DNA organization by the apicoplast-targeted bacterial histone-like protein of Plasmodium falciparum

Apicomplexans, including the pathogens Plasmodium and Toxoplasma, carry a nonphotosynthetic plastid of secondary endosymbiotic origin called the apicoplast. The P. falciparum apicoplast contains a 35 kb, circular DNA genome with limited coding capacity that lacks genes encoding proteins for DNA organization and replication. We report identification of a nuclear-encoded bacterial histone-like protein (PfHU) involved in DNA compaction in the apicoplast. PfHU is associated with apicoplast DNA and is expressed throughout the parasite's intra-erythocytic cycle. The protein binds DNA in a sequence nonspecific manner with a minimum binding site length of ~27 bp and a K_d of ~63 nM and displays a preference for supercoiled DNA. PfHU is capable of condensing Escherichia coli nucleoids in vivo indicating its role in DNA compaction. The unique 42 aa C-terminal extension of PfHU influences its DNA condensation properties. In contrast to bacterial HUs that bend DNA, PfHU promotes concatenation of linear DNA and inhibits DNA circularization. Atomic Force Microscopic study of PfHU–DNA complexes shows protein concentration-dependent DNA stiffening, intermolecular bundling and formation of DNA bridges followed by assembly of condensed DNA networks. Our results provide the first functional characterization of an apicomplexan HU protein and provide additional evidence for red algal ancestry of the apicoplast.     

Structural dynamics of dendritic spines: Molecular composition,geometry and functional regulation

The development of dendritic spines with specific geometry and membrane composition is critical for proper synaptic function. Specific spine membrane architecture, sub-spine microdomains and spine head and neck geometry allow for well-coordinated and compartmentalized signaling, disruption of which could lead to various neurological diseases. Research from neuronal cell culture, brain slices and direct in vivo imaging indicates that dendritic spine development is a dynamic process which includes transition from small dendritic filopodia through a series of structural refinements to elaborate spines of various morphologies. Despite intensive research, the precise coordination of this morphological transition, the changes in molecular composition, and the relation of spines of various morphologies to function remain a central enigma in the development of functional neuronal circuits. Here, we review research so far and aim to provide insight into the key events that drive structural change during transition from immature filopodia to fully functional spines and the relevance of spine geometry to function.     

Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3-S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.     

A Novel Mitochondrial Heteroplasmic C13806A Point Mutation Associated with Iranian Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by decreased expression of the protein Frataxin. Frataxin deficiency leads to excessive free radical production and dysfunction of chain complexes. Mitochondrial DNA (mtDNA) could be considered a candidate modifier factor for FRDA disease, since mitochondrial oxidative stress is thought to be involved in the pathogenesis of this disease. It prompted us to focus on the mtDNA and monitor the nucleotide changes of genome which are probably the cause of respiratory chain defects and reduced ATP generation. We searched about 46% of the entire mitochondrial genome by temporal temperature gradient gel electrophoresis (TTGE) and DNA fragments showing abnormal banding patterns were sequenced for the identification of exact mutations. In 18 patients, for the first time, we detected 26 mtDNA mutations; of which 5 (19.2%) was novel and 21 (80.8%) have been reported in other diseases. Heteroplasmic C13806A polymorphisms were associated with Iranian FRDA patients (55.5%). Our results showed that NADH dehydrogenase (ND) genes mutations in FRDA samples were higher than normal controls (P < 0.001) and we found statistically significant inverse correlation (r = −0.8) between number of mutation in ND genes and age of onset in FRDA patients. It is possible that mutations in ND genes could constitute a predisposing factor which in combination with environmental risk factors affects age of onset and disease progression.     

Aptamer-functionalized quantum dots as theranostic nanotools against cancer and bacterial infections: A comprehensive overview of recent trends

Aptamers (Apts) are synthetic nucleic acid ligands that can be engineered to target various molecules, including amino acids, proteins, and pharmaceuticals. Through a series of adsorption, recovery, and amplification steps, Apts are extracted from combinatorial libraries of synthesized nucleic acids. Using aptasensors in bioanalysis and biomedicine can be improved by combining them with nanomaterials. Moreover, Apt-associated nanomaterials, including liposomes, polymeric, dendrimers, carbon nanomaterials, silica, nanorods, magnetic NPs, and quantum dots (QDs), have been widely used as promising nanotools in biomedicine. Following surface modifications and conjugation with appropriate functional groups, these nanomaterials can be successfully used in aptasensing. Advanced biological assays can use Apts immobilized on QD surfaces through physical interaction and chemical bonding. Accordingly, modern QD aptasensing platforms rely on interactions between QDs, Apts, and targets to detect them. QD-Apt conjugates can be used to directly detect prostate, ovarian, colorectal, and lung cancers or simultaneously detect biomarkers associated with these malignancies. Tenascin-C, mucin 1, prostate-specific antigen, prostate-specific membrane antigen, nucleolin, growth factors, and exosomes are among the cancer biomarkers that can be sensitively detected using such bioconjugates. Furthermore, Apt-conjugated QDs have shown great potential for controlling bacterial infections such as Bacillus thuringiensis, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Campylobacter jejuni, Staphylococcus aureus, and Salmonella typhimurium. This comprehensive review discusses recent advancements in the design of QD-Apt bioconjugates and their applications in cancer and bacterial theranostics.     

Extremely high Tp53 mutation load in esophageal squamous cell carcinoma in Golestan Province, Iran

Background

Golestan Province in northeastern Iran has one of the highest incidences of esophageal squamous cell carcinoma (ESCC) in the world with rates over 50 per 100,000 person-years in both sexes. We have analyzed TP53 mutation patterns in tumors from this high-risk geographic area in search of clues to the mutagenic processes involved in causing ESCC.

Methodology/Principal Findings

Biopsies of 119 confirmed ESCC tumor tissue from subjects enrolled in a case-control study conducted in Golestan Province were analyzed by direct sequencing of TP53 exons 2 through 11. Immunohistochemical staining for p53 was carried out using two monoclonal antibodies, DO7 and 1801. A total of 120 TP53 mutations were detected in 107/119 cases (89.9%), including 11 patients with double or triple mutations. The mutation pattern was heterogeneous with infrequent mutations at common TP53 “hotspots” but frequent transversions potentially attributable to environmental carcinogens forming bulky DNA adducts, including 40% at bases known as site of mutagenesis by polycyclic aromatic hydrocarbons (PAHs). Mutations showed different patterns according to the reported temperature of tea consumption, but no variation was observed in relation to ethnicity, tobacco or opium use, and alcoholic beverage consumption or urban versus rural residence.

Conclusion/Significance

ESCC tumors in people from Golestan Province show the highest rate of TP53 mutations ever reported in any cancer anywhere. The heterogeneous mutation pattern is highly suggestive of a causative role for multiple environmental carcinogens, including PAHs. The temperature and composition of tea may also influence mutagenesis.     

Chemical modification of glucose oxidase: possible formation of molten globule-like intermediate structure

Chemical modification of lysine residues in glucose oxidase was carried out using citraconic anhydride. Modification brought about changes in the kinetic properties of the enzyme as evident by substantial lowering of V(max) and K(m). Enhancement of tryptophan fluorescence was observed with a dramatic change in its pH dependence due to modification. Near- and far-UV circular dichroism spectra of the native and modified forms suggested formation of molten globule-like structures, further supported by 8-anilino-1-naphthalenesulfonic acid fluorescence which indicated higher exposure of hydrophobic residues as a result of chemical modification.     

Kinetic properties of extracted lactate dehydrogenase and creatine kinase from mouse embryonic stem cell- and neonatal-derived cardiomyocytes

Embryonic stem cells (ESCs), representing a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics, are capable of spontaneous differentiation into cardiomyocytes. The present study sought to define the kinetic characterization of lactate dehydrogenase (LDH) and creatine kinase (CK) of ESC- and neonatal-derived cardiomyocytes. Spontaneously differentiated cardiomyocytes from embryoid bodies (EBs) derived from mouse ESC line (Royan B1) and neonatal cardiomyocytes were dispersed in a buffer solution. Enzymes were extracted by sonication and centrifugation for kinetic evaluation of LDH and CK with spectrophotometric methods. While a comparison between the kinetic properties of the LDH and CK of both groups revealed not only different Michaelis constants and optimum temperatures for LDH but also different Michaelis constants and optimum pH for CK, the pH profile of LDH and optimum temperature of CK were similar. In defining some kinetic properties of cardiac metabolic enzymes of ESC-derived cardiomyocytes, our results are expected to further facilitate the use of ESCs as an experimental model.     

Inferring copy number and genotype in tumour exome data

Background

Using whole exome sequencing to predict aberrations in tumours is a cost effective alternative to whole genome sequencing, however is predominantly used for variant detection and infrequently utilised for detection of somatic copy number variation.

Results

We propose a new method to infer copy number and genotypes using whole exome data from paired tumour/normal samples. Our algorithm uses two Hidden Markov Models to predict copy number and genotypes and computationally resolves polyploidy/aneuploidy, normal cell contamination and signal baseline shift. Our method makes explicit detection on chromosome arm level events, which are commonly found in tumour samples. The methods are combined into a package named ADTEx (Aberration Detection in Tumour Exome). We applied our algorithm to a cohort of 17 in-house generated and 18 TCGA paired ovarian cancer/normal exomes and evaluated the performance by comparing against the copy number variations and genotypes predicted using Affymetrix SNP 6.0 data of the same samples. Further, we carried out a comparison study to show that ADTEx outperformed its competitors in terms of precision and F-measure.

Conclusions

Our proposed method, ADTEx, uses both depth of coverage ratios and B allele frequencies calculated from whole exome sequencing data, to predict copy number variations along with their genotypes. ADTEx is implemented as a user friendly software package using Python and R statistical language. Source code and sample data are freely available under GNU license (GPLv3) at http://adtex.sourceforge.net/.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-732) contains supplementary material, which is available to authorized users.     

HspX protein as a candidate vaccine against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: an overview

Background

Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis (Mtb). This disease with two million deaths per year has the highest mortality rate among bacterial infections. The only available vaccine against TB is BCG vaccine. BCG is an effective vaccine against TB in childhood, however, due to some limitations, has not proper efficiency in adults. Also, BCG cannot produce an adequately protective response against reactivation of latent infections.

Objective

In the present study we will review the most recent findings about contribution of HspX protein in the vaccines against tuberculosis.

Methods

Therefore, many attempts have been made to improve BCG or to find its replacement. Most of the subunit vaccines for TB in various phases of clinical trials were constructed as prophylactic vaccines using Mtb proteins expressed in the replicating stage. These vaccines might prevent active TB but not reactivation of latent tuberculosis infection (LTBI). A literature search was performed on various online databases (PubMed, Scopus, and Google Scholar) regarding the roles of HspX protein in tuberculosis vaccines.

Results

Ideal subunit post-exposure vaccines should target all forms of TB infection, including active symptomatic and dormant (latent) asymptomatic forms. Among these subunit vaccines, HspX is the most important latent phase antigen of M. tuberculosis with a strong immunological response. There are many studies that have evaluated the immunogenicity of this protein to improve TB vaccine.

Conclusion

According to the studies, HspX protein is a good candidate for development of subunit vaccines against TB infection.