Functional characterization of enhancer activity during a long terminal repeat's evolution.

Many transposable elements (TEs) contain transcription factor binding sites and are implicated as potential regulatory elements. However, TEs are rarely functionally tested for regulatory activity, which in turn limits our understanding of how TE regulatory activity has evolved. We systematically tested the human LTR18A subfamily for regulatory activity using massively parallel reporter assay (MPRA) and found AP-1- and CEBP-related binding motifs as drivers of enhancer activity. Functional analysis of evolutionarily reconstructed ancestral sequences revealed that LTR18A elements have generally lost regulatory activity over time through sequence changes, with the largest effects occurring owing to mutations in the AP-1 and CEBP motifs. We observed that the two motifs are conserved at higher rates than expected based on neutral evolution. Finally, we identified LTR18A elements as potential enhancers in the human genome, primarily in epithelial cells. Together, our results provide a model for the origin, evolution, and co-option of TE-derived regulatory elements.

Killer cell immunoglobulin-like receptor (KIR) gene contents: Are they associated with cervical cancer?

Killer cell immunoglobulin-like receptors (KIRs) are required for natural killer cell function against virus-infected cells or tumor cells. KIR gene content polymorphisms in Indian women with cervical cancer (CaCx) remain unexplored. Hence, we analyzed the frequencies of KIR genes, KIR haplotypes, and Bx subsets to draw their association with CaCx. The polymerase chain reaction-sequence-specific primer method was used for KIR genotyping in three groups of women: healthy controls (n = 114), women with human papillomavirus (HPV) infection (n = 70), and women with CaCx (n = 120). The results showed that the frequency of KIR2DS5 was significantly higher in women with CaCx compared to women with HPV infection (p = 0.02) and healthy controls (p = 0.01). Whereas the frequency of KIR2DL5B was significantly higher in healthy controls than in women with HPV infection (p = 0.02). The total number of activating KIR genes was higher in women with CaCx than in healthy controls (p = 0.006), indicating their positive association with CaCx. Moreover, the C4T4 subset was higher in women with CaCx than in women with HPV infection, though not significant. In conclusion, our findings highlight KIR2DS5, the C4T4 subset, and activating KIR genes are susceptible factors or positively associated with CaCx. Besides KIR2DL5B, this study also reported for the first time significantly high frequency of KIR2DL1 in healthy controls, indicating its possible protective association against CaCx. Further, significantly high frequency of KIR2DL3 observed in HPV-infected women might be also a promising biomarker for viral infections. Thus, the study confirms the association of KIR genes with cervical cancer in women with HPV infection.

Human Papillomavirus E6/E7 oncogene transcripts as biomarkers for the early detection of cervical cancer.

Cancer of the cervix uteri is the fourth most common cancer worldwide with a high mortality rate. Due to limitations of the existing methods, alternative methods for triage are needed for early detection of cervical cancer precursors before progression to high-grade disease. The aim of this study was to evaluate human papillomavirus (HPV) E6/E7 oncogene expression as markers for early identification of cervical cancer risk in women with minor cytological abnormalities and in those with negative cytology. The detection of HPV was done using PCR and confirmed by southern hybridization. The high-risk (HR) and low-risk HPV types were identified by HPV typing. HPV DNA-positive patients were further tested for markers of oncogene expression by real-time PCR. Out of the women screened, 54/512 (10.54%) women tested positive for HPV infection. HR HPV DNA was found in 32/485 (6.60%) women with normal cytology (Pap negative) and 22/27 (81.5%) atypical squamous cells of undetermined significance/low-grade intraepithelial lesion cases. HR HPV E6/E7 oncogene transcripts were detected in 36/512 (7.03%) patients. The positivity rate of E6/E7 messenger RNA (mRNA) was 2.48% (12/485) in normal cervical cytology group and 88.9% (24/27) in abnormal cervical cytology group. The HPV E6/E7 mRNA test sensitivity was found to be 88.89% and specificity was 97.53%. In comparison, the sensitivity of the HPV DNA test was found to be 81.48% and specificity was 93.40%. In conclusion, E6 and E7 transcripts could provide a sensitive, early predictor of cervical cancer risk in women with normal cytology and minor cytological alterations.

Local sequence features that influence AP-1 cis-regulatory activity.

In the genome, most occurrences of transcription factor binding sites (TFBS) have no cis-regulatory activity, which suggests that flanking sequences contain information that distinguishes functional from nonfunctional TFBS. We interrogated the role of flanking sequences near Activator Protein 1 (AP-1) binding sites that reside in DNase I Hypersensitive Sites (DHS) and regions annotated as Enhancers. In these regions, we found that sequence features directly adjacent to the core motif distinguish high from low activity AP-1 sites. Some nearby features are motifs for other TFs that genetically interact with the AP-1 site. Other features are extensions of the AP-1 core motif, which cause the extended sites to match motifs of multiple AP-1 binding proteins. Computational models trained on these data distinguish between sequences with high and low activity AP-1 sites and also predict changes in cis-regulatory activity due to mutations in AP-1 core sites and their flanking sequences. Our results suggest that extended AP-1 binding sites, together with adjacent binding sites for additional TFs, encode part of the information that governs TFBS activity in the genome.

Polymorphism of interleukin-6 -174 G/C (rs1800795) & the corresponding interleukin-6 level as a prognostic marker of cervical cancer.

BACKGROUND & OBJECTIVES: Aetiology of cervical cancer (CaCx) is multifactorial. Besides human papillomavirus (HPV) infection, many immunogenetic factors are involved in this complex process. The present study was carried out to investigate one such factor, interleukin-6 (IL-6), a central pro-inflammatory cytokine and a polymorphism at its promoter region -174 G/C (rs1800795) with CaCx. METHODS: HPV-infected women with or without CaCx were enrolled in group I and II, respectively. Another group of uninfected healthy women was also included as group III for comparison. Polymorphism in IL-6-174 G/C and IL-6 levels were analyzed by sequence-specific primer PCR (PCR-SSP) and ELISA, respectively. RESULTS: Groups I (n=111) and II (n=87) had significantly higher frequency of IL-6-174 GG genotype [odds ratios (OR)=3.9; P<0.001 and OR=3.2; P<0.001, respectively] as compared to group III (n=163). Furthermore, individuals with GG or GC genotypes had high IL-6 levels than those with CC genotypes. IL-6 levels were significantly (P<0.001) elevated in group I. This was also significantly high in untreated cases as compared to treated (P<0.05) ones. IL-6 levels of treated group were comparable with groups II and III. INTERPRETATION & CONCLUSIONS: Our results suggested a possible association of IL-6-174 GG with CaCx, which was also associated with high IL-6 levels. Decreased levels of IL-6 following treatment indicate its possible prognostic use in CaCx cases.

High-throughput functional testing of ENCODE segmentation predictions.

The histone modification state of genomic regions is hypothesized to reflect the regulatory activity of the underlying genomic DNA. Based on this hypothesis, the ENCODE Project Consortium measured the status of multiple histone modifications across the genome in several cell types and used these data to segment the genome into regions with different predicted regulatory activities. We measured the cis-regulatory activity of more than 2000 of these predictions in the K562 leukemia cell line. We tested genomic segments predicted to be Enhancers, Weak Enhancers, or Repressed elements in K562 cells, along with other sequences predicted to be Enhancers specific to the H1 human embryonic stem cell line (H1-hESC). Both Enhancer and Weak Enhancer sequences in K562 cells were more active than negative controls, although surprisingly, Weak Enhancer segmentations drove expression higher than did Enhancer segmentations. Lower levels of the covalent histone modifications H3K36me3 and H3K27ac, thought to mark active enhancers and transcribed gene bodies, associate with higher expression and partly explain the higher activity of Weak Enhancers over Enhancer predictions. While DNase I hypersensitivity (HS) is a good predictor of active sequences in our assay, transcription factor (TF) binding models need to be included in order to accurately identify highly expressed sequences. Overall, our results show that a significant fraction (-26%) of the ENCODE enhancer predictions have regulatory activity, suggesting that histone modification states can reflect the cis-regulatory activity of sequences in the genome, but that specific sequence preferences, such as TF-binding sites, are the causal determinants of cis-regulatory activity.

Cervical cancer in Indian women reveals contrasting association among common sub-family of HLA class I alleles.

We studied the relationship between human leukocyte antigen (HLA) class I alleles and cervical cancer among Indian women. Seventy-five cervical cancer cases were compared with 175 noncancer controls. Cervical biopsy tissue specimen from cancer cases and cervical swab specimen from controls were collected for HPV detection and typing. Blood was taken for HLA typing by PCR-SSOP method. The impact of HLA class I alleles on cervical cancer risk was evaluated using StatCalc program (Epi Info version 6.0.4. CDC Atlanta, GA, USA), and confirmed with Bonferroni correction. Results revealed HLA-B*37, HLA-B*58 were associated significantly with increased risk while HLA-B*40 with decreased risk for cervical cancer. At high-resolution analysis after Bonferroni correction, HLA-B*37:01 allele was associated with increased risk, whereas HLA-B*40:06 was with decreased risk for cervical cancer. HLA-B*37:01 and HLA-B*40:06 belong to the same superfamily of HLA-B44. In silico analysis revealed different binding affinities of HLA-B*37:01 and HLA-B*40:06 for the epitopes predicted for E6 and L1 proteins of HPV16. The higher binding affinity of epitopes to B*40:06, as revealed by docking studies, supports the hypothesis that this allele is able to present the antigenic peptides more efficiently than B*37:01 and thereby can protect the carriers from the risk of cervical cancer. Thus, there is a clear indication that HLA plays an important role in the development of cervical cancer in HPV-infected women. Identification of these factors in high-risk HPV-infected women may help in reducing the cervical cancer burden in India.

Variations in immunogenetics, human papillomavirus (HPV) infection & predisposition to cervical cancer in Indian women.

BACKGROUND & OBJECTIVES: Human papillomavirus (HPV) is the main causative agent for cervical cancer. Variability in host immunogenetic factors is important in determining the overall cellular immune response to the HPV infection. This study was carried out to confirm the association between human leukocyte antigen (HLA) class II alleles and cervical cancer in HPV infected women. METHODS: Both low and high resolution methods were used to genotype HLA class II (DRB1 and DQB1) alleles in 75 women with cervical cancer (cases) and 75 HPV positive women and 100 HPV negative women with healthy cervix (controls). odds ratio and 95% confidence interval were calculated. Co-occurring HLA alleles (haplotype) across cases and controls were also studied. RESULTS: Significant association was found for HLA-DRB1*03(*13:01) and - DQB1*02(*02:01) with increased risk for cervical cancer. Also, HLA-DRB1*13(*13:01); -DQB1*06 and -DQB1*03:02 were significantly associated with decreased risk for cervical cancer. Haplotype analysis highlighted the significant association of HLA- DRB1*07:01-DQB1*02:02 and HLA DRB1*10:01-DQB1*05:01 with cervical cancer, while HLA-DRB1*14:04-DQB1*05:03 and DRB1*15:01-DQB1*06:01 conferred decreased risk for cervical cancer. Multivariate analysis highlighted the association of specific alleles with cervical cancer after adjusting for confounding factor age. INTERPRETATION & CONCLUSIONS: There were possible associations of specific HLA class II alleles either with risk of developing cervical cancer, or with its protection. Our results confirmed the assessment of DRB1*13 as a protective marker in HPV infection outcome. our study also revealed protective association of homozygous haplotype DRB1*15- DQB1*06 with cervical cancer.

Population length variability and nucleoid numbers in Escherichia coli.

MOTIVATION: Cell sizes and shapes are a fundamental defining characteristic of all cellular life. In bacteria like Escherichia coli, the machinery that determines cell length is complex and interconnected, spanning extracellular cues, biosynthesis and cell division. Few tools exist to study cell lengths in a population. We have developed and tested three automated image analysis routines on growing E.coli cultures to simultaneously measure cell lengths and nucleoid numbers in populations of bacteria. We find population profiles changing with culture density-higher density of culture leads to fewer long cells. Additionally, lab strains mutant for recA show a correlation between the number of nucleoids and cell length. CONTACT: cathale@iiserpune.ac.in; chaitanya.athale@gmail.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Evaluation of Homology-Independent CRISPR-Cas9 Off-Target Assessment Methods.

The ability to alter genomes specifically by CRISPR-Cas gene editing has revolutionized biological research, biotechnology, and medicine. Broad therapeutic application of this technology, however, will require thorough preclinical assessment of off-target editing by homology-based prediction coupled with reliable methods for detecting off-target editing. Several off-target site nomination assays exist, but careful comparison is needed to ascertain their relative strengths and weaknesses. In this study, HEK293T cells were treated with Streptococcus pyogenes Cas9 and eight guide RNAs with varying levels of predicted promiscuity in order to compare the performance of three homology-independent off-target nomination methods: the cell-based assay, GUIDE-seq, and the biochemical assays CIRCLE-seq and SITE-seq. The three methods were benchmarked by sequencing 75,000 homology-nominated sites using hybrid capture followed by high-throughput sequencing, providing the most comprehensive assessment of such methods to date. The three methods performed similarly in nominating sequence-confirmed off-target sites, but with large differences in the total number of sites nominated. When combined with homology-dependent nomination methods and confirmation by sequencing, all three off-target nomination methods provide a comprehensive assessment of off-target activity. GUIDE-seq's low false-positive rate and the high correlation of its signal with observed editing highlight its suitability for nominating off-target sites for ex vivo CRISPR-Cas therapies.

Isolation and characterization of indole acetic acid (IAA) producing Klebsiella pneumoniae strains from rhizosphere of wheat (Triticum aestivum) and their effect on plant growth.

The present study was undertaken for isolation of Klebsiella strains from rhizosphere of wheat (T. aestivum), screening and characterization of these strains for in vitro indole acetic acid (IAA) production and studying the effect of these strains on plant growth under gnotobiotic conditions. Nine strains of Klebsiella were isolated from rhizosphere of wheat (var. Lokwan) and identified as K. pneumoniae by 16S rRNA gene sequencing. Six K. pneumoniae strains showed in vitro IAA production. Colorimetric analysis showed that K8 produced maximum IAA (27.5 mg l(-1)) in the presence of tryptophan (1 mg ml(-1)) at 72 h of incubation with optimum conditions as pH 8.0, 37 degrees C and 0.5% (w/v) NaCl concentration. GC-MS analysis and IR studies confirmed presence of IAA in the cell filtrates of strain K8. Effect of six IAA producing Klebsiella strains on plant growth was studied by performing series of seed germination tests using moth bean seeds under axenic conditions and pot experiments using sterilized soil and wheat seeds (var. Lokwan). Strain K11 and K42 demonstrated increase in root length of inoculated moth beans (approximately 92.71% over the control). Results of pot experiments indicated that almost all the six IAA producing Klebsiella strains significantly increased the root length and shoot height of inoculated wheat seedlings over the control. The results suggest that these are promising isolates from wheat rhizosphere and merits research on appliance of these strains in agriculture.

Types of human papillomavirus observed in hospital-based population.

Background and Objectives: Human papillomavirus (HPV) is the causative agent of cervical cancer, a major cause of cancer mortality in Indian women. The current study was undertaken to add information to the existing data on HPV type distribution in Indians, in an attempt to document HPV types for future vaccination programme, if any. Materials and Methods: HPV infection was screened in 223 cervical cancer cases and 2408 healthy women without cancer and cervical intraepithelial neoplasia (control). HPV was typed using polymerase chain reaction, Southern hybridisation using specific probes and HPV GenoArray (Hybribio) test. Results: HPV DNA was found in 92.8% of cases and 7.3% of controls. Of the 383 HPV-infected women, 30.0% had single infection; 50.9% had multiple infections (two or more types) and 19.1% were infected with HPV types other than HPV-16, -18, -6 and -11. Besides HPV-16, HPV-51 and HPV-33 were also seen as single infection in cases. In cases, HPV-18 or its homologous HPV-45 was always present as co-infection with HPV-16 or with other high-risk type. Binary logistic regression (backward) analysis highlighted significant association of age, parity and socioeconomic status with HPV infection. The present study highlighted the presence of multiple HPV infection (186 of 207, 89.9%) along with HPV-16 in women with cervical cancer. In control, 27.3% were co-infected with other sexually transmitted infections, while Chlamydia trachomatis infection was seen in 13% of cases. Conclusions: The study highlighted the type of HPV infection seen among the hospital-based population. For better screening, HPV tests available in the market should include all the types seen in the population.

A massively parallel reporter assay dissects the influence of chromatin structure on cis-regulatory activity.

A gene's position in the genome can profoundly affect its expression because regional differences in chromatin modulate the activity of locally acting cis-regulatory sequences (CRSs). Here we study how CRSs and regional chromatin act in concert on a genome-wide scale. We present a massively parallel reporter gene assay that measures the activities of hundreds of different CRSs, each integrated at many specific genomic locations. Although genome location strongly affected CRS activity, the relative strengths of CRSs were maintained at all chromosomal locations. The intrinsic activities of CRSs also correlated with their activities in plasmid-based assays. We explain our data with a quantitative model in which expression levels are set by independent contributions from local CRSs and the regional chromatin environment, rather than by more complex sequence- or protein-specific interactions between these two factors. The methods we present will help investigators determine when regulatory information is integrated in a modular fashion and when regulatory sequences interact in more complex ways.

PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs.

RNA binding proteins (RBP) and microRNAs (miRNAs) often bind sequences in 3' untranslated regions (UTRs) of mRNAs, and regulate stability and translation efficiency. With the identification of numerous RBPs and miRNAs, there is an urgent need for new technologies to dissect the function of the cis-acting elements of RBPs and miRNAs. We describe post-transcriptional regulatory element sequencing (PTRE-seq), a massively parallel method for assaying the target sequences of miRNAs and RBPs. We use PTRE-seq to dissect sequence preferences and interactions between miRNAs and RBPs. The binding sites for these effector molecules influenced different aspects of the RNA lifecycle: RNA stability, translation efficiency, and translation initiation. In some cases, post-transcriptional control is modular, with different factors acting independently of each other, while in other cases factors show specific epistatic interactions. The throughput, flexibility, and reproducibility of PTRE-seq make it a valuable tool to study post-transcriptional regulation by 3'UTR elements.

Polymorphisms in cytotoxic T-lymphocyte associated antigen 4 gene does not affect scytotoxic T-lymphocyte associated antigen 4 levels in human papillomavirus-infected women with or without cervical cancer.

Background: Cervical cancer (CaCx) is the second most common cancer in Indian women. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) + 49 AA polymorphism is known to be associated with CaCx. Current attempt is to use immunotherapy for the treatment of metastatic melanoma and metastatic castration-resistant prostate cancer, i.e., blocking of CTLA-4 using a fully human monoclonal CTLA-4 antibody to disrupt its inhibitory signal. This allows the CTLs to destroy the cancer cells. There is no information available on the soluble level of CTLA-4 on which the immunotherapy is targeted. This is specifically in Indian population including cases with CaCx. Objective: The aim of this study is to evaluate the levels of soluble CTLA-4 (sCTLA-4) in human papillomavirus (HPV)-infected women with or without CaCx and their association with the polymorphism at CTLA-4 + 49 A/G and CTLA-4 -318 C/T genotypes. Materials and Methods: This is an exploratory case-control study involving two groups of HPV-infected women, the cases were with invasive CaCx and the control group was women with the healthy cervix. sCTLA-4 levels were measured using ELISA in 92 CaCx cases and 57 HPV-positive women with the healthy cervix. Results: Both cases and controls have similar sCTLA-4 levels. Comparison of CTLA-4 + 49A/G and -318 C/T genotypes with sCTLA-4 levels among cases and control also did not show any statistically significant difference. Conclusion: The present study suggests sCTLA-4 levels are not affected by a polymorphism at + 49 A>G CTLA-4. Hence, levels of CTLA-4 are similar in both CaCx cases and control group.

Scaling-Up Signaling Pathway Analysis.

A new technique for simultaneously measuring the activities of many signaling pathways unravels interconnected signaling networks.

Non-tenera Contamination and the Economic Impact of SHELL Genetic Testing in the Malaysian Independent Oil Palm Industry.

Oil palm (Elaeis guineensis) is the most productive oil bearing crop worldwide. It has three fruit forms, namely dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), which are controlled by the SHELL gene. The fruit forms exhibit monogenic co-dominant inheritance, where tenera is a hybrid obtained by crossing maternal dura and paternal pisifera palms. Commercial palm oil production is based on planting thin-shelled tenera palms, which typically yield 30% more oil than dura palms, while pisifera palms are female-sterile and have little to no palm oil yield. It is clear that tenera hybrids produce more oil than either parent due to single gene heterosis. The unintentional planting of dura or pisifera palms reduces overall yield and impacts land utilization that would otherwise be devoted to more productive tenera palms. Here, we identify three additional novel mutant alleles of the SHELL gene, which encode a type II MADS-box transcription factor, and determine oil yield via control of shell fruit form phenotype in a manner similar to two previously identified mutant SHELL alleles. Assays encompassing all five mutations account for all dura and pisifera palms analyzed. By assaying for these variants in 10,224 mature palms or seedlings, we report the first large scale accurate genotype-based determination of the fruit forms in independent oil palm planting sites and in the nurseries that supply them throughout Malaysia. The measured non-tenera contamination rate (10.9% overall on a weighted average basis) underscores the importance of SHELL genetic testing of seedlings prior to planting in production fields. By eliminating non-tenera contamination, comprehensive SHELL genetic testing can improve sustainability by increasing yield on existing planted lands. In addition, economic modeling demonstrates that SHELL gene testing will confer substantial annual economic gains to the oil palm industry, to Malaysian gross national income and to Malaysian government tax receipts.

Differential expression of collectins in human placenta and role in inflammation during spontaneous labor.

Collectins, collagen-containing Ca(2+) dependent C-type lectins and a class of secretory proteins including SP-A, SP-D and MBL, are integral to immunomodulation and innate immune defense. In the present study, we aimed to investigate their placental transcript synthesis, labor associated differential expression and localization at feto-maternal interface, and their functional implication in spontaneous labor. The study involved using feto-maternal interface (placental/decidual tissues) from two groups of healthy pregnant women at term (≥ 37 weeks of gestation), undergoing either elective C-section with no labor ('NLc' group, n = 5), or normal vaginal delivery with spontaneous labor ('SLv' group, n = 5). The immune function of SP-D, on term placental explants, was analyzed for cytokine profile using multiplexed cytokine array. SP-A, SP-D and MBL transcripts were observed in the term placenta. The 'SLv' group showed significant up-regulation of SP-D (p = 0.001), and down-regulation of SP-A (p = 0.005), transcripts and protein compared to the 'NLc' group. Significant increase in 43 kDa and 50 kDa SP-D forms in placental and decidual tissues was associated with the spontaneous labor (p<0.05). In addition, the MMP-9-cleaved form of SP-D (25 kDa) was significantly higher in the placentae of 'SLv' group compared to the 'NLc' group (p = 0.002). Labor associated cytokines IL-1α, IL-1ß, IL-6, IL-8, IL-10, TNF-α and MCP-1 showed significant increase (p<0.05) in a dose dependent manner in the placental explants treated with nSP-D and rhSP-D. In conclusion, the study emphasizes that SP-A and SP-D proteins associate with the spontaneous labor and SP-D plausibly contributes to the pro-inflammatory immune milieu of feto-maternal tissues.