जर्नल ऑफ़ क्लिनिकल एंड मेडिकल जीनोमिक्स

Genomic DNA isolation is an essential procedure to allow many genetic applications and analyses like that using molecular markers. Obtaining good quality DNA samples is the first step to succeed in such analysis and it depends on effective procedures for harvesting and preserving the plant material, and also for DNA extraction. The use of fresh material is ideal for DNA isolation, however, in studies that involve the harvesting of wild plants this is not always possible, since in most cases, populations are distant from the research laboratory. An alternative is freezing the plant material in liquid nitrogen in the field, but this practice is not feasible in many cases, given the difficulty and danger of transporting the liquid nitrogen tank on places of difficult access. In this study, we present four simple, efficient and low cost methodologies that have been successfully tested for a Myrtaceae species with medicinal potential collected at different biomes, Atlantic Rainforest and Brazilian Savanna (Cerrado). Among the main advantages observed are the reduced use of liquid nitrogen, the use of inexpensive materials and ease of transport and storage of samples. The methods presented here can potentially be applied to other species of this botanical family.

Reprogramming of mRNA translation by localized alterations in the standard translational rules is termed as recoding. Frameshifting is one class of recoding and defined as protein translations that start not at the first, but either at the second (+1 frameshift) or the third (-1 frameshift) nucleotide of the codon. Apparently, most frameshifts would yield nonfunctional proteins. Therefore frameshifts lead to waste of energy, resources and activity of the biosynthetic machinery. In addition, some peptides synthesized after frameshifts are most likely cytotoxic. Coding sequences lack stop codons, but many stop codons generated due to frameshifted sequences, termed off-frame stops or hidden stop codons. These hidden stops terminate frame-shifted translation, potentially decreasing energy, and resource waste on nonfunctional proteins. Frameshift mutations for the sake of mitochondrial genomes were evaluated and results support this putative ancient adaptive event for the selection of codons that can be part of hidden stop codons. Mitochondrial genomic data analyses support this fact as most of the correlations between hidden stops and various functional and evolutionary parameters were positive. Association of frameshift mutations with many diseases is also being studied to represent the biological significance of this putative event. It is estimated that this kind of study will reveal momentous correlation between frameshift mutations and their biological consequences.

Animal models are integral to our understanding of the cellular and molecular pathogenesis of human diseases and disorders. Functional genome-wide methods such as DNA microarray and RNA interference-based highthroughput screening have recently emerged as powerful tools for such studies. However, genomic results from animal models may not necessarily correspond to the pathogenesis in humans. Thus, there is a need for new methods that better correlate the data from these models with human disease and disorders. Here we describe the reverse direction method, which combines the in vitro data of genome-wide screening of animal models with the data from genome-wide association studies (GWAS) of human diseases and disorders to effectively link the results of the two. This review introduces the concept of the reverse direction method when applied to the study of the inflammation amplifier, a chemokine inducer in non-immune cells for the development of chronic inflammatory diseases.

Aim: The RAS/MAPK signaling pathway proteins with germline mutations in their respective genes are associated with several disorders such as Noonan, LEOPARD, neurofibromatosis type 1, Costello and cardio-facio-cutaneous syndromes. Some monogenic conditions are associated with the development of systemic lupus erythematosus (SLE), in medical literature are few reports that describe the association of RAS opathies and autoimmune disease. Our aim was to describe the clinical picture of a patient with diagnosis of Noonan and SLE.

Methods: We report a clinical case of a 24-year-old woman with Noonan syndrome who developed SLE according to American College of Rheumatology criteria for the classification of SLE. The patient had arthritis, serositis, lymphopenia, proteinuria, high levels of antinuclear antibodies and anti-ds DNA positive. This rare association then driven to search the medical literature for English articles on the subjects of Noonan and SLE in Pubmed.

Results: Our patient had oligoarthritis, serositis, lymphopenia, ISN/RPS Class IV lupus nephritis, ANA 1:1280 homogeneous pattern and anti-dsDNA antibodies very similar to the 8 patients already reported in literature.

Conclusion: There are nine cases reported with the association of two rare diseases, Noonan syndrome and SLE, this connection could suggests that RAS opathies may be a risk factor to the development of autoimmune disorders.

It is estimated that one-third of disease-causing mutations may induce aberrant splicing of pre-messenger RNA transcripts and a partially overlapping third to premature stop codons (PTC) and nonsense-mediated mRNA decay (NMD). In some diseases, the estimate even goes up to 50% and >70%, respectively. These highly prevalent effects of different mutations on mRNA processing have prompted much effort for the identification of compounds towards the therapy of a substantial number of diseases with mutation-specific, personalized medicine. Here I review the widespread occurrence of aberrant splicing, NMD and their association in human genetic diseases, and discuss the rationales underlying the corresponding therapeutic strategies and challenges.

The ability to sequence and analyze the human genome and transcriptomes of various sources at speeds unimaginable more than 20 years ago has had huge impacts on not only basic biological research but also the development of novel therapeutic strategies for human genetic diseases. Particularly this ability allows the screening of mutations in individuals at a genome/transcriptome scale for the design of different therapeutic strategies for mutation-specific, personalized medicine based on how the mutations take their toll.

For the genetic information-based disease therapy, RNA has also been targeted besides DNA and protein, with an accelerating speed of research in recent years. This development has mainly benefited from our understanding of different aspects of RNA processing and appreciation of their prevalence, such as the widespread presence of alternative pre-mRNA splicing in human transcriptomes, mRNA quality control by nonsense-mediated decay (NMD), as well as microRNA, long noncoding RNA and other non-coding RNAs. Their misregulation due to mutations has been linked to or associated with the development of human genetic diseases. Here I will discuss the therapeutic potential of targeting aberrant splicing and NMD, two widespread and related effects of a large number of genetic mutations that cause human diseases.

Nutrient deprivation conditions are capable to induce the endoplasmic reticulum stress and are responsible for regulation of the expression of numerous growth factors which control the cell proliferation and angiogenesis in malignant tumors, including glioma. The ERN1 mediated endoplasmic reticulum stress response-signalling pathway is tightly linked to the proliferation because the blockade of ERN1 function suppresses the tumor growth via specific changes in the cell transcriptome. Activation of the pentose phosphate pathway is an important factor of enhanced cell proliferation and tumor growthbecause it controls the synthesis of nucleotides and respectively nucleic acids. We studied the effect of blockade the ERN1 signaling enzyme function in glioma cells on the expression of phosphoribosyl pyrophosphate synthetase (PRPS), an important enzyme of pentose phosphate pathway, as well as its dependence of glucose and glutamine deprivation.

The expression of different genes of PRPS enzyme in glioma cells with complete suppression of ERN1 enzyme activity was measured by qPCR. For glucose and glutamine deprivation conditions the cells were exposurein the glucose or glutamine free medium for 16 hrs.

The expression of PRPS1 and PRPS2 genes as well as phosphoribosyl pyrophosphate synthetase associated protein 1(PRPSAP1) is significantly increased in U87 glioma cells with suppressed function of ERN1 gene, but the expression PRPSAP2 gene is decreased. It was also shown that glucose or glutamine deprivation leads to suppression of PRPS1 and PRPS2 gene expressions in U87 glioma cells; however, the blockade of ERN1 function in glioma cells modifies the effect of glutamine deprivation. Moreover, the expression of PRPSAP1 and PRPSAP2 genes was resistant to glucose deprivation condition in control glioma cells, but increased in glioma cells with ERN1 loss of function.

Results of this investigation demonstrated that the blockade of ERN1 signaling enzyme function affects the expression of genes encoding PRPS enzyme subunits in different ways and that dysregulation of the expression of important regulatory subunits of PRPS (PRPSAP1 and PRPSAP2) possibly changes the activity of this enzyme and participates in suppression of glioma growth via pentose phosphate pathway.

Platelet adhesion, activation and aggregation to the injured vessel wall are crucially involved in the pathogenesis of thrombus formation. Agents in theory thwarting these phases would have significant clinical value. The current antiplatelet drugs used in daily clinical practice include COX-1 inhibitor aspirin, ADP P2Y₁₂ receptor antagonist clopidogrel, and the GPIIb-IIIa antagonists (abciximab, eptifibatide and tirofiban). However, confined curative ratio along with unforeseen bleeding risk remains a major puzzle of antiplatelet therapy. With advances in understanding of the molecular basis of platelet in thrombosis, newer antiplatelet agents that targets different stage of thrombus formation have been recently developed, mostly including agents targeting platelet adhesion (GPIV, vWF), activation (GPVI, P2Y₁₂, TPα, PAR1, phosphodiesterase, cyclooxygenase), and aggregation (GPIIb/IIIa). In this article, we will review the advantages and limitations of various antiplatelet agents that have been approved by the US Food and Drug Administration (FDA) or under development.

Background: Neuropilin-1 (NRP1) is a membrane-bound co-receptor of vascular endothelial growth factor receptors (VEGFRs) and type-A plexins. These complexes mediate the effects of ligands such as vascular endothelial growth factor 165 (VEGF₁₆₅) and semaphorin 3A (SEMA-3A).

Aim: Our aims were to elucidate whether the expression of NRP1 is related to the proliferation or differentiation programme of keratinocytes and to identify putative downstream signaling pathways of NRP1 in keratinocytes.

Materials and methods: Synchronized HaCaT cells and paraffin-embedded tissue samples of psoriasis were used to modell highly proliferative stages of keratinocytes. Real-time RT-PCR-based expression array was used to analyse downstream signaling pathways in keratinocytes.

Results: Synchronized HaCaT cells revealed that the peak expression of NRP1 occurred 24 hours before the cells reached their highest proliferative activity. Immunohistochemical staining of the highly proliferative tissue samples demonstrated that the high level of NRP1 protein expression partially overlapped with the expression of Ki67-positive cells. Real-time RT-PCR-based expression array identified the NF-κB pathway as one putative downstream signaling pathway of NRP1-mediated signaling in HaCaT cells. VEGF₁₆₅ and SEMA-3A treatment of HaCaT cells stably transformed with a reporter construct for the assessment of NF-κB activation led to upregulation of the NF-κB activity.

Conclusion: Our results suggest that the NRP1 might be involved in the regulation of keratinocyte proliferation and might activate NF-κB pathway as a putative downstream signaling pathway in keratinocytes.

Aims: The present study focussed on deriving and validating a ‘genetic bleeding risk score’ (GBRS) based on genetic and non-genetic factors associated with bleeding in patients on long term anticoagulation therapy.

Patients and Methods: Patients on warfarin (n=53) or acenocoumarol (n=257) long-term therapy were genotyped for twenty one SNPs in six genes. Two GBRSs were developed and validated.

Results: The incidence rate was 16.86 and 4.46 per 100 person-years for minor and major bleeding respectively. The novel GBRS (positive predictive value = 83.3%, specificity = 97.4%) comprised of four parameters; age >65 years, F5 rs6025, VKORC1 rs9934438 and CYP2C9 rs1057911.

Conclusions: The present study is the first to devise and validate a genetic based score for predicting bleeding among first time users of oral anticoagulants.