER3, ER4 and ER5 was 65%, 26.7% and 61.7% in tissue respectively and 57.5%, 21.7% and 55.8% in serum respectively. The concordance between tumor and serum DNA methylation was 80%, 72% and 92% for ER3, ER4 and ER5 respectively.

Conclusions

This study demonstrated the potential utility of serum DNA methylation of ERα gene promoter as a non-invasive diagnostic and/or prognostic marker in patients with breast cancer.     

CYP1A1 gene polymorphisms and smoking status as modifier factors for lung cancer risk

Objective

Lung cancer remains the most prevalent malignancy worldwide. Susceptibility to lung cancer has been shown to be modulated by inheritance of polymorphic genes. Several metabolic enzymes are currently under investigation for their possible role in lung cancer susceptibility, including members of the cytochrome P450 (CYP) superfamily. The aim of this work was to identify the correlation between CYP1A1 m1 and m2 polymorphisms and lung cancer risk and figure its interactions with smoking as genetic modifiers in the etiology of lung cancer in the Egyptian population.

Materials and methods

One hundred and ten patients with lung cancer and one hundred and ten controls were enrolled in the study. CYP1A1 m1 and m2 polymorphisms were determined using polymerase chain reaction restriction fragment length polymorphism.

Results

Subjects carrying TC and CC genotypes of CYP1A1 m1 and AG and GG genotypes of CYP1A1 m2 were significantly more likely to develop lung cancer especially squamous cell carcinoma. The proportion of lung cancer attributable to the interaction of smoking and CYP1A1 m1 and CYP1A1 m2 polymorphisms was 32% and 52% respectively.

Conclusion

Our results revealed that CYP1A1 m1 and m2 polymorphisms contribute to smoking related lung cancer risk in the Egyptian population.