Patient navigation pathway and barriers to treatment seeking in cancer in India: A qualitative inquiry

Cancer is a leading cause of mortality worldwide. Early diagnosis and treatment of cancer may curb the growing burden of the disease. Understanding cancer patients’ navigation pathways for seeking treatment is important in order to facilitate early diagnosis and treatment. With this background we conducted a hospital-based cross-sectional study comprising 68 randomly selected cancer inpatients in a tertiary cancer specialty hospital in Odisha, India, to explore the treatment-seeking pathways of the cancer patients and the barriers and enablers in seeking treatment. Financial constraint is one of the major reasons for the delay in accessing treatment, even when patients are suspected of or diagnosed with cancer. Low awareness of the presenting signs and symptoms of cancer and limited knowledge of the availability of cancer diagnosis and treatment facilities are major factors contributing to delay. Family and friends’ support is found to be the major enabling factor toward seeking treatment. Generation of awareness of cancer among the general population and primary-care practitioners – including those in alternative systems of medicine – is important. Information on diagnostic and treatment services appears to be a felt need.     

ProDeGe: a computational protocol for fully automated decontamination of genomes

Single amplified genomes and genomes assembled from metagenomes have enabled the exploration of uncultured microorganisms at an unprecedented scale. However, both these types of products are plagued by contamination. Since these genomes are now being generated in a high-throughput manner and sequences from them are propagating into public databases to drive novel scientific discoveries, rigorous quality controls and decontamination protocols are urgently needed. Here, we present ProDeGe (Protocol for fully automated Decontamination of Genomes), the first computational protocol for fully automated decontamination of draft genomes. ProDeGe classifies sequences into two classes—clean and contaminant—using a combination of homology and feature-based methodologies. On average, 84% of sequence from the non-target organism is removed from the data set (specificity) and 84% of the sequence from the target organism is retained (sensitivity). The procedure operates successfully at a rate of ~0.30 CPU core hours per megabase of sequence and can be applied to any type of genome sequence.Recent technological advancements have enabled the large-scale sampling of genomes from uncultured microbial taxa, through the high-throughput sequencing of single amplified genomes (SAGs; Rinke et al., 2013; Swan et al., 2013) and assembly and binning of genomes from metagenomes (GMGs; Cuvelier et al., 2010; Sharon and Banfield, 2013). The importance of these products in assessing community structure and function has been established beyond doubt (Kalisky and Quake, 2011). Multiple Displacement Amplification (MDA) and sequencing of single cells has been immensely successful in capturing rare and novel phyla, generating valuable references for phylogenetic anchoring. However, efforts to conduct MDA and sequencing in a high-throughput manner have been heavily impaired by contamination from DNA introduced by the environmental sample, as well as introduced during the MDA or sequencing process (Woyke et al., 2011; Engel et al., 2014; Field et al., 2014). Similarly, metagenome binning and assembly often carries various errors and artifacts depending on the methods used (Nielsen et al., 2014). Even cultured isolate genomes have been shown to lack immunity to contamination with other species (Parks et al., 2014; Mukherjee et al., 2015). As sequencing of these genome product types rapidly increases, contaminant sequences are finding their way into public databases as reference sequences. It is therefore extremely important to define standardized and automated protocols for quality control and decontamination, which would go a long way towards establishing quality standards for all microbial genome product types.Current procedures for decontamination and quality control of genome sequences in single cells and metagenome bins are heavily manual and can consume hours/megabase when performed by expert biologists. Supervised decontamination typically involves homology-based inspection of ribosomal RNA sequences and protein coding genes, as well as visual analysis of k-mer frequency plots and guanine–cytosine content (Clingenpeel, 2015). Manual decontamination is also possible through the software SmashCell (Harrington et al., 2010), which contains a tool for visual identification of contaminants from a self-organizing map and corresponding U-matrix. Another existing software tool, DeconSeq (Schmieder and Edwards, 2011), automatically removes contaminant sequences, however, the contaminant databases are required input. The former lacks automation, whereas the latter requires prior knowledge of contaminants, rendering both applications impractical for high-throughput decontamination.Here, we introduce ProDeGe, the first fully automated computational protocol for decontamination of genomes. ProDeGe uses a combination of homology-based and sequence composition-based approaches to separate contaminant sequences from the target genome draft. It has been pre-calibrated to discard at least 84% of the contaminant sequence, which results in retention of a median 84% of the target sequence. The standalone software is freely available at http://prodege.jgi-psf.org//downloads/src and can be run on any system that has Perl, R (R Core Team, 2014), Prodigal (Hyatt et al., 2010) and NCBI Blast (Camacho et al., 2009) installed. A graphical viewer allowing further exploration of data sets and exporting of contigs accompanies the web application for ProDeGe at http://prodege.jgi-psf.org, which is open to the wider scientific community as a decontamination service (Supplementary Figure S1).The assembly and corresponding NCBI taxonomy of the data set to be decontaminated are required inputs to ProDeGe (Figure 1a). Contigs are annotated with genes following which, eukaryotic contamination is removed based on homology of genes at the nucleotide level using the eukaryotic subset of NCBI''s Nucleotide database as the reference. For detecting prokaryotic contamination, a curated database of reference contigs from the set of high-quality genomes within the Integrated Microbial Genomes (IMG; Markowitz et al., 2014) system is used as the reference. This ensures that errors in public reference databases due to poor quality of sequencing, assembly and annotation do not negatively impact the decontamination process. Contigs determined as belonging to the target organism based on nucleotide level homology to sequences in the above database are defined as ‘Clean'', whereas those aligned to other organisms are defined as ‘Contaminant''. Contigs whose origin cannot be determined based on alignment are classified as ‘Undecided''. Classified clean and contaminated contigs are used to calibrate the separation in the subsequent 5-mer based binning module, which classifies undecided contigs as ‘Clean'' or ‘Contaminant'' using principal components analysis (PCA) of 5-mer frequencies. This parameter can also be specified by the user. When data sets do not have taxonomy deeper than phylum level, or a single confident taxonomic bin cannot be detected using sequence alignment, solely 9-mer based binning is used due to more accurate overall classification. In the absence of a user-defined cutoff, a pre-calibrated cutoff for 80% or more specificity separates the clean contigs from contaminated sequences in the resulting PCA of the 9-mer frequency matrix. Details on ProDeGe''s custom database, evaluation of the performance of the system and exploration of the parameter space to calibrate ProDeGe for a high accurate classification rate are provided in the Supplementary Material.Open in a separate windowFigure 1(a) Schematic overview of the ProDeGe engine. (b) Features of data sets used to validate ProDeGe: SAGs from the Arabidopsis endophyte sequencing project, MDM project, public data sets found in IMG but not sequenced at the JGI, as well as genomes from metagenomes. All the data and results can be found in Supplementary Table S3.The performance of ProDeGe was evaluated using 182 manually screened SAGs (Figure 1b,Supplementary Table S1) from two studies whose data sets are publicly available within the IMG system: genomes of 107 SAGs from an Arabidopsis endophyte sequencing project and 75 SAGs from the Microbial Dark Matter (MDM) project* (only 75/201 SAGs from the MDM project had 1:1 mapping between contigs in the unscreened and the manually screened versions, hence these were used; Rinke et al., 2013). Manual curation of these SAGs demonstrated that the use of ProDeGe prevented 5311 potentially contaminated contigs in these data sets from entering public databases. Figure 2a demonstrates the sensitivity vs specificity plot of ProDeGe results for the above data sets. Most of the data points in Figure 2a cluster in the top right of the box reflecting a median retention of 89% of the clean sequence (sensitivity) and a median rejection of 100% of the sequence of contaminant origin (specificity). In addition, on average, 84% of the bases of a data set are accurately classified. ProDeGe performs best when the target organism has sequenced homologs at the class level or deeper in its high-quality prokaryotic nucleotide reference database. If the target organism''s taxonomy is unknown or not deeper than domain level, or there are few contigs with taxonomic assignments, a target bin cannot be assessed and thus ProDeGe removes contaminant contigs using sequence composition only. The few samples in Figure 2a that demonstrate a higher rate of false positives (lower specificity) and/or reduced sensitivity typically occur when the data set contains few contaminant contigs or ProDeGe incorrectly assumes that the largest bin is the target bin. Some data sets contain a higher proportion of contamination than target sequence and ProDeGe''s performance can suffer under this condition. However, under all other conditions, ProDeGe demonstrates high speed, specificity and sensitivity (Figure 2). In addition, ProDeGe demonstrates better performance in overall classification when nucleotides are considered than when contigs are considered, illustrating that longer contigs are more accurately classified (Supplementary Table S1).Open in a separate windowFigure 2ProDeGe accuracy and performance scatterplots of 182 manually curated single amplified genomes (SAGs), where each symbol represents one SAG data set. (a) Accuracy shown by sensitivity (proportion of bases confirmed ‘Clean'') vs specificity (proportion of bases confirmed ‘Contaminant'') from the Endophyte and Microbial Dark Matter (MDM) data sets. Symbol size reflects input data set size in megabases. Most points cluster in the top right of the plot, showing ProDeGe''s high accuracy. Median and average overall results are shown in Supplementary Table S1. (b) ProDeGe completion time in central processing unit (CPU) core hours for the 182 SAGs. ProDeGe operates successfully at an average rate of 0.30 CPU core hours per megabase of sequence. Principal components analysis (PCA) of a 9-mer frequency matrix costs more computationally than PCA of a 5-mer frequency matrix used with blast-binning. The lack of known taxonomy for the MDM data sets prevents blast-binning, thus showing longer finishing times than the endophyte data sets, which have known taxonomy for use in blast-binning.All SAGs used in the evaluation of ProDeGe were assembled using SPAdes (Bankevich et al., 2012). In-house testing has shown that reads assembled with SPAdes from different strains or even slightly divergent species of the same genera may be combined into the same contig (Personal communications, KT and Robert Bowers). Ideally, the DNA in a well that gets sequenced belongs to a single cell. In the best case, contaminant sequences need to be at least from a different species to be recognized as such by the homology-based screening stage. In the absence of closely related sequenced organisms, contaminant sequences need to be at least from a different genus to be recognized as such by the composition-based screening stage (Supplementary Material). Thus, there is little risk of ProDeGe separating sequences from clonal populations or strains. We have found species- and genus-level contamination in MDA samples to be rare.To evaluate the quality of publicly available uncultured genomes, ProDeGe was used to screen 185 SAGs and 14 GMGs (Figure 1b). Compared with CheckM (Parks et al., 2014), a tool which calculates an estimate of genome sequence contamination using marker genes, ProDeGe generally marks a higher proportion of sequence as ‘Contaminant'' (Supplementary Table S2). This is because ProDeGe has been calibrated to perform at high specificity levels. The command line version of ProDeGe allows users to conduct their own calibration and specify a user-defined distance cutoff. Further, CheckM only outputs the proportion of contamination, but ProDeGe actually labels each contig as ‘Clean'' or ‘Contaminant'' during the process of automated removal.The web application for ProDeGe allows users to export clean and contaminant contigs, examine contig gene calls with their corresponding taxonomies, and discover contig clusters in the first three components of their k-dimensional space. Non-linear approaches for dimensionality reduction of k-mer vectors are gaining popularity (van der Maaten and Hinton, 2008), but we observed no systematic advantage of using t-Distributed Stochastic Neighbor Embedding over PCA (Supplementary Figure S2).ProDeGe is the first step towards establishing a standard for quality control of genomes from both cultured and uncultured microorganisms. It is valuable for preventing the dissemination of contaminated sequence data into public databases, avoiding resulting misleading analyses. The fully automated nature of the pipeline relieves scientists of hours of manual screening, producing reliably clean data sets and enabling the high-throughput screening of data sets for the first time. ProDeGe, therefore, represents a critical component in our toolkit during an era of next-generation DNA sequencing and cultivation-independent microbial genomics.     

Modelling expected trout ranges under current and future water temperature regimes in the Eastern Cape,South Africa

Different values have resulted in conflicts between anglers and conservation lobbies in the management of trout in South Africa. Key to the conflict is the demarcation of boundaries to areas in which brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss currently occur, or are likely to establish following stocking for angling. To provide a longer-term perspective on these areas, we developed models to link salmonid biological thermal thresholds to elevation. These, when applied spatially using a digital elevation model with a probability of occurrence model, provided the basis for estimating potentially available thermal habitat for these two cold water species. Here, we acknowledge that other variables (stocking history; river connectivity) also play a role in understanding trout distributions. Using a simple scenario of an increase in mean daily water temperatures of 2 °C, we demonstrated that both brown and rainbow trout are likely to exhibit considerable range reductions in the future. Because it is possible that these range restrictions will result in an increasing desire to introduce trout into areas above their current distribution limits for the maintenance of angling opportunities, conservation managers should prioritise these areas, with management interventions seeking to understand what will help to limit introductions.     

Genome sequence of Ensifer medicae strain WSM1115; an acid-tolerant Medicago-nodulating microsymbiont from Samothraki,Greece

Ensifer medicae strain WSM1115 forms effective nitrogen fixing symbioses with a range of annual Medicago species and is used in commercial inoculants in Australia. WSM1115 is an aerobic, motile, Gram-negative, non-spore-forming rod. It was isolated from a nodule recovered from the root of burr medic (Medicago polymorpha) collected on the Greek Island of Samothraki. WSM1115 has a broad host range for nodulation and N₂ fixation capacity within the genus Medicago, although this does not extend to all medic species. WSM1115 is considered saprophytically competent in moderately acid soils (pH(CaCl₂) 5.0), but it has failed to persist at field sites where soil salinity exceeded 10 ECe (dS/m). Here we describe the features of E. medicae strain WSM1115, together with genome sequence information and its annotation. The 6,861,065 bp high-quality-draft genome is arranged into 7 scaffolds of 28 contigs, contains 6,789 protein-coding genes and 83 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

Complete genome sequence of the orange-red pigmented, radioresistant Deinococcus proteolyticus type strain (MRP(T))

Deinococcus proteolyticus (ex Kobatake et al. 1973) Brook and Murray 1981 is one of currently 47 species in the genus Deinococcus within the family Deinococcaceae. Strain MRP(T) was isolated from feces of Lama glama and possesses extreme radiation resistance, a trait is shares with various other species of the genus Deinococcus, with D. proteolyticus being resistant up to 1.5 Mrad of gamma radiation. Strain MRP(T) is of further interest for its carotenoid pigment. The genome presented here is only the fifth completed genome sequence of a member of the genus Deinococcus (and the forth type strain) to be published, and will hopefully contribute to a better understanding of how members of this genus adapted to high gamma- or UV ionizing-radiation. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,886,836 bp long genome with its four large plasmids of lengths 97 kbp, 132 kbp, 196 kbp and 315 kbp harbors 2,741 protein-coding and 58 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Predicted warming and browning affect timing and magnitude of plankton phenological events in lakes: a mesocosm study

1. Aquatic ecosystems in Northern Europe are expected to face increases in temperature and water colour (TB) in future. While effects of these factors have been studied separately, it is unknown whether and how a combination of them might affect phenological events and trophic interactions. 2. In a mesocosm study, we combined both factors to create conditions expected to arise during the coming century. We focused on quantifying effects on timing and magnitude of plankton spring phenological events and identifying possible mismatches between resources (phytoplankton) and consumers (zooplankton). 3. We found that the increases in TB had important effects on timing and abundance of different plankton groups. While increased temperature led to an earlier peak in phytoplankton and zooplankton and a change in the relative timing of different zooplankton groups, increased water colour reduced chlorophyll‐a concentrations. 4. Increased TB together benefitted cladocerans and calanoid copepods and led to stronger top‐down control of algae by zooplankton. There was no sign of a mismatch between primary producers and grazers as reported from other studies. 5. Our results point towards an earlier onset of plankton spring growth in shallow lakes in future with a stronger top‐down control of phytoplankton by zooplankton grazers.     

A review on fisheries and conservation status of Asian horseshoe crabs

Horseshoe crabs are the only extant xiphosurans and are believed to be morphologically unchanged for more than 200 million years. Of the four extant species namely, Limulus polyphemus, Tachypleus tridentatus, Tapinauchenius gigas and Carcinoscorpius rotundicauda, the latter three are found in Asian waters. Recent evidences showed that Asian horseshoe crabs are facing serious threats such as degradation of their spawning grounds and habitat, environmental pollution, overexploitation as a culinary delicacy and biomedical bleeding practices. Baseline data on the distribution and existing population of the wild horseshoe crabs remain poorly known in several Asian regions. Several studies have clearly revealed that pressure due to over-fishing of wild stock has increased tremendously in the last decade. Due to an increase in demand for Tachypleus Amebocyte Lysate (TAL) analogous to Limulus Amebocyte Lysate (LAL) in the United States, there is an urgent need to comprehensively address their fishing and conservation measures in the Asian region. This review addresses the overall studies on three species of Asian horseshoe crabs in relation to their fishing practices, local exploitation of their wild stock either for human consumption (or) by biomedical industries. The authors have structured the discussion on an international scale to address the existing problems in fishing and conservation of horseshoe crabs. Since no specific regulatory force or legislative protection act or a policy to preserve their natural stock are available to this date, this paper strongly recommends representative countries to include horseshoe crabs under their wildlife protection act to avoid further unsustainable exploitation of their wild populations.     

Pattern of shift rota modulates oral temperature circadian rhythm and sleep-wakefulness profiles in shift workers

Twenty four shift workers (8 from a steel industry and 16 from a Government hospital) participated in the study. The subjects were instructed to self-measure oral temperature, 4 6 times a day for about three weeks. Sleep quantity and quality for each subject were analysed with the help of an appropriate inventory. The data were analysed by cosinor and power spectrum methods. The frequency of circadian rhythm detection was in the order of 48% in senior nurses, 29% in steel plant workers and 14% in junior nurses. These were also complemented by the results of power spectrum analysis. Present results suggest that rhythms of subjective fatigue and subjective drowsiness are governed neither by oral temperature oscillator nor by the sleep/wake cycle oscillator. The results show that shift rotation pattern chiefly modulates the circadian time structure of shift workers. It is also suggested that the phenomenon of circadian rhythm desynchronization in oral temperature appears to be independent of per day total sleep length.     

Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family

Mucin-type O-glycosylation is initiated by a large family of UDP- GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc- transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.      

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agents

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence of NaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv strain and the α-glucosidase and glycogen phosphorylase enzymes. Few of the compounds have shown interesting in vitro activity with MIC up to 3.12 μg/mL against M. tuberculosis and very good inhibition of α-glucosidase and glycogen phosphorylase enzymes. The most potent non toxic compound 40 exhibited about 58% ex vivo activity at MIC of 3.12 μg/mL. The present study opens a new gate to synthesize antitubercular agents for diabetic TB patients. In silico docking studies indicate that mycobacterial dihydrofolate reductase is the possible target of these compounds.