Characterization of rodent selenoprotein W promoter

Rat selenoprotein W (SeW) promoter activity was investigated using different concentrations of cadmium, copper, and zinc. Two fragments (404bp and 1265bp) of the SeW promoter, containing a single metal response element (MRE), were ligated into the multiple cloning site of a pGL3-Basic reporter plasmid. The constructs were transfected into cultured rat C6 (glial) and L8 (myoblast) cells and promoter activity measured by means of luciferase reporter gene fused to the SeW promoter fragments in the reporter plasmid. With post-transfection exposure of these cell lines to these metals, copper and zinc, but not cadmium, significantly increased promoter activity of the unmutated 1265bp (not 404bp) construct (p<0.05) only in the C6 cells. Mutation of the MRE sequence abolished promoter response to metal exposure but did not eliminate promoter activity. The results suggest that SeW expression in glial cells can be increased on exposure to copper and zinc and that this response is dependent on the MRE sequence present in the SeW promoter. To understand transcriptional regulation of the SeW gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter, and antibodies, we were able to show that the specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as the MRE. However, the MRE, GRE, AP-1 and LF-A1 did not yield any specific binding. Although, competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore the SeW gene may be a target for Sp1 whose interaction with the SeW promoter may activate or repress the transcription of SeW

Antiviral and Neuroprotective Role of Octaguanidinium Dendrimer-Conjugated Morpholino Oligomers in Japanese Encephalitis

Japanese encephalitis (JE) is caused by a flavivirus that is transmitted to humans by mosquitoes belonging to the Culex sp. The threat of JE looms over a vast geographical realm, encompassing approximately 10 billion people. The disease is feared because currently there are no specific antiviral drugs available. There have been reports where other investigators have shown that agents that block viral replication can be used as effective therapeutic countermeasures. Vivo-Morpholinos (MOs) are synthetically produced analogs of DNA or RNA that can be modified to bind with specific targeted regions in a genome. In this study the authors propose that in an animal model of JE, MOs specifically designed to bind with specific region of JE virus (JEV) genome, blocks virus production in cells of living organisms. This results in reduced mortality of infected animals. As the major target of JEV is the nerve cells, analysis of brain of experimental animals, post treatment with MOs, showed neuroprotection. Studies in cultured cells were also supportive of the antiviral role of the MOs. The potent anti-sense effect in animals and lack of obvious toxicity at the effective dosage make these MOs good research reagents with future therapeutic applications in JE

Pre-mRNA Splicing Modulation by Antisense Oligonucleotides

Pre-mRNA splicing, a dynamic process of intron removal and exon joining, is governed by a combinatorial control exerted by overlapping cis-elements that are unique to each exon and its flanking intronic sequences. Splicing cis-elements are usually 4-to-8-nucleotide-long linear motifs that provide binding sites for specific proteins. Pre-mRNA splicing is also influenced by secondary and higher order RNA structures that affect accessibility of splicing cis-elements. Antisense oligonucleotides (ASOs) that block splicing cis-elements and/or affect RNA structure have been shown to modulate splicing in vivo. Therefore, ASO-based strategies have emerged as a powerful tool for therapeutic manipulation of splicing in pathological conditions. Here we describe an ASO-based approach to increase the production of the full-length SMN2 mRNA in spinal muscular atrophy patient cells

Comparison of the Safety and Pharmacokinetics of ST-246® after IV Infusion or Oral Administration in Mice, Rabbits and Monkeys

ST-246® is an antiviral, orally bioavailable small molecule in clinical development for treatment of orthopoxvirus infections. An intravenous (IV) formulation may be required for some hospitalized patients who are unable to take oral medication. An IV formulation has been evaluated in three species previously used in evaluation of both efficacy and toxicology of the oral formulation. plasma concentrations. These effects were eliminated using slower IV infusions. associated toxicity. Shorter infusions at higher doses in NHP resulted in decreased clearance, suggesting saturated distribution or elimination. Elimination half-lives in all species were similar between oral and IV administration. The administration of ST-246 was well tolerated as a slow IV infusion

Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides

The potential use of antisense and siRNA oligonucleotides as therapeutic agents has elicited a great deal of interest. However, a major issue for oligonucleotide-based therapeutics involves effective intracellular delivery of the active molecules. In this Survey and Summary, we review recent reports on delivery strategies, including conjugates of oligonucleotides with various ligands, as well as use of nanocarrier approaches. These are discussed in the context of intracellular trafficking pathways and issues regarding in vivo biodistribution of molecules and nanoparticles. Molecular-sized chemical conjugates and supramolecular nanocarriers each display advantages and disadvantages in terms of effective and nontoxic delivery. Thus, choice of an optimal delivery modality will likely depend on the therapeutic context

Selenium biochemistry and its role for human health

Despite its very low level in humans, selenium plays an important and unique role among the (semi)metal trace essential elements because it is the only one for which incorporation into proteins is genetically encoded, as the constitutive part of the 21st amino acid, selenocysteine. Twenty-five selenoproteins have been identified so far in the human proteome. The biological functions of some of them are still unknown, whereas for others there is evidence for a role in antioxidant defence, redox state regulation and a wide variety of specific metabolic pathways. In relation to these functions, the selenoproteins emerged in recent years as possible biomarkers of several diseases such as diabetes and several forms of cancer. Comprehension of the selenium biochemical pathways under normal physiological conditions is therefore an important requisite to elucidate its preventing/therapeutic effect for human diseases. This review summarizes the most recent findings on the biochemistry of active selenium species in humans, and addresses the latest evidence on the link between selenium intake, selenoproteins functionality and beneficial health effects. Primary emphasis is given to the interpretation of biochemical mechanisms rather than epidemiological/observational data. In this context, the review includes the following sections: (1) brief introduction; (2) general nutritional aspects of selenium; (3) global view of selenium metabolic routes; (4) detailed characterization of all human selenoproteins; (5) detailed discussion of the relation between selenoproteins and a variety of human diseases

Tingkat Walkability Berdasarkan Persepsi Pejalan Kaki di Koridor Jalan MT. Haryono Kota Malang

Berjalan merupakan salah satu cara berlalu lintas dalam sistem transportasi, dan sangat dominan di daerah perkotaan atau lokasi yang memiliki permintaan tinggi dengan periode pendek. Jalur pejalan kaki berupa trotoar merupakan wadah atau ruang untuk kegiatan pejalan kaki melakukan aktivitas dan untuk memberikan pelayanan kepada pejalan kaki. Walkability menjadi salah satu metode yang digunakan untuk menjelaskan dan mengukur kenyamanan konektifitas dan kualitas jalur pejalan kaki. Pengukurannya dilakukan melalui penilaian komprehensif terhadap infrastruktur yang tersedia untuk pejalan kaki dimana kajian walkability ini menghubungkan sisi penyediaan (supply), dan sisi kebutuhan (demand). Jalan MT. Haryono di Kota Malang termasuk salah satu kawasan yang memiliki intensitas pergerakan yang cukup tinggi dengan karakter guna lahan mixed use antara perdagangan dan jasa yang membentuk pola linear di sepanjang jalan, kawasan permukiman, serta keberadaan kawasan pendidikan berupa Universitas Brawijaya. Universitas Brawijaya yang merupakan fasilitas pendidikan dengan skala nasional menjadikan kawasan ini memiliki tarikan dan bangkitan pergerakan yang besar di Kota Malang. Namun, tingginya pergerakan dari dan menuju Universitas Brawijaya itu tidak didukung dengan adanya prasarana pergerakan seperti jalur pejalan kaki yang memadai. Tujuan dari penelitian ini adalah untuk mengetahui tingkat walkability di koridor Jalan MT. Haryono berdasarkan persepsi dari pengguna jalur pejalan kaki. Pengukuran dilakukan dengan menggunakan 9 variabel walkability berdasarkan Global Walkability Index (Leather et. al, 2011). Dalam hal ini persepsi dari pejalan kaki sebagai pihak yang memiliki demand akan jalur pejalan kaki diperlukan agar dapat memberi arahan yang tepat dan sesuai dengan pejalan kaki di koridor Jalan MT. Haryono. Variabel yang digunakan dalam penelitian ini adalah konflik jalur pejalan kaki dengan moda transportasi lain, ketersediaan jalur pejalan kaki, ketersediaan penyeberangan, keamanan penyeberangan, sikap pengendara motor, amenities (kelengkapan pendukung), infrastruktur penunjang kelompok penyandang cacat, kendala/hambatan dan keamanan terhadap kejahatan. Metode yang digunakan dalam penelitian ini adalah metode deskriptif dan analisis regresi linier berganda. Hasil dari analisis, diketahui bahwa 9 variabel walkability seluruhnya berpengaruh terhadap tingkat walkability di koridor Jalan MT. Haryono Kota Malang. Tingkat walkability di koridor Jalan MT Haryono menurut persepsi pengguna didapat penilaian bahwa segmen yang mendapat nilai walkability terbaik adalah pada segmen 4A dengan nilai 58,63 yang masuk dalam kategori “Somewhat Walkable” yang berarti segmen yang beberapa kegiatannya dapat dicapai dengan berjalan kaki, sementara segmen dengan walkability index terendah adalah pada segmen 5B dengan nilai walkability sebesar 24,11 dengan kategori “Car Dependent” yang berarti segmen yang kegiatannya kebanyakan menggunakan kendaraan bermotor