Variability in genome size of Trigonella foenum-graecum, Trigonella corniculata and Trigonella caerulea as estimated by flow cytometry indicates complex evolutionary history of fenugreek.

BACKGROUND: The determination of genome size is a fundamental step which provides a basis to initiate studies aimed at deciphering the genetic similarity of a species and to carry out other genomics based investigations. Fenugreek (Trigonella spp.) is an important spice crop which has numerous health promoting phytochemicals. Many species within this genus are known for their various health benefits owing to the presence of a wide diversity of important phytochemicals like diosgenin, trigonelline, fenugreekine, galactomannan, 4-hydroxy isoleucine, etc. It is a multipurpose crop being cultivated for food, animal feed and industrial purposes. Despite its importance, research on the genomics aspect of fenugreek remains scant. In the absence of sufficient genomic information, crop improvement in fenugreek is severely lagging. METHODS AND RESULTS: Estimation of genome size of a species is the preliminary step for initiation of any genomic studies and therefore in the present study we have estimated the genome size for fenugreek. Here, we have determined the genome sizes of three different Trigonella spp. namely T. foenum-graecum, T. corniculata and T. caerulea through flow cytometry (FC). The 2 C DNA content values were found to be 6.05 pg (T. foenum-graecum), 1.83 pg (T. corniculata) and 1.96 pg (T. caerulea). The genome size of T. foenum-graecum is approximately three times the genome size of T. corniculata and T. caerulea. This variation in genome size of more than three-fold indicates the level of genetic divergence among the three species, though within the same genus. CONCLUSIONS: The differences observed in the genome sizes of the three species provide conclusive evidence of their genetic divergence. Additionally, the information about the genome size would provide an impetus to the structural and functional genomics-based research in this crop.

Small cardamom genome: development and utilization of microsatellite markers from a draft genome sequence of Elettaria cardamomum Maton.

Small cardamom (Elettaria cardamomum Maton), the queen of spices, is the third most expensive spice in the world after saffron and vanilla, valued highly for its aroma and taste. This perennial herbaceous plant is a native of coastal parts of Southern India and displays a significant amount of morphological diversity. Its genetic potential has not been exploited due to lack of genomic resources limiting our understanding of the genome and important metabolic pathways which give it the economic advantage in the spice industry. Here, we report upon the de novo assembled, draft whole genome sequence of cardamom variety, Njallani Green Gold. We used a hybrid assembly strategy using the reads from the Oxford Nanopore, Illumina and 10x Genomics GemCode sequencing chemistries. The assembled genome length was 1.06 Gb (gigabases) which is close to the estimated genome size of cardamom. More than 75% of the genome was captured in 8000 scaffolds with a N50 of 0.15 Mb. The genome appears to have a high repeat content and 68055 gene models were predicted. The genome is close to Musa species and displays an expansion and contraction in different gene families. The draft assembly was used for in silico mining of simple sequence repeats (SSRs). A total of 2,50,571 SSRs were identified of which 2,18,270 were perfect SSRs and 32,301 were compound SSRs. Among the perfect SSRs, trinucleotides were most abundant (1,25,329) and hexanucleotide repeats appear least (2,380). From the 2,50,571 SSRs mined, 2,27,808 primer pairs were designed based on flanking sequence information. Wet lab validation was performed for 246 SSR loci and based on their amplification profiles, 60 SSR markers were used for diversity analysis of a set of 60 diverse cardamom accessions. The average number of alleles detected per locus were 14.57 with a minimum of 4 and maximum of 30 alleles. Population structure analysis revealed the presence of high degree of admixtures which could primarily be due to cross-pollination prevalent in this species. The SSR markers identified would help in the development of gene or trait-linked markers which can be subsequently used for marker-assisted breeding for crop improvement in cardamom. The information on utilization of the SSR loci for generation of markers has been developed into a public database, 'cardamomSSRdb' that is freely available for use by the cardamom community.

Clinical spectrum and neuroimaging findings in children with seizures: A five-year retrospective study.

Objectives: Seizures are the most common neurological illness in the pediatric population and account for 1% of all emergency department (ED) visits and 2% of all visits to children's hospital EDs. Pediatric epilepsy presents with various diagnostic challenges. Neuroimaging, especially structural neuroimaging and preferably MRI brain, plays an essential role in diagnosing, managing, and guiding pediatric epilepsy treatment.The study aimed to estimate the clinical spectrum of seizures in children and examine the neuroimaging findings in children with seizures. Materials & Methods: The study was a hospital-based retrospective observational study. The hospital case records of all children belonging to the age group 1 month to 12 years with 'seizures' were reviewed for 5 years from Jan 2016 to Dec 2020. Clinicodemographic profiles and neuroimaging (CT/MRI) findings were obtained, and descriptive statistics were applied. Results: A total of 838(11%) children in the age group 1 to 144 months (mean±SD: 32.57±32.65) presented with seizures, of whom 515(61.5%) were boys and 323(38.5%) girls. Of 596(71.1%) children under five years, 409(68.6%) had febrile seizures. Generalized onset-motor seizures were the predominant type of seizures seen in 666(79.4%) children, of whom 434(65.1%) had febrile seizures.Neuroimaging (CT/MRI) was normal in 335(40%) and abnormal in 124(14.8%) children. Perinatal insult (7%) was the most common abnormality, followed by CNS infections (2.8%). Conclusion: Neuroimaging, preferably MRI brain, is the most helpful tool for the etiological diagnosis of afebrile seizures.In our study, seizures secondary to perinatal insult/hypoxic insult followed by infections were major causes. Improvement in peripartum and perinatal care coupled with a targeted Tuberculosis control program may help in reducing these potentially preventable causes.

The chloroplast genome of black pepper (Piper nigrum L.) and its comparative analysis with related Piper species.

Piper nigrum, also known as black pepper, is an economically and ecologically important crop of the genus Piper. It has been titled as the king of spices due to its wide consumption throughout the world. In the present investigation, the chloroplast genome of P. nigrum has been assembled from a whole genome sequence by integrating the short and long reads generated through Illumina and PacBio platforms, respectively. The chloroplast genome was observed to be 161,522 bp in size, having a quadripartite structure with a large single copy (LSC) region of 89,153 bp and a small single copy (SSC) region of 18,255 bp separated by a copy of inverted repeats (IRs), each 27,057 bp in length. Taking into consideration all the duplicated genes, a total of 131 genes were observed, which included 81 protein-coding genes, 37 tRNAs, 4 rRNAs, and 1 pseudogene. Individually, the LSC region consisted of 83 genes, the SSC region had 13 genes, and 18 genes were present in each IR region. Additionally, 216 SSRs were detected and 11 of these were validated through amplification in 12 species of Piper. The features of the chloroplast genome have been compared with those of the genus Piper. Our results provide useful insights into evolutionary and molecular studies of black pepper which will contribute to its further genetic improvement and breeding.

Fatty changes associated with N-Nitrosodiethylamine (DEN) induced hepatocellular carcinoma: a role of sonic hedgehog signaling pathway.

BACKGROUNDS AND AIMS: Hepatocellular Carcinoma (HCC) is the leading cause of cancer-related mortality across the world. Non-viral etiological factors including obesity and metabolic syndrome have now become prevalent cause of hepatocellular carcinoma. Sonic Hedgehog (SHH) pathway is activated in hepatocellular carcinoma but its role in regulation of lipogenic molecules during the hepatocarcinogenesis is not known. The aim of present study is to explore the role of SHH pathway in fatty changes associated with hepatocarcinogenesis at different stages and to further correlate the expression of SHH with lipogenic pathways. RESULTS: Our results demonstrated significant increase in lipidosis and fibrosis in DEN+CCl4 treated animals. It was simultaneously associated with the enhanced expression level of SHH, E2F1, adiponectin, and lipogenic molecules in DEN+CCl4 treated animals. These results were also corroborated with the similar findings in higher stage patients' biospecimens. METHODS: N-Nitrosodiethylamine (DEN) and Carbon TetraChloride (CCl4) induced hepatocellular acrcinoma model in male Wistar rats were established to study the expression level of SHH pathway and associated fatty changes during different stages of hepatocarcinogenesis. The expression levels of SHH, E2F1, and lipogenic molecules were checked at different stages of hepatocellular carcinoma. These results were further compared with biospecimens of hepatocellular carcinoma patients of different stages. CONCLUSIONS: Our results revealed an unknown aspect of SHH pathway in hepatocarcinogenesis via its control over lipogenesis. It gives insight into the lipogenic properties of DEN+CCl4 induced rodent hepatocarcinogenesis model and how SHH pathway operate to arbitrate this response.

Genome wide identification and characterization of microsatellite markers in black pepper (Piper nigrum): A valuable resource for boosting genomics applications.

Black pepper is one of the most valued and widely used spices in the world and dominates multi-billion dollar global spices trade. India is amongst the major producers, consumers and exporters of black pepper. In spite of its commercial and cultural importance, black pepper has received meagre attention in terms of generation of genomic resources. Availability of markers distributed throughout the genome would facilitate and accelerate genetic studies, QTL identification, genetic enhancement and crop improvement in black pepper. In this perspective, the sequence information from the recently sequenced black pepper (Piper nigrum) genome has been used for identification and characterisation of Simple Sequence Repeats (SSRs). Total 69,126 SSRs were identified from assembled genomic sequence of P. nigrum. The SSR frequency was 158 per MB making it, one SSR for every 6.3 kb in the assembled genome. Among the different types of microsatellite repeat motifs, dinucleotides were the most abundant (48.6%), followed by trinucleotide (23.7%) and compound repeats (20.62%). A set of 85 SSRs were used for validation, of which 74 produced amplification products of expected size. Genetic diversity of 30 black pepper accessions using 50 SSRs revealed four distinct clusters. Further, the cross species transferability of the SSRs was checked in nine other Piper species. Out of 50 SSRs used, 19 and 31 SSRs were amplified in nine and seven species, respectively. Thus the identified SSRs may have application in other species of the genus Piper where genome sequence is not available yet. Present study reports the first NGS based genomic SSRs in black pepper and thus constitute a valuable resource for a whole fleet of applications in genetics and plant breeding studies such as genetic map construction, QTL identification, map-based gene cloning, marker-assisted selection and evolutionary studies in Piper nigrum and related species.

Morphophysiological and biochemical response of mungbean [Vigna radiata (L.) Wilczek] varieties at different developmental stages under drought stress.

The present study was conducted to assess the morphophysiological and biochemical responses during different developmental stages in mungbean varieties subjected to drought stress, and to screen the varieties for drought tolerance. A field experiment was performed according to a completely randomized design on 25 mungbean varieties with 3 replicates per variety. Stress treatment was applied at 3 levels: control (no stress), vegetative stage (25 days after sowing), and reproductive stage (35 days after sowing). According to combined analysis of variance, there were significant effects from drought stress on relative water content (RWC), membrane stability index (MSI), protein and proline content of leaves, leaf area, plant height, and yield traits. MSI, RWC, protein content, leaf area, plant height, and yield traits were decreased during drought stress, while proline content was increased under drought stress conditions. The results showed that the vegetative stage was more sensitive to drought stress, which was further supported by correlation analysis. Taken together, Vigna sublobata, MCV-1, PLM-32, LGG-407, LGG-450, TM-96-2, and Sattya varieties were identified as drought tolerant as they maintained the higher values of RWC, MSI, protein, proline content, leaf area, plant height, and yield traits. These varieties could be used in breeding programs for better physiological drought tolerance traits.

The molecular connection of histopathological heterogeneity in hepatocellular carcinoma: A role of Wnt and Hedgehog signaling pathways.

BACKGROUND: Hepatocellular carcinoma (HCC) is leading cause of cancer-related mortality and is categorized among the most common malignancies around the world. It is a heterogeneous tumor, which shows significant degree of histopathological heterogeneity. Despite the apparent histopathological diversity there has been very little distinct correlation between histopathological features and molecular aberrations particularly when it comes to the expression level of Wnt and Hh pathway molecules. The role of Wnt and Hh pathways in relation to HCC behavior viz. histopathological heterogeneity and aggressiveness is not known. Determining the sequential molecular changes and associated histopathological characteristic during HCC initiation, promotion, and progression would probably lead to a better treatment and prognosis. METHODS: N-Nitrosodiethylamine (DEN) induced HCC model in male Wistar rats were established to study the expression level of Wnt and Hh pathway molecules during different stages of hepatocarcinogenesis. Their expression levels were checked at mRNA and protein levels at initiation, promotion, and progression stages of HCC. The expression levels of Wnt and Hh pathway molecules were correlated with biospecimens of HCC patients of different stages. RESULTS: In the present study we identified the comprehensive change in the expression pattern of Wnt and Hh pathway molecules in DEN induced rodent hepatocarcinogenesis model. Our results demonstrate that ß-catenin /CTNNB1 plays important role in tumor initiation and promotion by stimulating tumor cell proliferation. The activated Wnt signaling in early stage of HCC is associated with well-differentiated histological pattern. The Hh activity although activated during the initiation stage but is significantly increased during the early promotion stage of hepatocarcinogenesis. The increased activity of both Wnt & Hh pathways during promotion stage is associated with moderately-differentiated histological pattern and was simultaneously linked with an increased expression of MMP9. Furthermore, our data demonstrated that during the progression stage Wnt pathway is modestly down-regulated but the Hh pathway activity sustained which in turn is associated with aggressive and invasive phenotype and poorly-differentiated histopathology. CONCLUSION: Our data uncovers the grade related expression of Wnt and Hh pathway molecules and the potential utility of these molecular signatures in daily clinical practice is to decide best therapy according to patients characteristic. Additionally, our data offer insight into the interaction between Wnt and Hh pathways which triggers HCC development and progression.

Identification of genes associated with stress tolerance in moth bean [Vigna aconitifolia (Jacq.) Marechal], a stress hardy crop.

Moth bean is the most drought and heat tolerant cultigens among Asian Vigna. We performed comparative transcriptome analysis of moth bean cultivar "Marumoth" under control and stress condition. De novo transcriptome assembly was carried out by using Velvet followed by Oases softwares. Differential expression analyses, SSR identification and validation and mapping of pathways and transcription factors were conducted. A total of 179,979 and 201,888 reads were generated on Roche 454 platform and 48,617,205 and 45,449,053 reads were generated on ABI Solid platform for the control and stressed samples. Combined assembly from Roche and ABI Solid platforms generated 16,090 and 15,096 transcripts for control and stressed samples. We found 1287 SSRs and 5606 transcripts involved in 179 pathways. The 55 transcription factor families represented 19.42% of total mothbean transcripts. In expression profiling, ten transcripts were found to be up-regulated and 41 down-regulated while 490 showed no major change under moisture stress condition. Stress inducible genes like Catalase, Cyt P450 monooxygenase, heat shock proteins (HSP 90 and HSP 70), oxidoreductase, protein kinases, dehydration responsive protein (DRP), universal stress protein and ferridoxin NADH oxidoreductase genes were up-regulated in stressed sample. Genes which might be involved in moisture stress tolerance in moth bean were identified and these might be useful for stress tolerance breeding in moth bean and other related crops.

Hepatocellular carcinoma treatment: hurdles, advances and prospects.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related mortality and is particularly refractory to the available chemotherapeutic drugs. Among various etiologies of HCC, viral etiology is the most common, and, along with alcoholic liver disease and nonalcoholic steatohepatitis, accounts for almost 90% of all HCC cases. HCC is a heterogeneous tumor associated with multiple signaling pathway alterations and its complex patho-physiology has made the treatment decision challenging. The potential curative treatment options are effective only in small group of patients, while palliative treatments are associated with improved survival and quality of life for intermediate/advanced stage HCC patients. This review article focuses on the currently available treatment strategies and hurdles encountered for HCC therapy. The curative treatment options discussed are surgical resection, liver transplantation, and local ablative therapies which are effective for early stage HCC patients. The palliative treatment options discussed are embolizing therapies, systemic therapies, and molecular targeted therapies. Besides, the review also focuses on hurdles to be conquered for successful treatment of HCC and specifies the future prospects for HCC treatment. It also discusses the multi-modal approach for HCC management which maximizes the chances of better clinical outcome after treatment and identifies that selection of a particular treatment regimen based on patients' disease stage, patients' ages, and other underlying factors will certainly lead to a better prognosis.

Analysis of spatial distribution of genetic diversity and validation of Indian foxtail millet core collection.

Foxtail millet [Setaria italica (L.) P. Beauv.] is an important small millet, grown as a short duration, drought tolerant crop across the world. This crop can be grown on wide ranges of soil conditions and has an immense potential for food and fodder in rainfed and arid regions of the India. In the present study, 31 primer pairs (27 SSR and 4 EST-SSR) were used to analyse the genetic diversity in 223 core collection accessions. Analysis resulted in detection of a total of 136 alleles with an average of 4.38 alleles per locus. Among these 136 alleles, 22 were rare, 70 were common and 44 were frequent. The PIC value ranged from 0.01 to 0.86 with an average of 0.31. The average number of observed alleles ranged from 2.0 (northern hills of India accessions) to 4.06 (exotic) with an average of 2.72. The mean Shannon's Information Index ranged from 0.44 (northern hills of India) to 0.69 (exotic) with an average of 0.52. Pair-wise Fst values indicated little to moderate genetic differentiation among the group of accessions. UPGMA clustering grouped the accessions into two major groups while analysis for population substructure indicated presence of four subpopulations. However there was no statistically well supported grouping of the accessions based on eco-geographic specificities. The core collection designated here represented substantial genetic diversity at molecular level, hence may be a good source of diversity for use in foxtail improvement programs in the region.

Constitutively activated ERK sensitizes cancer cells to doxorubicin: Involvement of p53-EGFR-ERK pathway.

The tumour suppressor gene p53 is mutated in approximately 50% of the human cancers. p53 is involved in genotoxic stress-induced cellular responses. The role of EGFR and ERK in DNA-damage-induced apoptosis is well known. We investigated the involvement of activation of ERK signalling as a consequence of non-functional p53, in sensitivity of cells to doxorubicin. We performed cell survival assays in cancer cell lines with varying p53 status: MCF-7 (wild-type p53, WTp53), MDA MB-468 (mutant p53, MUTp53), H1299 (absence of p53, NULLp53) and an isogenic cell line MCF-7As (WTp53 abrogated). Our results indicate that enhanced chemosensitivity of cells lacking wild-type p53 function is because of elevated levels of EGFR which activates ERK. Additionally, we noted that independent of p53 status, pERK contributes to doxorubicin-induced cell death.

Value addition in sesame: A perspective on bioactive components for enhancing utility and profitability.

Sesame seed is a reservoir of nutritional components with numerous beneficial effects along with health promotion in humans. The bioactive components present in the seed include vital minerals, vitamins, phytosterols, polyunsaturated fatty acids, tocopherols and unique class of lignans such as sesamin and sesamolin. The presence of phenylpropanoid compounds namely lignans along with tocopherols and phytosterols provide defense mechanism against reactive oxygen species and increases keeping quality of oil by preventing oxidative rancidity. In this article, we have reviewed the nutraceutical, pharmacological, traditional and industrial value of sesame seeds with respect to bioactive components that hold high antioxidant value. Valuable information on superior functional components of sesame will strongly promote the use of sesame seeds in the daily diet world-wide. In spite of huge repertoire of sesame germplasm collection, limited research efforts on the use of conventional and biotechnological methodologies have resulted in minimal success in developing nutritionally superior cultivars. In consequence, value addition efforts in sesame would enable development of genotypes with high antioxidant activity and subsequently prevention of free radical related diseases. Modification of bioactive components in sesame would enable production of stabilized sesame oil with enhanced shelf life and better market value.

Cyclin-dependent kinase 9 inhibition protects cartilage from the catabolic effects of proinflammatory cytokines.

OBJECTIVE: Cyclin-dependent kinase 9 (CDK-9) controls the activation of primary inflammatory response genes. The purpose of this study was to determine whether CDK-9 inhibition protects cartilage from the catabolic effects of proinflammatory cytokines. METHODS: Human chondrocytes were challenged with different proinflammatory stimuli (interleukin-1ß [IL-1ß], lipopolysaccharides, and tumor necrosis factor α) in the presence or absence of either the CDK-9 inhibitor flavopiridol or small interfering RNA (siRNA). The expression of messenger RNA (mRNA) for inflammatory mediator genes, catabolic genes, and anabolic genes were determined by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Cartilage explants were incubated for 6 days with IL-1ß in the presence or absence of flavopiridol. Cartilage matrix degradation was assessed by the release of glycosaminoglycan (GAG) and cleaved type II collagen (COL2A) peptides. RESULTS: CDK-9 inhibition by flavopiridol or knockdown by siRNA effectively suppressed the induction of mRNA for inducible nitric oxide synthase by all 3 proinflammatory stimuli. Results from NF-κB-targeted PCR array analysis showed that flavopiridol suppressed IL-1ß induction of a broad range of inflammatory mediator genes (59 of 67 tested). CDK-9 inhibition also suppressed the induction of catabolic genes (matrix metalloproteinase 1 [MMP-1], MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5), but did not affect the basal expression of anabolic genes (COL2A, aggrecan, and cartilage oligomeric matrix protein) and housekeeping genes. Flavopiridol had no apparent short-term cytotoxicity, as assessed by G6PDH activity. Finally, in IL-1ß-treated cartilage explants, flavopiridol reduced the release of the matrix degradation product GAG and cleaved COL2A peptides, but did not affect long-term chondrocyte viability. CONCLUSION: CDK-9 activity is required for the primary inflammatory response in chondrocytes. Flavopiridol suppresses the induction of inflammatory mediator genes and catabolic genes to protect cartilage from the deleterious effects of proinflammatory cytokines, without affecting cell viability and functions.

Inherent and acquired resistance to paclitaxel in hepatocellular carcinoma: molecular events involved.

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and is a major cause of cancer related deaths worldwide. Only 10 to 20% of HCC can be surgically excised. Therefore, chemotherapeutic intervention and treatment is essential for achieving favorable prognosis. However, therapeutic outcome of chemotherapy is generally poor owing to inherent resistance of cancer cells to the treatment or due to development of acquired resistance. To differentiate and delineate the molecular events, we developed drug resistant Hep3B cells (DRC) by treating cells with the increasing concentration of paclitaxel. We also developed a unique single cell clone of Hep3B cells (SCC) by selecting single cell colonies and screening them for resistant phenotype. Interestingly, both DRC and SCC were resistant to paclitaxel in comparison to parental Hep3B cells. We analyzed the contributory factors that may be involved in the development of resistance. As expected, level of P-glycoprotein (P-gp) was elevated in DRC. In addition, Caveolin-1 (Cav-1), Fatty acid synthase (FASN) and Cytochrome P450 (CYP450) protein levels were elevated in DRC whereas in SCC, FASN and CYP450 levels were elevated. Downregulation of these molecules by respective siRNAs and/or by specific pharmacological inhibitors resensitized cells to paclitaxel. Interestingly, these drug resistant cells were also less sensitive to vinblastine, doxorubicin and methotrexate with the exception of cisplatin. Our results suggested that differential levels of P-gp, Cav-1 and FASN play a major role in acquired resistant phenotype whereas FASN level was associated with the presentation of inherent resistant phenotype in HCC.

The Oncogene LRF Stimulates Proliferation of Mesenchymal Stem Cells and Inhibits Their Chondrogenic Differentiation.

OBJECTIVE: The oncogene leukemia/lymphoma-related factor (LRF) enhances chondrosarcoma proliferation and malignancy. This study aimed to investigate the roles of LRF in chondrogenic differentiation of primary human bone marrow-derived mesenchymal stem cells (BMSCs). DESIGN: LRF was overexpressed in BMSC by lentiviral transduction. Chondrogenic differentiation of BMSC was induced by high-density pellet culture. Western blotting and real-time polymerase chain reaction were used to investigate changes in protein and mRNA levels, respectively, during chondrogenesis. Safranin-O staining, immunohistochemistry, and glycoaminoglycan contents were used to assess cartilage matrix deposition. BMSC proliferation was determined by mitochondrial dehydrogenase activity and cell counting. Cell cycle profiling was performed by flow cytometry. RESULTS: LRF overexpression effectively inhibited protein and mRNA expression of chondrocyte markers and cartilage matrix deposition during chondrogenesis of BMSC. Endogenous LRF expression was constitutively high in undifferentiated BMSC but remained low in primary articular chondrocytes. Endogenous LRF protein was downregulated in a time-dependent manner during chondrogenesis. BMSCs overexpressing LRF had higher proliferation rates and cell population in the S phase. LRF suppressed p53 expression during chondrogenesis and this might prevent differentiating chondrocytes from entering a quiescent state. CONCLUSION: Our data showed that LRF is important for stimulating stem cell proliferation and cell cycle progression. It is known that LRF is highly expressed in the mouse limb buds prior to overt chondrogenesis; thus, LRF might function to prevent premature chondrogenic differentiation of stem cells.

The oncogene LRF is a survival factor in chondrosarcoma and contributes to tumor malignancy and drug resistance.

Chondrosarcoma is a form of malignant skeletal tumor of cartilaginous origin. The non-malignant form of the disease is termed chondroma. Correctly distinguishing between the two forms is essential for making therapeutic decisions. However, due to their similar histological appearances and the lack of a reliable diagnostic marker, it is often difficult to distinguish benign tumors from low-grade chondrosarcoma. Therefore, it is necessary to search for a potential marker that has diagnostic and prognostic values in chondrosarcoma. In this study, we demonstrated by immunohistochemistry that elevated leukemia/lymphoma-related factor (LRF) expression was associated with increased malignancy in human chondrosarcoma tissue microarrays. Moreover, siRNA depletion of LRF drastically reduced proliferation of chondrosarcoma cell lines and effectively induced senescence in these cells. This could be attributed to the observation that LRF-depleted cells were arrested at the G(1) phase, and had increased p53 and p21 expression. Moreover, LRF depletion not only drastically reduces the cellular migration and invasion potentials of chondrosarcoma cells but also sensitized these cells to the apoptosis-inducing chemotherapeutic agent doxorubicin. We conclude that LRF is a survival factor in chondrosarcomas and its expression correlates with tumor malignancy and chemoresistance. Our data implicate the potential role of LRF as both a diagnostic marker and therapeutic target for chondrosarcomas.

Analysis of molecular genetic diversity in a representative collection of foxtail millet [Setaria italica (L.) P. Beauv.] from different agro-ecological regions of India.

Foxtail millet [Setaria italica (L.) P. Beauv.], an important crop of East Asia is known for its drought tolerance and was once an indispensible crop of vast rainfed areas in semi-arid regions in India. In India it is cultivated in Andhra Pradesh, Karnataka, Maharashtra, Tamil Nadu, Rajasthan, Madhya Pradesh, Uttar Pradesh and north eastern states. The grain finds use in several local recipes such as roti (bread), jaula, singal, sirol. Foxtail millet grain contains 12.3 % protein, 4.7 % fat, 60.6 % carbohydrates, and 3.2 % ash. The present study was conducted to analyse the genetic diversity among foxtail accessions from different states of India and a few exotic accessions using RAPD and ISSR techniques and identify diverse accessions for use in variety improvement programmes. A set of 125 foxtail millet accessions selected from 11 different agro-ecological regions of India were analyzed using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker techniques. A total of 146 (115 RAPD and 31 ISSR) scoreable markers were generated with 16 RAPD and four ISSR primers. The dendrogram generated using Nei's genetic distances and principal component analyses revealed presence of two clusters and two subclusters in group I. The accessions from Andhra Pradesh, Karnataka, Maharashtra and Uttarakhand were more diverse since they were distributed in both the clusters. There was no clear geographical differentiation observable. The bootstrap support for the major groups identified was strong (above 80 %) indicating good statistical support. The average value of Nei and Li's genetic distance was lowest (0.081) for accessions from West Bengal while the collections from Karnataka showed highest dissimilarity (average genetic distance = 0.239). The average genetic distance for all 125 accessions together was 0.177 indicating presence of only moderate genetic diversity in the collections. The analysis of molecular variance indicated that only 2.76 % variation was explained by variations among the groups and 11.55 % among populations within groups. However the percentage of variation observed within populations was high (85.68). The value of Fst was observed to be very low (0.028) indicating low differentiation of the accessions analysed. The population genetic analysis carried out indicates that highest number of alleles per locus (1.745 ± 0.438) was observed for Andhra Pradesh with 35 accessions. When four eco-geographic regions were considered, the southern region comprising AP, Karnataka and TN showed the highest number of alleles per locus (1.787 ± 0.411). The value of Gst was lowest for south (0.123) and highest for central west (0.455). This indicated that all the landraces from south share common alleles. The gene flow between the accessions from different regions was also observed to be high with the highest migration (3.557) recorded for south.

Cdk5 phosphorylates non-genotoxically overexpressed p53 following inhibition of PP2A to induce cell cycle arrest/apoptosis and inhibits tumor progression.

BACKGROUND: p53 is the most studied tumor suppressor and its overexpression may or may not cause cell death depending upon the genetic background of the cells. p53 is degraded by human papillomavirus (HPV) E6 protein in cervical carcinoma. Several stress activated kinases are known to phosphorylate p53 and, among them cyclin dependent kinase 5 (Cdk5) is one of the kinase studied in neuronal cell system. Recently, the involvement of Cdk5 in phosphorylating p53 has been shown in certain cancer types. Phosphorylation at specific serine residues in p53 is essential for it to cause cell growth inhibition. Activation of p53 under non stress conditions is poorly understood. Therefore, the activation of p53 and detection of upstream kinases that phosphorylate non-genotoxically overexpressed p53 will be of therapeutic importance for cancer treatment. RESULTS: To determine the non-genotoxic effect of p53; Tet-On system was utilized and p53 inducible HPV-positive HeLa cells were developed. p53 overexpression in HPV-positive cells did not induce cell cycle arrest or apoptosis. However, we demonstrate that overexpressed p53 can be activated to upregulate p21 and Bax which causes G2 arrest and apoptosis, by inhibiting protein phosphatase 2A. Additionally, we report that the upstream kinase cyclin dependent kinase 5 interacts with p53 to phosphorylate it at Serine20 and Serine46 residues thereby promoting its recruitment on p21 and bax promoters. Upregulation and translocation of Bax causes apoptosis through intrinsic mitochondrial pathway. Interestingly, overexpressed activated p53 specifically inhibits cell-growth and causes regression in vivo tumor growth as well. CONCLUSION: Present study details the mechanism of activation of p53 and puts forth the possibility of p53 gene therapy to work in HPV positive cervical carcinoma.

Mitomycin C induces bystander killing in homogeneous and heterogeneous hepatoma cellular models.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide that is particularly refractory to chemotherapy. Several studies have proposed combination chemotherapy regimen for HCC treatment. However, these therapies are not effective in regressing tumor and prolonging survival of patient's suffering from HCC. Therefore, the development of more effective therapeutic tools and new strategies for the treatment of HCC are urgently needed. Over the last decade much attention has been focused on "bystander effect" as a possible therapeutic strategy for the treatment of certain human tumors. Interest in this therapeutic approach originated from numerous reports describing the radiation induced bystander effect. However, the knowledge about chemotherapy induced bystander effect is still limited. Hence, chemotherapy induced bystander phenomenon in hepatoma cells was explored by utilizing Mitomycin C (MMC). RESULTS: MMC induced bystander killing was observed only in hepatoma cells and it did not occur in cervical cancer cells. MMC induced bystander killing was transferable via medium. It occurred in co-cultured cells indicating the involvement of secreted as well as membrane bound factors. FasL and TRAIL were detected in the conditioned medium from treated cells. In medium transfer experiment, pre-treatment with EDTA (a broad range protease inhibitor) diminished MMC induced bystander killing. Following drug exposure, expression of Fas and TRAIL receptors increased and treatment with neutralizing antibodies against FasL and TRAIL inhibited bystander killing. CONCLUSION: Our results highlight the therapeutic importance of MMC in the treatment of HCC and implicate role of membrane bound and secreted forms of FasL and TRAIL in MMC induced bystander killing.