Cell cycle and DNA content of mitotic cells in brain ganglia of drosophila larvae

The programmes of replication of hetero- and euchromatin regions, mitotic cell cycle and the DNA content in metaphases in brain ganglia from late third instar larvae of Drosophila melanogaster (wild type and a tumour bearing mutant, 1(2)gl, strain) and of Drosophila nasuta were examined by autoradiography of [3H]thymidine labelled (continuous or pulse) cells and by cytophotometry, respectively. Brain ganglia labelled continuously with [3H]thymidine for 24 h in vitro showed a significantly high proportion of cells with incorporation of radioactivity restricted to heterochromatin only. Pulse labelling of brain ganglia from larvae of Drosophila melanogaster and Drosophila nasuta followed by chase for different time intervals showed that (i) the frequency of labelled metaphases was more than 50% within 15 to 30 min of chase and remained higher than 50% in nearly all the chase samples till 24 h, (ii) euchromatin labelled metaphases appeared with a low frequency within 1 to 4 h chase period but the heterochromatin labelled metaphases continued to be more common in the later chase samples also, (iii) single chromatid labelled second cycle metaphases were seen within 1 to 4 h after the pulse, but their frequency did not increase in the later samples. Cytophotometry of feulgen-DNA and Hoechst 33258 stained metaphases in late third instar larval brain ganglia revealed a greater variation in the DNA content of individual metaphases, although the means were close to the expected 4 C content. It appears that in relation to the known asymmetric cell divisions of neuroblast and other neural cells, the mitotically active cells in brain ganglia comprise a heterogenous population with widely varying lengths of the different phases of cell cycle; some of them may not cycle regularly and may possibly have a discontinuous S-phase

A Widespread Distribution of Genomic CeMyoD Binding Sites Revealed and Cross Validated by ChIP-Chip and ChIP-Seq Techniques

Identifying transcription factor binding sites genome-wide using chromatin immunoprecipitation (ChIP)-based technology is becoming an increasingly important tool in addressing developmental questions. However, technical problems associated with factor abundance and suitable ChIP reagents are common obstacles to these studies in many biological systems. We have used two completely different, widely applicable methods to determine by ChIP the genome-wide binding sites of the master myogenic regulatory transcription factor HLH-1 (CeMyoD) in C. elegans embryos. The two approaches, ChIP-seq and ChIP-chip, yield strongly overlapping results revealing that HLH-1 preferentially binds to promoter regions of genes enriched for E-box sequences (CANNTG), known binding sites for this well-studied class of transcription factors. HLH-1 binding sites were enriched upstream of genes known to be expressed in muscle, consistent with its role as a direct transcriptional regulator. HLH-1 binding was also detected at numerous sites unassociated with muscle gene expression, as has been previously described for its mouse homolog MyoD. These binding sites may reflect several additional functions for HLH-1, including its interactions with one or more co-factors to activate (or repress) gene expression or a role in chromatin organization distinct from direct transcriptional regulation of target genes. Our results also provide a comparison of ChIP methodologies that can overcome limitations commonly encountered in these types of studies while highlighting the complications of assigning in vivo functions to identified target sites

The HLH-6 Transcription Factor Regulates C. elegans Pharyngeal Gland Development and Function

The Caenorhabditis elegans pharynx (or foregut) functions as a pump that draws in food (bacteria) from the environment. While the “organ identity factor” PHA-4 is critical for formation of the C. elegans pharynx as a whole, little is known about the specification of distinct cell types within the pharynx. Here, we use a combination of bioinformatics, molecular biology, and genetics to identify a helix-loop-helix transcription factor (HLH-6) as a critical regulator of pharyngeal gland development. HLH-6 is required for expression of a number of gland-specific genes, acting through a discrete cis-regulatory element named PGM1 (Pharyngeal Gland Motif 1). hlh-6 mutants exhibit a frequent loss of a subset of glands, while the remaining glands have impaired activity, indicating a role for hlh-6 in both gland development and function. Interestingly, hlh-6 mutants are also feeding defective, ascribing a biological function for the glands. Pharyngeal pumping in hlh-6 mutants is normal, but hlh-6 mutants lack expression of a class of mucin-related proteins that are normally secreted by pharyngeal glands and line the pharyngeal cuticle. An interesting possibility is that one function of pharyngeal glands is to secrete a pharyngeal lining that ensures efficient transport of food along the pharyngeal lumen

Analysis of Pre-Analytic Factors Affecting the Success of Clinical Next-Generation Sequencing of Solid Organ Malignancies

Application of next-generation sequencing (NGS) technology to routine clinical practice has enabled characterization of personalized cancer genomes to identify patients likely to have a response to targeted therapy. The proper selection of tumor sample for downstream NGS based mutational analysis is critical to generate accurate results and to guide therapeutic intervention. However, multiple pre-analytic factors come into play in determining the success of NGS testing. In this review, we discuss pre-analytic requirements for AmpliSeq PCR-based sequencing using Ion Torrent Personal Genome Machine (PGM) (Life Technologies), a NGS sequencing platform that is often used by clinical laboratories for sequencing solid tumors because of its low input DNA requirement from formalin fixed and paraffin embedded tissue. The success of NGS mutational analysis is affected not only by the input DNA quantity but also by several other factors, including the specimen type, the DNA quality, and the tumor cellularity. Here, we review tissue requirements for solid tumor NGS based mutational analysis, including procedure types, tissue types, tumor volume and fraction, decalcification, and treatment effects

Pancreatic neuroendocrine tumor masquerading as metastasis in a patient with esophageal cancer: Diagnosis by endoscopic ultrasound-guided fine-needle aspiration

The role of endoscopic ultrasound (EUS) and guided biopsies has been well established for the locoregional staging of esophageal cancers. However, their role in posttreatment surveillance is unclear. Here, we describe a case of a pancreatic mass diagnosed on the follow-up positron emission tomography scan, concerning for a metastatic lesion. EUS-guided fine-needle aspiration (FNA) helped in establishing the diagnosis of neuroendocrine tumor, which tends to have a similar sonographic appearance. Therefore, it is imperative to evaluate a suspicious mass seen on computed tomography/positron emission tomography scan. EUS and EUS-guided FNA can serve as useful modalities to confirm the diagnosis by cytopathology

Novel ensemble of multivariate adaptive regression spline with spatial logistic regression and boosted regression tree for gully erosion susceptibility

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. The extreme form of land degradation through different forms of erosion is one of the major problems in sub-tropical monsoon dominated region. The formation and development of gullies is the dominant form or active process of erosion in this region. So, identification of erosion prone regions is necessary for escaping this type of situation and maintaining the correspondence between different spheres of the environment. The major goal of this study is to evaluate the gully erosion susceptibility in the rugged topography of the Hinglo River Basin of eastern India, which ultimately contributes to sustainable land management practices. Due to the nature of data instability, the weakness of the classifier and the ability to handle data, the accuracy of a single method is not very high. Thus, in this study, a novel resampling algorithm was considered to increase the robustness of the classifier and its accuracy. Gully erosion susceptibility maps have been prepared using boosted regression trees (BRT), multivariate adaptive regression spline (MARS) and spatial logistic regression (SLR) with proposed resampling techniques. The re-sampling algorithm was able to increase the efficiency of all predicted models by improving the nature of the classifier. Each variable in the gully inventory map was randomly allocated with 5-fold cross validation, 10-fold cross validation, bootstrap and optimism bootstrap, while each consisted of 30% of the database. The ensemble model was tested using 70% and validated with the other 30% using the K-fold cross validation (CV) method to evaluate the influence of the random selection of training and validation database. Here, all resampling methods are associated with higher accuracy, but SLR bootstrap optimism is more optimal than any other methods according to its robust nature. The AUC values of BRT optimism bootstrap, MARS optimism bootstrap and SLR optimism bootstrap are 87.40%, 90.40% and 90.60%, respectively. According to the SLR optimism bootstrap, the 107,771 km2 (27.51%) area of this region is associated with a very high to high susceptible to gully erosion. This potential developmental area of the gully was found primarily in the Hinglo River Basin, where lateral exposure was mainly observed with scarce vegetation. The outcome of this work can help policy-makers to implement remedial measures to minimize the damage caused by erosion of the gully

Invited review—next-generation sequencing: a modern tool in cytopathology

In recent years, cytopathology has established itself as an independent diagnostic modality to guide clinical management in many different settings. The application of molecular techniques to cytological samples to identify prognostic and predictive biomarkers has played a crucial role in achieving this goal. While earlier studies have demonstrated that single biomarker testing is feasible on cytological samples, currently, this provides only limited and increasingly insufficient information in an era where an increasing number of biomarkers are required to guide patient care. More recently, multigene mutational assays, such as next-generation sequencing (NGS), have gained popularity because of their ability to provide genomic information on multiple genes. The cytopathologist plays a key role in ensuring success of NGS in cytological samples by influencing the pre-analytical steps, optimizing preparation types and adequacy requirement in terms of cellularity and tumor fraction, and ensuring optimal nucleic acid extraction for DNA input requirements. General principles of the role and potential of NGS in molecular cytopathology in the universal healthcare (UHC) European environment and examples of principal clinical applications were discussed in the workshop that took place at the 30th European Congress of Pathology in Bilbao, European Society of Pathology, whose content is here comprehensively described