Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA

CONTEXT:

Amplification of Guanine-Cytosine (GC) -rich sequences becomes important in screening and diagnosis of certain genetic diseases such as diseases arising due to expansion of GC-rich trinucleotide repeat regions. However, GC-rich sequences in the genome are refractory to standard polymerase chain reaction (PCR) amplification and require a special reaction conditions and/or modified PCR cycle parameters.

AIM:

Optimize a cost effective PCR assay to amplify the GC-rich DNA templates.

SETTINGS AND DESIGN:

Fragile X mental retardation gene (FMR 1) is an ideal candidate for PCR optimization as its GC content is more than 80%. Primers designed to amplify the GC rich 5’ untranslated region of the FMR 1 gene, was selected for the optimization of amplification using DNA extracted from buccal mucosal cells.

MATERIALS AND METHODS:

A simple and rapid protocol was used to extract DNA from buccal cells. PCR optimization was carried out using three methods, (a) substituting a substrate analog 7-deaza-dGTP to dGTP (b) in the presence of a single PCR additive and (c) using a combination of PCR additives. All PCR amplifications were carried out using a low-cost thermostable polymerase.

RESULTS:

Optimum PCR conditions were achieved when a combination of 1M betaine and 5% dimethyl sulfoxide (DMSO) was used.

CONCLUSIONS:

It was possible to amplify the GC rich region of FMR 1 gene with reproducibility in the presence of betaine and DMSO as additives without the use of commercially available kits for DNA extraction and the expensive thermostable polymerases.     

Binding and Translocation of Termination Factor Rho Studied at the Single-Molecule Level

Rho termination factor is an essential hexameric helicase responsible for terminating 20-50% of all mRNA synthesis in Escherichia coli. We used single-molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho utilization site of the λtR1 terminator. Our results are consistent with Rho complexes adopting two states: one that binds 57 ± 2 nt of RNA across all six of the Rho primary binding sites, and another that binds 85 ± 2 nt at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5′ → 3′ toward RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the Rho utilization site and RNAP. These findings lead to a general model for Rho binding and translocation and establish a novel experimental approach that should facilitate additional single-molecule studies of RNA-binding proteins.     

Identification and functional characterization of novel compound heterozygotic mutations in the TECTA gene

Mutations of the TECTA gene, which encodes alpha-tectorin, are associated with both dominant (DFNA8/A12) and recessive (DFNB 21) modes of inherited nonsyndromic sensorineural hearing loss, respectively. Although clinical data and genetic analysis for TECTA gene have been reported from different groups, there is no report that compound heterozygous mutations in the TECTA gene result in nonsyndromic sensorineural hearing loss. Here, we identified a missense mutation (p.C1691F) and a splicing mutation (c.6162 + 3insT), one in each TECTA allele, in the patient with hearing loss. Also, we demonstrated that the splicing mutation results in the abnormal skipping of an exon, which leads to a truncated protein as determined by exon-trapping analysis. To the best of our knowledge, this is the first report of an in vitro functional study of splice site mutations in the TECTA gene.     

Monitoring Autophagy Flux and Activity: Principles and Applications

Macroautophagy is a major degradation mechanism of cell components via the lysosome. Macroautophagy greatly contributes to not only cell homeostasis but also the prevention of various diseases. Because macroautophagy proceeds through multi-step reactions, researchers often face a persistent question of how macroautophagic activity can be measured correctly. To make a straightforward determination of macroautophagic activity, diverse monitoring assays have been developed. Direct measurement of lysosome-dependent degradation of radioisotopically labeled cell proteins has long been applied. Meanwhile, indirect monitoring procedures have been developed. In these assays, autophagosome marker proteins, microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) and gamma-aminobutyric acid receptor-associated protein-II (GABARAP-II) have been analyzed and the validity of the assays strongly depends on appropriate assessment of the fluctuation of LC3-II and/or GABARAP-II levels in the presence or absence of lysosomal inhibitors. This article describes these monitoring methods, paying special attention to the principles and characteristics of each procedure.     

Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients

A hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a central role in the pathogenesis of AE. Nevertheless, the precise causes of AE-associated barrier dysfunction are largely unknown. In this study, a comprehensive analysis of ceramide composition and lipid organization in nonlesional SC of AE patients and control subjects was performed by means of mass spectrometry, infrared spectroscopy, and X-ray diffraction. In addition, the skin barrier and clinical state of the disease were examined. The level of ceramides with an extreme short chain length is drastically increased in SC of AE patients, which leads to an aberrant lipid organization and a decreased skin barrier function. Changes in SC lipid properties correlate with disease severity but are independent of filaggrin mutations. We demonstrate for the first time that changes in ceramide chain length and lipid organization are directly correlated with the skin barrier defects in nonlesional skin of AE patients. We envisage that these insights will provide a new therapeutic entry in therapy and prevention of AE.     

Imaging mass spectroscopy delineates the thinned and thickened walls of intracranial aneurysms

Object

The wall thickness of intracranial aneurysms (IAs) is heterogeneous. Although thinning of the IA wall is thought to contribute to IA rupture, the underlying mechanism remains poorly understood. Recently, imaging mass spectroscopy (IMS) has been used to reveal the distribution of phospholipids in vascular diseases. To investigate the feature of phospholipid composition of IA walls, we conducted IMS in a rat model of experimentally induced IA.

Liver-type fatty acid binding protein interacts with hepatocyte nuclear factor 4α

Hepatocyte nuclear factor 4α (HNF4α) regulates liver type fatty acid binding protein (L-FABP) gene expression. Conversely as shown herein, L-FABP structurally and functionally also interacts with HNF4α. Fluorescence resonance energy transfer (FRET) between Cy3-HNF4α (donor) and Cy5-L-FABP (acceptor) as well as FRET microscopy detected L-FABP in close proximity (∼80 Å) to HNF4α, binding with high affinity K_d ∼250–300 nM. Circular dichroism (CD) determined that the HNF4α/L-FABP interaction altered protein secondary structure. Finally, L-FABP potentiated transactivation of HNF4α in COS7 cells. Taken together, these data suggest that L-FABP provides a signaling path to HNF4α activation in the nucleus.     

The nitrite-oxidizing community in activated sludge from a municipal wastewater treatment plant determined by fatty acid methyl ester-stable isotope probing

Metabolically-active autotrophic nitrite oxidizers from activated sludge were labeled with ¹³C-bicarbonate under exposure to different temperatures and nitrite concentrations. The labeled samples were characterized by FAME-SIP (fatty acid methyl ester-stable isotope probing). The compound cis-11-palmitoleic acid, which is the major lipid of the most abundant nitrite oxidizer in activated sludge, Candidatus Nitrospira defluvii, showed ¹³C-incorporation in all samples exposed to 3 mM nitrite. Subsequently, the lipid cis-7-palmitoleic acid was labeled, and it indicated the activity of a nitrite oxidizer that was different from the known Nitrospira taxa in activated sludge. The highest incorporation of cis-7-palmitoleic acid label was found after incubation with a nitrite concentration of 0.3 mM at 17 and 22 °C. While activity of Nitrobacter populations could not be detected by the FAME-SIP approach, an unknown nitrite oxidizer with the major lipid cis-9 isomer of palmitoleic acid exhibited ¹³C-incorporation at 28 °C with 30 mM nitrite. These results indicated flexibility of nitrite-oxidizing guilds in a complex community responding to different conditions. Labeled lipids so far not described for activated sludge-associated nitrifiers indicated the presence of unknown nitrite oxidizers in this habitat. The FAME-SIP-based information can be used to define appropriate conditions for the enrichment of nitrite-oxidizing guilds from complex samples.