Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

Genetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA.A sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed.The assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA.This SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts

Influence of fowl uropygial gland and its secretory lipid components on the growth of skin surface fungi of fowl

1218-1222Fungal species, which were shown to colonize consistently on the skin surface of the breast region of adult (1year old)white leghorn fowl, were identified as Aspergillus sydowii, A. tamarii , A. rugulosus and Absidia corymbifera.Of these,A sydowii and A. tamarii were the dominant forms. Two species of fungi, namely, Aspergillus niger and Scopulariopsis brevicaulis were shown to be present in the cultures of the scrubbings from breast skin surface after 60 days or captivity of the fowls. Extirpation of the uropygiai gland resulted in encouragement of the in vitro population growth of all specices of fungi except that of A.rugulosus. The effect was found to be very conspicuous for A. sydowii and A. tamarii, particularly after 60 days of gland removal. Addition of total lipids and the wax diester component of free- flowing uropygial secretion as 0.2% suspension in Sabouraud 's agar medium of individual fungal isolates caused marked suppression of the population growth of A. sydowii, A. tamarii, Absidia corymbifera and to some extent of S. brevicaulis. Other components of secretory lipids, such as wax alcohols (2,3-alkane-diols ), wax acids, triglycerides and hydrocarbons (including squalene) when supplemented separately to culture medium of individual fungi at identical concentration, were also shown to cause inhibition of the growth of most of fungal species at different degrees

Ab initio study of boron hydroxy species: B(OH), B(OH)4 and B(OH)3O-

553-557Ab initio SCF studies at STO-3G and 4-31G levels have been performed on B(OH) as well as on the recently reported radicals B(OH)4 and B(OH)3O-. The RHF calculations for B(OH) lead to Td  structure, which is in agreement with the experimental results. The B(OH) radical is found to prefer a C2v geometry rather than Td or D2d structures according to UHF calculations. The Td and D2d structures are 23.7 and 12.6 kcal/mol higher respectively in energy than the C2v structure at the STO-3G level. STO 4-31G calculations show that the corresponding values are 16.3 and 11.5 kcal/ mol respectively for the Td and D2d structures. The UHF calculations using both the basis sets indicate that the two geometries considered for the B(OH)3O- radical anion, Td and C3v, are very close energetically

Further studies on Norrish type II reactions including a reaction in the first excited singlet state and cyclization of 1:4 biradicals

The Norrish type II processes of methyl-2,2-dimethyl- cyclopropyl ketone, alpha-alkoxy acetones and alkyl pyruvates have been examined using the AM1 semi-empirical molecular orbital method with complete geometry optimization at the partial configuration interaction level in the restricted Hartree-Fock (RHF) frame. The results reveal that the methyl-substituted cyclopropyl ketone has a constrained geometry favourable for hydrogen abstraction from the gamma-position relative to the carbonyl group in the excited singlet state. The presence of the ether oxygen atom in the beta-position relative to the carbonyl group in alkoxy acetones and alkyl pyruvates leads to increased reactivity relative to alkyl monoketones and diketones respectively. The cyclization of 1:4 biradicals has been studied in the unrestricted Hartree-Fock (UHF) frame, and the results reveal that the 1:4 biradical derived from alkoxy acetones readily cyclizes to form oxetanols. On the other hand, in the 1:4 biradicals derived from methyl-substituted cyclopropyl ketone, the three-membered ring breaks readily to form an enol intermediate. Delocalization of an odd electron in 1:4 biradicals derived from alkyl pyruvates is thought to make cyclization difficult

Leiomyoma of the mandible in a child

Leiomyomas are the benign tumors of the smooth muscle that usually arise in the gastrointestinal system and in the uterus. Oral leiomyomas are uncommon due to the paucity of the smooth muscles in the mouth (except in blood vessels) and those of the mandible are extremely rare. Leiomyomas have been classified as solid, angiomyoma (vascular leiomyoma), and epithelioid variants. Here, we report a rare case of leiomyoma of the mandible in a 9-year-old child, together with conventional histopathologic and immunohistochemical findings

Genetic transformation of a hepatoprotective plant, Phyllanthus amarus

Phyllanthus amarus Schum & Thonn. is a source of various pharmacologically active compounds such as phyllanthin, hypophyllanthin, gallic acid, catechin, and nirurin, a flavone glycoside. A genetic transformation method using Agrobacterium tumefaciens was developed for this plant species for the first time. Shoot tips of full grown plants were used as explants for Agrobacteriummediated transformation. Transgenic plants were obtained by co-cultivation of shoot tips explants and A. tumefaciens strain LBA4404 containing the pCAMBIA 2301 plasmid harboring neomycin phosphotransferase II (NPT II) and β- glucuronidase encoding (GUS) genes in the T-DNA region in the presence of 200 μM acetosyringone. Integration of the NPT II gene into the genome of transgenic plants was verified by PCR and Southern blot analyses. Expression of the NPT II gene was confirmed by RT-PCR analysis. An average of 25 explants was used, out of which an average of 19 explants produced kanamycin-resistant shoots, which rooted to produce 13 complete transgenic plants