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Next Generation Sequencing (NGS) has become an important tool in the biological sciences and has a growing number of applications across medical fields. Currently, few undergraduate programs provide training in the design and implementation of NGS applications. Here, we describe an inquiry-based laboratory exercise for a college-level molecular biology laboratory course that uses real-time MinION deep sequencing and bioinformatics to investigate characteristic genetic variants found in cancer cell-lines. The overall goal for students was to identify non-small cell lung cancer (NSCLC) cell-lines based on their unique genomic profiles. The units described in this laboratory highlight core principles in multiplex PCR primer design, real-time deep sequencing, and bioinformatics analysis for genetic variants. We found that the MinION device is an appropriate, feasible tool that provides a comprehensive, hands-on NGS experience for undergraduates. Student evaluations demonstrated increased confidence in using molecular techniques and enhanced understanding of NGS concepts. Overall, this exercise provides a pedagogical tool for incorporating NGS approaches in the teaching laboratory as way of enhancing students' comprehension of genomic sequence analysis. Further, this NGS lab module can easily be added to a variety of lab-based courses to help undergraduate students learn current DNA sequencing methods with limited effort and cost.

Cerebral malaria (CM), a fatal complication of Plasmodium infection that affects children, especially under the age of five, in sub-Saharan Africa and adults in South-East Asia, results from incompletely understood pathogenetic mechanisms. Increased release of circulating miRNA, proteins, lipids and extracellular vesicles has been found in CM patients and experimental mouse models. We compared lipid profiles derived from the plasma of CBA mice infected with Plasmodium berghei ANKA (PbA), which causes CM, to those from Plasmodium yoelii (Py), which does not. We previously showed that platelet-free plasma (18k fractions enriched from plasma) contains a high number of extracellular vesicles (EVs). Here, we found that this fraction produced at the time of CM differed dramatically from those of non-CM mice, despite identical levels of parasitaemia. Using high-resolution liquid chromatography–mass spectrometry (LCMS), we identified over 300 lipid species within 12 lipid classes. We identified 45 and 75 lipid species, mostly including glycerolipids and phospholipids, with significantly altered concentrations in PbA-infected mice compared to Py-infected and uninfected mice, respectively. Total lysophosphatidylethanolamine (LPE) levels were significantly lower in PbA infection compared to Py infection and controls. These results suggest that experimental CM could be characterised by specific changes in the lipid composition of the 18k fraction containing circulating EVs and can be considered an appropriate model to study the role of lipids in the pathophysiology of CM. [ABSTRACT FROM AUTHOR]

Byline: Matthew J. Simpson (a), Dong C. Zhang (b), Michael Mariani (b), Kerry A. Landman (a), Donald F. Newgreen (b) Keywords: Cell invasion; Cell proliferation; Cell motility; Mathematical modeling; Neural crest cell; Enteric nervous system; Development Abstract: A general mathematical model of cell invasion is developed and validated with an experimental system. The model incorporates two basic cell functions: non-directed (diffusive) motility and proliferation to a carrying capacity limit. The model is used here to investigate cell proliferation and motility differences along the axis of an invasion wave. Mathematical simulations yield surprising and counterintuitive predictions. In this general scenario, cells at the invasive front are proliferative and migrate into previously unoccupied tissues while those behind the front are essentially nonproliferative and do not directly migrate into unoccupied tissues. These differences are not innate to the cells, but are a function of proximity to uninvaded tissue. Therefore, proliferation at the invading front is the critical mechanism driving apparently directed invasion. An appropriate system to experimentally validate these predictions is the directional invasion and colonization of the gut by vagal neural crest cells that establish the enteric nervous system. An assay using gut organ culture with chick-quail grafting is used for this purpose. The experimental results are entirely concordant with the mathematical predictions. We conclude that proliferation at the wavefront is a key mechanism driving the invasive process. This has important implications not just for the neural crest, but for other invasion systems such as epidermal wound healing, carcinoma invasion and other developmental cell migrations. Author Affiliation: (a) Department of Mathematics and Statistics, University of Melbourne, Victoria 3010, Australia (b) The Murdoch Childrens Research Institute, Department of Embryology, Flemington Road, Parkville, Victoria 3052, Australia Article History: Received 17 May 2006; Revised 12 September 2006; Accepted 9 October 2006

Human herpesvirus -6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes. Viral integration can occur in several cell types, including germinal cells, resulting in individuals that harbor the viral genome in every cell of their body. The integrated genome is efficiently silenced but can sporadically reactivate resulting in various clinical symptoms. To date, the integration mechanism and the subsequent silencing of HHV-6A/B genes remains poorly understood. Here we investigate the genome-wide chromatin contacts of the integrated HHV-6A in latently-infected cells. We show that HHV-6A becomes transcriptionally silent upon infection of these cells over the course of seven days. In addition, we established an HHV-6–specific 4C-seq approach, revealing that the HHV-6A 3D interactome is associated with quiescent chromatin states in cells harboring integrated virus. Furthermore, we observed that the majority of virus chromatin interactions occur toward the distal ends of specific human chromosomes. Exploiting this finding, we established a 4C-seq method that accurately detects the chromosomal integration sites. We further implement long-read minION sequencing in the 4C-seq assay and developed a method to identify HHV-6A/B integration sites in clinical samples. [ABSTRACT FROM AUTHOR]

A new resonator for X-band in vivo EPR nail dosimetry, the dielectric-backed aperture resonator (DAR), is developed based on rectangular TE102 geometry. This novel geometry for surface spectroscopy improves at least a factor of 20 compared to a traditional non-backed aperture resonator. Such an increase in EPR sensitivity is achieved by using a non-resonant dielectric slab, placed on the aperture inside the cavity. The dielectric slab provides an increased magnetic field at the aperture and sample, while minimizing sensitive aperture resonance conditions. This work also introduces a DAR semi-spherical (SS)-TE011 geometry. The SS-TE011 geometry is attractive due to having twice the incident magnetic field at the aperture for a fixed input power. It has been shown that DAR provides sufficient sensitivity to make biologically relevant measurements both in vitro and in vivo. Although in vivo tests have shown some effects of physiological motions that suggest the necessity of a more robust finger holder, equivalent dosimetry sensitivity of approximately 1.4 Gy has been demonstrated. [ABSTRACT FROM AUTHOR]

There is an imperative need to develop methods that can rapidly and accurately determine individual exposure to radiation for screening (triage) populations and guiding medical treatment in an emergency response to a large-scale radiological/nuclear event. To this end, a number of methods that rely on dose-dependent chemical and/or physical alterations in biomaterials or biological responses are in various stages of development. One such method, ex vivo electron paramagnetic resonance (EPR) nail dosimetry using human nail clippings, is a physical biodosimetry technique that takes advantage of a stable radiation-induced signal (RIS) in the keratin matrix of fingernails and toenails. This dosimetry method has the advantages of ubiquitous availability of the dosimetric material, easy and non-invasive sampling, and the potential for immediate and rapid dose assessment. The major challenge for ex vivo EPR nail dosimetry is the overlap of mechanically induced signals and the RIS. The difficulties of analysing the mixed EPR spectra of a clipped irradiated nail were addressed in the work described here. The following key factors lead to successful spectral analysis and dose assessment in ex vivo EPR nail dosimetry: (1) obtaining a thorough understanding of the chemical nature, the decay behaviour, and the microwave power dependence of the EPR signals, as well as the influence of variation in temperature, humidity, water content, and O2 level; (2) control of the variability among individual samples to achieve consistent shape and kinetics of the EPR spectra; (3) use of correlations between the multiple spectral components; and (4) use of optimised modelling and fitting of the EPR spectra to improve the accuracy and precision of the dose estimates derived from the nail spectra. In the work described here, two large clipped nail datasets were used to test the procedures and the spectral fitting model of the results obtained with it. A 15-donor nail set with 90 nail samples from 15 donors was used to validate the sample handling and spectral analysis methods that have been developed but without the interference of a native background signal. Good consistency has been obtained between the actual RIS and the estimated RIS computed from spectral analysis. In addition to the success in RIS estimation, a linear dose response has also been achieved for all individuals in this study, where the radiation dose ranges from 0 to 6 Gy. A second 16-donor nail set with 96 nail samples was used to test the spectral fitting model where the background signal was included during the fitting of the clipped nail spectra data. Although the dose response for the estimated and actual RIS calculated in both donor nail sets was similar, there was an increased variability in the RIS values that was likely due to the variability in the background signal between donors. Although the current methods of sample handling and spectral analysis show good potential for estimating the RIS in the EPR spectra of nail clippings, there is a remaining degree of variability in the RIS estimate that needs to be addressed; this should be achieved by identifying and accounting for demographic sources of variability in the background nail signal and the composition of the nail matrix. [ABSTRACT FROM AUTHOR]

Over the past few decades, waste management authorities have had to face up to the changing social, environmental and economic circumstances of putting waste to landfill. The responses to these changes can be placed into three general categories: first, state legislation to reduce packaging or use of non-environmentally friendly materials; secondly, encouraging the consumer to separate waste into recyclable and non-recyclable streams; and thirdly, utilisation of current alternative waste technologies (AWTs) to turn waste into useful commodities. AWTs often require different or no separation of waste materials by the consumer. Local waste management authorities therefore find themselves in the unenviable position of having to make strategic decisions on waste separation based on the perceived future improvements in AWTs, achievable recycling rates through `separation at source', future costs and the relative values of the recycled products produced. This paper provides a case study of the introduction of electronic bin tagging and weighing systems and how this technology could be utilised to increase recycling rates further. It is argued that such technology is required to break through the recycling rate ceiling that many waste management authorities are now encountering, having already explored the separation by consumer option. [ABSTRACT FROM AUTHOR]

A novel switchable coplanar waveguide (CPW) notch filter is developed using a microelectromechanical system (MEMS) switch element to perturb a patterned CPW centre-conductor line. The switch element is easily integrated with the CPW due to isolation of DC bias from RF signal paths. An equivalent lumped-element model has been developed for simulation of the electrical response of the filter. The measured notch frequency was shifted from 24.75 to 22.25 GHz, with greater then 20-dB rejection in both states. © 2006 Wiley Periodicals, Inc. Microwave Opt Technol Lett 48: 935–938, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.21524 [ABSTRACT FROM AUTHOR]

This paper describes a simple yet effective distributed database model that simulates database usage and buffer management in the distributed data processing environment. The model is table driven such that database access requirements, file location, and other information defining the database environment are set up internally in several tables, and linked lists represent the directory and data blocks. Each database transaction is defined and represented by a transaction flow diagram (TED) and a sequence of TED's representing an operational scenario is input to the model. The model "executes" input TED's by looking up tables, and performs buffer management for directory and file data while logging history and gathering various statistics on database usage and buffer management. The performance data are used for database access overhead measurement, database workload characterization, and buffer allocation. Disk access frequency and response time are used to validate the simulation results. The model has been implemented in Fortran on the VAX 11/780 and has been used for both measuring the given database performance and analyzing sensitivity to design changes. The results have proven that, despite its simplicity, the database model presented is an effective systems engineering tool for the design of distributed databases. [ABSTRACT FROM AUTHOR]

The small heat-shock protein, α-crystallin, has chaperone ability whereby it stabilises proteins under stress conditions. In this study, alterations in the structure of α-crystallin during its interaction with a variety of substrate proteins (insulin, α-lactalbumin, ovotransferrin and serum albumin) under stress conditions have been examined using visible absorption, 31P-NMR and 1H-NMR and fluorescence spectroscopy. The fluorescence and 31P-NMR data imply that during the chaperone action of α-crystallin under reducing conditions, there is a slight increase in hydrophilicity of its N-terminal region and an alteration in flexibility of its C-terminal region, but overall, α-crystallin does not undergo a gross structural change. The fluorescence data suggest that substrate proteins interact with α-crystallin in a molten globule or intermediately folded state. The same conclusion is made from 1H-NMR spectroscopic monitoring of the interaction of α-crystallin with substrate proteins, e.g. the insulin B chain. The stoichiometry of interaction between α-crystallin and the various substrate proteins reveals that steric factors are important in determining the efficiency of interaction between the two proteins, i.e. on a molar subunit basis, α-crystallin is a more efficient chaperone protein with smaller substrate proteins. Comparison is also made between the high-molecular-mass (HMM) complexes formed between α-crystallin and ovotransferrin when reduced and heat stressed. Under heating conditions, fluorescence spectroscopy indicates that the HMM complex has a greater exposure of hydrophobicity to solution than that formed by reduction. Furthermore, in interacting with heated ovotransferrin, the C-terminal extension of the αB-crystallin subunit preferentially loses its flexibility suggesting that it is involved in stabilising bound ovotransferrin. By contrast, this extension... [ABSTRACT FROM AUTHOR]

The cellular insulator protein CTCF plays a role in HSV-1 latency through the establishment and regulation of chromatin boundaries. We previously found that the CTRL2 regulatory element downstream from the LAT enhancer was bound by CTCF during latency and underwent CTCF eviction at early times post-reactivation in mice latent with 17syn+. We also showed that CTRL2 was a functional enhancer-blocking insulator in both epithelial and neuronal cell lines. We hypothesized that CTRL2 played a direct role in silencing lytic gene expression during the establishment of HSV-1 latency. To test this hypothesis, we used a recombinant virus with a 135-bp deletion spanning only the core CTRL2 insulator domain (ΔCTRL2) in the 17syn+ background. Deletion of CTRL2 resulted in restricted viral replication in epithelial cells, but not neuronal cells. Following ocular infection, mouse survival decreased in the ΔCTRL2 cohort and we found a significant decrease in the number of viral genomes in mouse trigeminal ganglia (TG) infected with ΔCTRL2, indicating that the CTRL2 insulator was required for the efficient establishment of latency. Immediate early (IE) gene expression significantly increased in the ganglia infected with ΔCTRL2 by 31 days post-infection, relative to 17syn+, indicating that deletion of the CTRL2 insulator disrupted the organization of chromatin domains during HSV-1 latency. Finally, ChIP-seq analyses of TG from ΔCTRL2 infected mice confirmed that the distribution of the repressive H3K27me3 mark on the ΔCTRL2 recombinant genomes was altered compared to the wild-type, indicating that CTRL2 site modulates the repression of IE genes during latency. [ABSTRACT FROM AUTHOR]

Python for Population Genomics (PyPop) is a software package that processes genotype and allele data and performs large-scale population genetic analyses on highly polymorphic multi-locus genotype data. In particular, PyPop tests data conformity to Hardy-Weinberg equilibrium expectations, performs Ewens-Watterson tests for selection, estimates haplotype frequencies, measures linkage disequilibrium, and tests significance. Standardized means of performing these tests is key for contemporary studies of evolutionary biology and population genetics, and these tests are central to genetic studies of disease association as well. Here, we present PyPop 1.0.0, a new major release of the package, which implements new features using the more robust infrastructure of GitHub, and is distributed via the industry-standard Python Package Index. New features include implementation of the asymmetric linkage disequilibrium measures and, of particular interest to the immunogenetics research communities, support for modern nomenclature, including colon-delimited allele names, and improvements to meta-analysis features for aggregating outputs for multiple populations. Code available at: https://zenodo.org/records/10080668 and https://github.com/alexlancaster/pypop., Competing Interests: Authors AL and GW are co-owners of Amber Biology LLC. Author MM is the owner of Mariani Systems LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lancaster, Single, Mack, Sochat, Mariani and Webster.)

Cerebral malaria (CM), a fatal complication of Plasmodium infection that affects children, especially under the age of five, in sub-Saharan Africa and adults in South-East Asia, results from incompletely understood pathogenetic mechanisms. Increased release of circulating miRNA, proteins, lipids and extracellular vesicles has been found in CM patients and experimental mouse models. We compared lipid profiles derived from the plasma of CBA mice infected with Plasmodium berghei ANKA (PbA), which causes CM, to those from Plasmodium yoelii (Py), which does not. We previously showed that platelet-free plasma (18k fractions enriched from plasma) contains a high number of extracellular vesicles (EVs). Here, we found that this fraction produced at the time of CM differed dramatically from those of non-CM mice, despite identical levels of parasitaemia. Using high-resolution liquid chromatography-mass spectrometry (LCMS), we identified over 300 lipid species within 12 lipid classes. We identified 45 and 75 lipid species, mostly including glycerolipids and phospholipids, with significantly altered concentrations in PbA-infected mice compared to Py-infected and uninfected mice, respectively. Total lysophosphatidylethanolamine (LPE) levels were significantly lower in PbA infection compared to Py infection and controls. These results suggest that experimental CM could be characterised by specific changes in the lipid composition of the 18k fraction containing circulating EVs and can be considered an appropriate model to study the role of lipids in the pathophysiology of CM.

DNA methylation-based copy number variation (CNV) calling software offers the advantages of providing both genetic (copy-number) and epigenetic (methylation) state information from a single genomic library. This method is advantageous when looking at large-scale chromosomal rearrangements such as the loss of the short arm of chromosome 3 (3p) in renal cell carcinoma and the codeletion of the short arm of chromosome 1 and the long arm of chromosome 19 (1p/19q) commonly seen in histologically defined oligodendrogliomas. Herein, we present MethylMasteR: a software framework that facilitates the standardization and customization of methylation-based CNV calling algorithms in a single R package deployed using the Docker software framework. This framework allows for the easy comparison of the performance and the large-scale CNV event identification capability of four common methylation-based CNV callers. Additionally, we incorporated our custom routine, which was among the best performing routines. We employed the Affymetrix 6.0 SNP Chip results as a gold standard against which to compare large-scale event recall. As there are disparities within the software calling algorithms themselves, no single software is likely to perform best for all samples and all combinations of parameters. The employment of a standardized software framework via creating a Docker image and its subsequent deployment as a Docker container allows researchers to efficiently compare algorithms and lends itself to the development of modified workflows such as the custom workflow we have developed. Researchers can now use the MethylMasteR software for their methylation-based CNV calling needs and follow our software deployment framework. We will continue to refine our methodology in the future with a specific focus on identifying large-scale chromosomal rearrangements in cancer methylation data., Competing Interests: Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

During development, tissues often undergo rapid physical expansion due to cell proliferation. Continuous and discrete models of one- and two-dimensional tissue growth are developed and applied to observational data of the developing avian gut, where the gut tissue cells undergo dramatic proliferation. The discrete cellular automata model provides results at the level of individual cells that reflect a realistic stochasticity and nonuniformity expected in cellular systems. Averaging the discrete results predicts population-level properties of the system, which match those of the continuous model. This dual approach provides an understanding of the interaction between the individual-level and population-level aspects of a developmental growth process. Both models are applied to a case study involving the developing intestinal tract of a quail embryo. A nonuniform growth model accurately predicts the positions of measurable biological landmarks within the growing tissue. Furthermore, the discrete model provides a framework for modeling the interactions between growing tissues and other biological mechanisms, such as cell motility and proliferation on an expanding tissue.