The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor–containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline–dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4), and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia–stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2) oligomerization domain and the NHR3 protein–protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen–activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti–tumor effect.
The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis.
The introduction of next generation sequencing methods in genome studies has made it possible to shift research from a gene-centric approach to a genome wide view. Although methods and tools to detect single nucleotide polymorphisms are becoming more mature, methods to identify and visualize structural variation (SV) are still in their infancy. Most genome browsers can only compare a given sequence to a reference genome; therefore, direct comparison of multiple individuals still remains a challenge. Therefore, the implementation of efficient approaches to explore and visualize SVs and directly compare two or more individuals is desirable. In this article, we present a visualization approach that uses space-filling Hilbert curves to explore SVs based on both read-depth and pair-end information. An interactive open-source Java application, called Meander, implements the proposed methodology, and its functionality is demonstrated using two cases. With Meander, users can explore variations at different levels of resolution and simultaneously compare up to four different individuals against a common reference. The application was developed using Java version 1.6 and Processing.org and can be run on any platform. It can be found at http://homes.esat.kuleuven.be/~bioiuser/meander.
The present study was designed to evaluate the effect of aqueous extract of Trigonella foenum-graecum(AqE-TFG) seeds on monosodium glutamate (MSG)-induced dyslipidemia and oxidative stress in Wistar rats.
Materials and Methods:
Neonatal Wistar rats were treated subcutaneously with MSG (4 g/kg b.w.) from day 2 to 14 after birth, on alternate days. After attaining six-weeks of age, MSG-treated rats were administered with AqE-TFG (0.5 and 1 g/kg b.w., orally) or orlistat (10 mg/kg b.w., orally) for 28 days, respectively. Serum chemistry and relevant enzymes in hepato-cardiac tissues were assessed on day 29.
AqE-TFG produced significant reduction in serum total cholesterol (TC), triglycerides (TGs), lactate dehydrogenase (LDH), aspartate amino transferase (AST), alanine amino transferase (ALT), hepatic and cardiac lipid peroxides (MDA) levels and elevation in serum high density lipoprotein cholesterol (HDL-C), hepatic and cardiac antioxidant enzymes [glutathione (GSH), and superoxide dismutase (SOD) and catalase (CAT)] levels.
Results were comparable with orlistat, a standard anti-obesity drug, and provide clear evidence that the AqE-TFG treatment offered significant protection against MSG-induced dyslipidemia and oxidative stress, and may play an important role in amelioration of the free radical generated consequences like dyslipidemia and atherosclerosis.
Antihyperlipidemic; monosodium glutamate; neonatal; oxidative stress; Trigonella foenum-graecum
Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell’s copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes.
Multidrug-resistant tuberculosis is an increasing public health concern in many parts of the world, especially in low-income countries, where most cases occur. Traditional mycobacteria culture and drug susceptibility testing (DST) is either time-consuming or expensive and for that reason uptake of these technologies has remained limited in many resource-constrained settings. However, several non-commercial culture and DST methods that do not require sophisticated infrastructure and techniques have been developed. One such method is the microscopic observation drug susceptibility assay (MODS). In this method microcolonies that form in the liquid culture medium after specimen inoculation to drug-free and drug-containing micro-wells are detected by visual observation with a simple inverted microscope. The identification and drug susceptibility results can be obtained in 7-15 days. This standard operating procedure document has been developed through the culture and DST subgroup of the STOP TB Partnership, New Diagnostic Working Group. It is intended for laboratories that would want to use or already using this rapid non-commercial method for culture identification and DST of Mycobacterium tuberculosis, notably in resource-constraint settings in Asia and Africa.
Tuberculosis; rapid; drug susceptibility testing; Stop-TB; Training
In the previous part, we presented the standard operating procedure (SOP) of the microscopic observation drug susceptibility assay drug susceptibility testing (DST) for Mycobacterium tuberculosis. The present SOP is devoted to another non-commercial culture and DST method known as nitrate reductase assay (NRA). As the name implies, the NRA detects the ability of M. tuberculosis to reduce nitrate to nitrite. In the assay, the presence of nitrite is detected by the addition of p-nitrobenzoate into the growth yield. The reaction is detected by the naked eye. The incorporation of drugs in the medium allows to use the test for DST, which can be interpreted with naked eyes. The identification and drug susceptibility results can be obtained in 2-3 weeks. This SOP document has been developed through the culture and DST subgroup of the STOP tuberculosis (TB) Partnership New Diagnostic Working Group. It is intended for laboratories that would want to use or already using this rapid non-commercial method for culture identification and DST of M. tuberculosis, notably in resource-constraint settings in Asia and Africa.
Mycobacterium; tuberculosis; drugs; susceptibility; testing; training
The previous two standard operating procedures (SOPs) related to the culture and drug susceptibility testing (DST) of Mycobacterium tuberculosis with the microscopic observation drug susceptibility assay (Part 1) and nitrate reductase assay (Part 2). The present SOP is devoted to a third non-commercial culture and DST method known as colorimetric redox indicator assay (CRI). As its name indicates, the CRI detects the ability of the M. tuberculosis to reduce the colored oxidation-reduction indicator when added to a liquid culture of M. tuberculosis, after exposing the growth to different anti-mycobacterial drugs. The change in the color of the indicator denotes the proportionate number of viable Mycobacteria in the medium. The identification and DST results can be obtained in 7-8 days. This SOP document has been developed through the culture and DST subgroup of the STOP tuberculosis (TB) Partnership New Diagnostic Working Group. It is intended for laboratories that would want to use or already use this rapid non-commercial method for culture identification and DST of M. tuberculosis, notably in resource-constraint settings in Asia and Africa.
Mycobacterium; tuberculosis; drugs; susceptibility; testing; training
MTG16, MTGR1 and ETO are nuclear transcriptional corepressors of the human ETO protein family. MTG16 is implicated in hematopoietic development and in controlling erythropoiesis/megakaryopoiesis. Furthermore, ETO homologue genes are 3'participants in leukemia fusions generated by chromosomal translocations responsible of hematopoietic dysregulation. We tried to identify structural and functional promoter elements of MTG16 and MTGR1 genes in order to find associations between their regulation and hematopoiesis.
5' deletion examinations and luciferase reporter gene studies indicated that a 492 bp sequence upstream of the transcription start site is essential for transcriptional activity by the MTG16 promoter. The TATA- and CCAAT-less promoter with a GC box close to the start site showed strong reporter activity when examined in erythroid/megakaryocytic cells. Mutation of an evolutionary conserved GATA -301 consensus binding site repressed promoter function. Furthermore, results from in vitro antibody-enhanced electrophoretic mobility shift assay and in vivo chromatin immunoprecipitation indicated binding of GATA-1 to the GATA -301 site. A role of GATA-1 was also supported by transfection of small interfering RNA, which diminished MTG16 expression. Furthermore, expression of the transcription factor HERP2, which represses GATA-1, produced strong inhibition of the MTG16 promoter reporter consistent with a role of GATA-1 in transcriptional activation. The TATA-less and CCAAT-less MTGR1 promoter retained most of the transcriptional activity within a -308 to -207 bp region with a GC-box-rich sequence containing multiple SP1 binding sites reminiscent of a housekeeping gene with constitutive expression. However, mutations of individual SP1 binding sites did not repress promoter function; multiple active SP1 binding sites may be required to safeguard constitutive MTGR1 transcriptional activity. The observed repression of MTG16/MTGR1 promoters by the leukemia associated AML1-ETO fusion gene may have a role in hematopoietic dysfunction of leukemia.
An evolutionary conserved GATA binding site is critical in transcriptional regulation of the MTG16 promoter. In contrast, the MTGR1 gene depends on a GC-box-rich sequence for transcriptional regulation and possible ubiquitous expression. Our results demonstrate that the ETO homologue promoters are regulated differently consistent with hematopoietic cell-type- specific expression and function.
Analysis of chromatin-immunoprecipitation followed by sequencing (ChIP-seq) usually disregards sequence reads that do not map within binding positions (peaks). Using an unbiased approach, we analysed all reads, both that mapped and ones that were not included as part of peaks. ChIP-seq experiments were performed in human lung adenocarcinoma and fibrosarcoma cells for the metastasis suppressor non-metastatic 2 (NME2). Surprisingly, we identified sequence reads that uniquely represented human telomere ends in both cases. In vivo presence of NME2 at telomere ends was validated using independent methods and as further evidence we found intranuclear association of NME2 and the telomere repeat binding factor 2. Most remarkably, results demonstrate that NME2 associates with telomerase and reduces telomerase activity in vitro and in vivo, and sustained NME2 expression resulted in reduced telomere length in aggressive human cancer cells. Anti-metastatic function of NME2 has been demonstrated in human cancers, however, mechanisms are poorly understood. Together, findings reported here suggest a novel role for NME2 as a telomere binding protein that can alter telomerase function and telomere length. This presents an opportunity to investigate telomere-related interactions in metastasis suppression.
Background & objectives:
The severe toxicity, exorbitant cost and emerging resistance of Leishmania species against most of the currently used drugs underscores the urgent need for the alternative drugs. The present study evaluates in vitro anti-leishmanial activity of Plumeria bicolor and its isolated compounds.
The in vitro anti-parasitic activity of chloroform extract of Plumeria bicolor, plumericin and isoplumericin were tested alongwith appropriate controls against promastigote and amastigote forms of Leishmania donovani using 96 well microtiter plate. The concentration used for assessing the anti-leishmanial activity of extract of Plumeria bicolor and both isolated compounds were 100 μg/ml and 15 μM, respectively. The viability of the cells was assessed by MTT assay. The cytotoxicity of these compounds was performed against J774G8 murine macrophage cells lines at the concentration of 30 μM.
The Plumeria bicolor extract showed activity with the IC50 of 21±2.2 and 14±1.6 μg/ml against promastigote and amastigote forms of L. donovani, respectively. Plumericin consistently showed high activity with the IC50 of 3.17±0.12 and 1.41±0.03 μM whereas isoplumericin showed the IC50 of 7.2±0.08 μM and 4.1±0.02 μM against promastigote and amastigote forms, respectively. Cytotoxic effect of the chloroform extract of P. bicolor, plumericin and isoplumericin was evaluated in murine macrophage (J774G8) model with CC50 value of 75±5.3 μg/ml, 20.6±0.5 and 24±0.7 μM, respectively.
Interpretation & conclusions:
Our results indicated that plumericin showed more potent activity than isoplumericin and might be a promising anti-leishmanial agent against L. donovani.
Isoplumericin; Leishmania donovani; Plumeria bicolor; plumericin
A randomized, double-blind, placebo-controlled study was conducted to evaluate the efficacy of OciBest, an extract of Ocimum tenuiflorum Linn. in symptomatic control of general stress. The participants received either placebo (n = 79) or OciBest (n = 71; 1200 mg of actives per day) for six weeks. The severity of stress-related symptoms was self-evaluated by patients at weeks 0, 2, 4 and 6 of the trial period using a symptom rating scale. After six weeks of intervention, scores of symptoms such as forgetfulness, sexual problems of recent origin, frequent feeling of exhaustion, and frequent sleep problems of recent origin decreased significantly (P ≤ 0.05) in OciBest group as compared with placebo group. Also, the total symptom scores of OciBest group revealed significant reduction
(P ≤ 0.05) as compared to placebo group. The overall improvement in OciBest group was found to be 1.6 times or 39% more in the control of general stress symptoms with respect to placebo. No adverse events were reported during the study. The findings revealed that OciBest was found to be effective and well tolerated by all the patients over the six weeks of study period.
Function of non-B DNA structures are poorly understood though several bioinformatics studies predict role of the G-quadruplex DNA structure in transcription. Earlier, using transcriptome profiling we found evidence of widespread G-quadruplex-mediated gene regulation. Herein, we asked whether potential G-quadruplex (PG4) motifs associate with transcription factors (TF). This was analyzed using 220 position weight matrices [designated as transcription factor binding sites (TFBS)], representing 187 unique TF, in >75 000 genes in human, chimpanzee, mouse and rat. Results show binding sites of nine TFs, including that of AP-2, SP1, MAZ and VDR, occurred significantly within 100 bases of the PG4 motif (P < 1.24E-10). PG4–TFBS combinations were conserved in ‘orthologously’ related promoters across all four organisms and were associated with >850 genes in each genome. Remarkably, seven of the nine TFs were zinc-finger binding proteins indicating a novel characteristic of PG4 motifs. To test these findings, transcriptome profiles from human cell lines treated with G-quadruplex-specific molecules were used; 66 genes were significantly differentially expressed across both cell-types, which also harbored conserved PG4 motifs along with one/more of the nine TFBS. In addition, genes regulated by PG4–TFBS combinations were found to be co-regulated in human tissues, further emphasizing the regulatory significance of the associations.
Indian potato (Solanum tuberosum L.) processing industry has emerged fast due to economic liberalization coupled with growing urbanization, expanding market options and development of indegenous processing varieties. India’s first potato processing varieties ‘Kufri Chipsona-1’ and ‘Kufri Chipsona-2’ were developed in 1998, followed by an improved processing variety ‘Kufri Chipsona-3’ in 2005 for the Indian plains and first chipping variety ‘Kufri Himsona’ for the hills. These varieties have >21% tuber dry matter content, contain low reducing sugars (<0.1% on fresh wt) and are most suitable for producing chips, French fries and dehydrated products. The availability of these varieties and standardization of storage techniques for processing potatoes at 10–12°C with sprout suppressant isopropyl N-(3-chlorophenyl) carbamate have revolutionized the processing scenario within a short span of 10 years. Currently about 4% of total potato produce is being processed in organized and unorganized sector. Potato processing industry mainly comprises 4 segments: potato chips, French fries, potato flakes/powder and other processed products. However, potato chips still continue to be the most popular processed product. The major challenge facing the industries lies in arranging round the year supply of processing varieties at reasonable price for their uninterrupted operation, besides several others which have been discussed at length and addressed with concrete solutions.
Potato; Tuber quality; Indian processing varieties; Products; Storage
Antineoplastic drug cisplatin remains the drug of choice for various solid tumours including breast cancer. But dose dependent nephrotoxicity is the major drawback in majority of platinum based chemotherapy regimens. Recent reports have shown that inflammatory pathways are the main offender for cisplatin induced nephrotoxicity. The present study was undertaken to assess the effect of rosiglitazone, a PPARγ agonist and an anti-inflammatory agent, on cisplatin induced nephrotoxicity, and its anticancer activity in DMBA induced breast cancer rats.
Mammary tumours were induced in female Sprague-Dawley rats by feeding orally with dimethylbenz [a]anthracene (DMBA) (60 mg/kg). Cisplatin induced nephropathy was assessed by measurements of blood urea nitrogen, albumin and creatinine levels. Posttranslational modifications of histone H3, mitogen-activated protein (MAP) kinase p38 expression and PPAR-γ expression were examined by western blotting.
Our data shows involvement of TNF-α in preventing cisplatin induced nephrotoxicity by rosiglitazone. Rosiglitazone pre-treatment to cisplatin increases the expression of p38, PPAR-γ in mammary tumours and shows maximum tumour reduction. Furthermore, cisplatin induced changes in histone acetylation, phosphorylation and methylation of histone H3 in mammary tumours was ameliorated by pre-treatment of rosiglitazone. Suggesting, PPAR-γ directly or indirectly alters aberrant gene expression in mammary tumours by changing histone modifications.
To best of our knowledge this is the first report which shows that pre-treatment of rosiglitazone synergizes the anticancer activity of cisplatin and minimizes cisplatin induced nephrotoxicity in DMBA induced breast cancer.
We tested 310,605 single-nucleotide polymorphisms for association in 778 celiac disease cases and 1422 controls. Outside the HLA, the most significant finding (rs13119723, P=2.0 × 10−7, empirical genome-wide significance P=0.045) was in the KIAA1109/Tenr/IL2/IL21 linkage disequilibrium block. Association was independently confirmed in two further collections (strongest at rs6822844, 24kB 5' of IL21, meta-analysis P=1.3 × 10−14, OR 0.63), suggesting genetic variation in this region predisposes to celiac disease.
Immunoglobulin-containing plasma cell densities in the jejunal mucosa and serum and jejunal fluid immunoglobulins have been measured in patients with adult coeliac disease and dermatitis herpetiformis with and without jejunal mucosal abnormality. Studies were performed in patients before and after treatment of the jejunal lesion.
Total immunofluorescent plasma cells were increased in untreated adult coeliac disease and dermatitis herpetiformis patients with jejunal lesions, but in general the normal predominance of IgA > IgM > IgG was found. There was no difference from controls in IgA-containing cells in the two conditions before or after treatment. The numbers of IgM-containing cells were significantly increased both before and after treatment in groups of patients with adult coeliac disease and dermatitis herpetiformis who had jejunal lesions. IgG-containing cells were significantly raised in only the before-treatment groups. Patients with dermatitis herpetiformis without jejunal lesions, even whilst on gluten-containing diets, had normal numbers of immunoglobulin-containing cells. IgA and IgM jejunal fluid immunoglobulins were significantly raised in dermatitis herpetiformis and adult coeliac disease.
It is concluded that patients with dermatitis herpetiformis with jejunal morphological abnormality have a comparable immunological disturbance of the jejunal mucosa to that found in adult coeliac disease.
A double-lumen perfusion technique has been used to study amino acid and peptide absorption in eight normal control subjects, 13 patients with untreated adult coeliac disease, and 16 patients with dermatitis herpetiformis who had varying morphological abnormalities of the small bowel. All subjects were perfused with isotonic solutions containing 10 mM glycyl-L-alanine and 10 mM glycine + 10 mM L-alanine.
Patients with adult coeliac disease had impaired absorption of glycine (p < 0·01) and L-alanine (p < 0·05) from the amino acid solution compared with the control subjects. Amino acid uptake from the dipeptide solution was not significantly impaired, although four individual patients had impaired uptake of both amino acids. In contrast to these findings, very few patients with dermatitis herpetiformis had impaired amino acid absorption from either solution.
Sodium absorption was impaired from both solutions when the groups of patients with adult coeliac disease and dermatitis herpetiformis with subtotal villous atrophy and partial villous atrophy were studied, and there were patients in each group who secreted sodium and water.
The results suggest that malabsorption of dietary protein is unlikely to occur in dermatitis herpetiformis but may occur and contribute to protein deficiency seen in some severe cases of adult coeliac disease. The impairment of sodium and water absorption provides evidence that there may be functional impairment of the jejunal mucosa in dermatitis herpetiformis as well as in adult coeliac disease.
The jejunal morphological and functional response to either a gluten-free diet or corticosteroid therapy was studied in six patients with dermatitis herpetiformis. In each treatment group there were two patients with subtotal villous atrophy and one with partial villous atrophys. After treatment, in both groups, morphological improvement was seen in villous and surface cell heights, mucosal thickness, and intraepithelial lymphocyte counts. Jejunal function was assessed using the perfusion technique. Absorption of glucose from a 56 mM solution was shown to improve. Water movement from this solution was in a secretory state in four patients (two in each group) before treatment and moved into the normal absorptive range after treatment. These results further outline the similarity between the mucosal lesion of dermatitis herpetiformis and true coeliac disease.