JournalTOCs

Biology Methods and Protocols
Number of Followers: 0

This is an Open Access Journal

Open Access journal
ISSN (Online) 2396-8923
Published by Oxford University Press

[419 journals]

Risk factors and early cardiovascular outcomes in cancer patients treated
with anthracycline-based chemotherapy in Tanzania: a protocol for a
quasi-experimental study
- Authors: Vicent B; Meda J, Ngoma M, et al.
  Abstract: AbstractThe objective of this quasi-experimental study is to assess the risk factors and early cardiovascular outcomes in cancer patients treated with anthracycline-based chemotherapy in Tanzania. The study will be conducted at Ocean Road Cancer Institute in Dar es salaam, Tanzania. The study will have three phases: baseline survey, follow-up, and end-line survey. Participants will be newly diagnosed adult cancer patients who are eligible for anthracycline-based chemotherapy. A total of 427 participants will be involved. At baseline, participants will be evaluated for the cardiovascular risk factors before commencing chemotherapy. During follow-up, participants will commence their prescribed anthracycline and the cardiovascular indices monitored throughout until the patient completes the prescribed anthracycline cycles. After completing the prescribed anthracycline cycles, an end line survey will be conducted to evaluate any change in cardiovascular indices. The outcome variable in this study will be the change in biochemical data (high-density lipoprotein, low-density lipoprotein, triglyceride, and troponin I), blood pressure, and electrocardiographic information (heart rate and Bazett QT interval). Independent variables will be demographic characteristics, risk factors for cardiovascular disorders, current dietary practices, and body mass index. Descriptive statistics will be used to describe the participants. Independent and paired t-tests will be performed to make comparisons between and within groups. P-values <0.05 will be considered statistically significant. The results of this study will help clinicians and policymakers to understand the burden of early cardiovascular outcomes and plan for appropriate preventive strategies.
  PubDate: Wed, 09 Mar 2022 00:00:00 GMT
  DOI: 10.1093/biomethods/bpac006
  Issue No: Vol. 7, No. 1 (2022)
PSSMCOOL: a comprehensive R package for generating evolutionary-based
descriptors of protein sequences from PSSM profiles
- Authors: Mohammadi A; Zahiri J, Mohammadi S, et al.
  Abstract: AbstractPosition-specific scoring matrix (PSSM), also called profile, is broadly used for representing the evolutionary history of a given protein sequence. Several investigations reported that the PSSM-based feature descriptors can improve the prediction of various protein attributes such as interaction, function, subcellular localization, secondary structure, disorder regions, and accessible surface area. While plenty of algorithms have been suggested for extracting evolutionary features from PSSM in recent years, there is not any integrated standalone tool for providing these descriptors. Here, we introduce PSSMCOOL, a flexible comprehensive R package that generates 38 PSSM-based feature vectors. To our best knowledge, PSSMCOOL is the first PSSM-based feature extraction tool implemented in R. With the growing demand for exploiting machine-learning algorithms in computational biology, this package would be a practical tool for machine-learning predictions.
  PubDate: Wed, 30 Mar 2022 00:00:00 GMT
Semi-automatic detection of honeybee brood hygiene—an example of
artificial learning to facilitate ethological studies on social insects
- Authors: Batz P; Ruttor A, Thiel S, et al.
  Abstract: AbstractMachine-learning techniques are shifting the boundaries of feasibility in many fields of ethological research. Here, we describe an application of machine learning to the detection/measurement of hygienic behaviour, an important breeding trait in the honey bee (Apis mellifera). Hygienic worker bees are able to detect and destroy diseased brood, thereby reducing the reproduction of economically important pathogens and parasites such as the Varroa mite (Varroa destructor). Video observation of this behaviour on infested combs has many advantages over other methods of measurement, but analysing the recorded material is extremely time-consuming. We approached this problem by combining automatic tracking of bees in the video recordings, extracting relevant features, and training a multi-layer discriminator on positive and negative examples of the behaviour of interest. Including expert knowledge into the design of the features lead to an efficient model for identifying the uninteresting parts of the video which can be safely skipped. This algorithm was then used to semiautomatically identify individual worker bees involved in the behaviour. Application of the machine-learning method allowed to save 70% of the time required for manual analysis, and substantially increased the number of cell openings correctly identified. It thereby turns video-observation of individual cell opening events into an economically competitive method for selecting potentially resistant bees. This method presents an example of how machine learning can be used to boost ethological research, and how it can generate new knowledge by explaining the learned decision rule in form of meaningful parameters.
  PubDate: Wed, 16 Feb 2022 00:00:00 GMT
  DOI: 10.1093/biomethods/bpac005
Combining tangential flow filtration and size fractionation of mesocosm
water as a method for the investigation of waterborne coral diseases
- Authors: Evans J; Paul V, Ushijima B, et al.
  Abstract: AbstractThe causative agents of most coral diseases today remain unknown, complicating disease response and restoration efforts. Pathogen identifications can be hampered by complex microbial communities naturally associated with corals and seawater, which create complicating “background noise” that can potentially obscure a pathogen’s signal. Here, we outline an approach to investigate waterborne coral diseases that use a combination of coral mesocosms, tangential flow filtration, and size fractionation to reduce the impact of this background microbial diversity, compensate for unknown infectious dose, and further narrow the suspect pool of potential pathogens. As proof of concept, we use this method to compare the bacterial communities shed into six Montastraea cavernosa coral mesocosms and demonstrate this method effectively detects differences between diseased and healthy coral colonies. We found several amplicon sequence variants (ASVs) in the diseased mesocosms that represented 100% matches with ASVs identified in prior studies of diseased coral tissue, further illustrating the effectiveness of our approach. Our described method is an effective alternative to using coral tissue or mucus to investigate waterborne coral diseases of unknown etiology and can help more quickly narrow the pool of possible pathogens to better aid in disease response efforts. Additionally, this versatile method can be easily adapted to characterize either the entire microbial community associated with a coral or target-specific microbial groups, making it a beneficial approach regardless of whether a causative agent is suspected or is completely unknown.
  PubDate: Tue, 08 Feb 2022 00:00:00 GMT
  DOI: 10.1093/biomethods/bpac007
Surgical method to prevent early death of neonatal rat pups with
Hirschsprung disease, thus permitting development of long-term therapeutic
approaches
- Authors: Stamp L; Lei E, Liew J, et al.
  Abstract: AbstractHirschsprung disease occurs when children are born with no intrinsic nerve cells in varying lengths of the large intestine. In the most severe cases, neurons are also missing from the distal part of the small intestine. Nerve-mediated relaxation of the aganglionic bowel fails and fecal matter accumulates in the more proximal regions of the intestine. This is life threatening. Perforation of the bowel can ensue, causing sepsis and in some cases, death of the infant. Repopulation of the colon with neural stem cells is a potential therapy, but for this to be successful the patient or experimental animal needs to survive long enough for neural precursors to differentiate and make appropriate connections. We have developed a surgical procedure that can be applied to rats with Hirschsprung disease. A stoma was created to allow the normal bowel to empty and a second stoma leading to the aganglionic bowel was also created. This allowed homozygous mutants that would usually die at less than 3 weeks of age to survive into adulthood. During this time, the rats also required post-operative care of their stomas. The interventions we describe provide an animal model of Hirschsprung disease that is suited to assess the effectiveness of cell therapies in the treatment of this condition.
  PubDate: Thu, 27 Jan 2022 00:00:00 GMT
  DOI: 10.1093/biomethods/bpac004
Dissolution improvement of binary solid dispersions of erlotinib prepared
by one-step electrospray method
- Authors: Jahangiri A; Khalilzad F, Barghi L.
  Abstract: AbstractErlotinib hydrochloride, a selective tyrosine kinase inhibitor approved for treatment of non-small cell lung cancer firstly. Erlotinib classified as class II drugs in the Biopharmaceutical Classification System (BCS), which characterized by low solubility and high permeability. The aim of this study was to enhance the dissolution rate of this drug. The binary solid dispersions of erlotinib: PVP prepared at different ratios (1:3, 1:5, and 1:8) by electrospray technique. The characterization of formulations performed using differential scanning calorimetery (DSC), Fourier transform infrared spectroscopy (FT-IR) and dissolution rate test. The dissolution results showed that the dissolution rate of erlotinib from binary solid dispersions improved in comparison to pure drug. FTIR spectrum results showed that all peaks of erlotinib functional groups are also observable in the prepared solid dispersions. The FTIR results demonstrated that there was no interaction between drug and polymer. DSC thermograms of the prepared solid dispersions showed no drug-related peak, which is probably related to reduced crystallinity and drug amorphization. Based on the obtained results, it can be concluded that the erlotinib solid dispersion systems displayed improved dissolution rate compared to the pure drug. This will likely lead to increased drug bioavailability.
  PubDate: Thu, 27 Jan 2022 00:00:00 GMT
  DOI: 10.1093/biomethods/bpac001
Improving one-step scarless genome editing in Drosophila melanogaster by
combining ovoD co-CRISPR selection with sgRNA target site masking
- Authors: Götze K; Mrestani A, Beckmann P, et al.
  Abstract: AbstractThe precise and rapid construction of alleles through CRISPR/Cas9-mediated genome engineering renders Drosophila melanogaster a powerful animal system for molecular structure–function analyses and human disease models. Application of the ovoD co-selection method offers expedited generation and enrichment of scarlessly edited alleles without the need for linked transformation markers, which specifically in the case of exon editing can impact allele usability. However, we found that knockin procedures by homology-directed repair (HDR) under ovoD co-selection resulted in low transformation efficiency. This is likely due to repeated rounds of Cas9 cleavage of HDR donor and/or engineered genomic locus DNA, as noted for other CRISPR/Cas9 editing strategies before, impeding the recovery of correctly edited alleles. Here we provide a one-step protocol to improve the generation of scarless alleles by ovoD-co-selection with single-guide RNA (sgRNA) binding site masking. Using this workflow, we constructed human disease alleles for two Drosophila genes, unc-13/CG2999 and armadillo/CG11579. We show and quantify how a known countermeasure, the insertion of silent point mutations into protospacer adjacent motif (PAM) or sgRNA homology regions, can potently suppress unintended sequence modifications during CRISPR/Cas9 genome editing of D. melanogaster under ovoD co-selection. This strongly increased the recovery frequency of disease alleles.
  PubDate: Wed, 12 Jan 2022 00:00:00 GMT
  DOI: 10.1093/biomethods/bpac003
Effectiveness of a community-based intervention (Konga model) to address
the factors contributing to viral load suppression among children living
with HIV in Tanzania: a cluster-randomized clinical trial protocol
- Authors: Kihulya M; Katalambula L, Kapologwe N, et al.
  Abstract: AbstractThis study aims to test the effectiveness of a community-based intervention (Konga model) to improve viral-load suppression in children living with human immunodeficiency virus (HIV) and enrolled in care and treatment centers in Tanzania mainland. The study will be a cluster-randomized clinical trial study designed with both intervention and control arms. The study will involve 268 children with a viral load of >1000 copies/ml who are aged between 2 and 14 years. The children will be randomly allocated into the intervention and control arms. The intervention will include three distinct activities: adherence and retention counseling, psychosocial support, and comorbidity screening (i.e. tuberculosis). The outcome of the study will be assessment of the success of the intervention to increase medication adherence with the immediate result of reducing the viral load below 1000 copies/ml. Descriptive statistics will be used to calculate the mean, median, standard deviation, and interquartile range of continuous data. We will use frequencies and percentages to summarize categorical data. As for the primary outcome (proportion of HIV-infected children with viral suppression), we will compare the proportion of successful participants in the intervention and control arms. Proportions and tests for different proportions will be used as a measure of improvement. All statistical tests will be two-sided and P < 0.05 will be considered statistically significant.
  PubDate: Fri, 07 Jan 2022 00:00:00 GMT
  DOI: 10.1093/biomethods/bpac002
In vitro fluorescence assay to measure GDP/GTP exchange of guanine
nucleotide exchange factors of Rho family GTPases
- Authors: Blaise A; Corcoran E, Wattenberg E, et al.
  Abstract: AbstractGuanine nucleotide exchange factors (GEFs) are enzymes that promote the activation of GTPases through GTP loading. Whole exome sequencing has identified rare variants in GEFs that are associated with disease, demonstrating that GEFs play critical roles in human development. However, the consequences of these rare variants can only be understood through measuring their effects on cellular activity. Here, we provide a detailed, user-friendly protocol for purification and fluorescence-based analysis of the two GEF domains within the protein, Trio. This analysis offers a straight-forward, quantitative tool to test the activity of GEF domains on their respective GTPases, as well as utilize high-throughput screening to identify regulators and inhibitors. This protocol can be adapted for characterization of other Rho family GEFs. Such analyses are crucial for the complete understanding of the roles of GEF genetic variants in human development and disease.
  PubDate: Fri, 31 Dec 2021 00:00:00 GMT
  DOI: 10.1093/biomethods/bpab024
Structure-based discovery of selective CYP17A1 inhibitors for
Castration-resistant prostate cancer treatment
- Authors: Omoboyowa D; Balogun T, Saibu O, et al.
  Abstract: AbstractProstate cancer (PCa) is the most common malignancy found in men and the second leading cause of cancer-related death worldwide. Castration-resistant PCa (CRPC) is defined by PCa cells that stop responding to hormone therapy. Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) plays a critical role in the biosynthesis of androgens in humans. Androgen signaling cascade is a principal survival pathway for PCa cells and androgen-deprivation therapy (ADT) remains the key treatment for patients marked with locally advanced and metastatic PCa cells. Available synthetic drugs have been reported for toxicity, drug resistance, and decreasing efficacy. Thus, the design of novel selective inhibitors of CYP17A1 lyase would help circumvent associated side effects and improve pharmacological activities. Therefore, we employed structural bioinformatics techniques via molecular docking; molecular mechanics generalized born surface area (MM-GBSA), molecular dynamics (MD) simulation, and pharmacokinetic study to identify putative CYP17A1 lyase inhibitors. The results of the computational investigation showed that the Prunus dulcis compounds exhibited higher binding energy than the clinically approved abiraterone acetate. The stability of the ligand with the highest binding affinity (quercetin-3-o-rutinoside) was observed during MD simulation for 10 ns. Quercetin-3-o-rutinoside was observed to be stable within the active site of CYP17A1Lyase throughout the simulation period. The result of the pharmacokinetic study revealed that these compounds are promising therapeutic agents. Collectively, this study proposed that bioactive compounds from P. dulcis may be potential selective inhibitors of CYP17A1Lyase in CRPC treatments.
  PubDate: Sat, 25 Dec 2021 00:00:00 GMT
  DOI: 10.1093/biomethods/bpab026
Preparation of fatty acid solutions exerts significant impact on
experimental outcomes in cell culture models of lipotoxicity
- Authors: Römer A; Rawat D, Linn T, et al.
  Abstract: AbstractFree fatty acids are essentially involved in the pathogenesis of chronic diseases such as diabetes mellitus, non-alcoholic fatty liver disease, and cardiovascular disease. They promote mitochondrial dysfunction, oxidative stress, respiratory chain uncoupling, and endoplasmic reticulum stress and modulate stress-sensitive pathways. These detrimental biological effects summarized as lipotoxicity mainly depend on fatty acid carbon chain length, degree of unsaturation, concentration, and treatment time. Preparation of fatty acid solutions involves dissolving and complexing. Solvent toxicity and concentration, amount of bovine serum albumin (BSA), and ratio of albumin to fatty acids can vary significantly between equal concentrations, mediating considerable harmful effects and/or interference with certain assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Herein, we studied the impact of commonly used solvents ethanol and dimethyl sulfoxide and varying concentrations of BSA directly and in solution with oleic acid on MTT to formazan conversion, adenosine triphosphate level, and insulin content and secretion of murine β-cell line MIN6 employing different treatment duration. Our data show that experimental outcomes and assay readouts can be significantly affected by mere preparation of fatty acid solutions and should thus be carefully considered and described in detail to ensure comparability and distinct evaluation of data.
  PubDate: Fri, 03 Dec 2021 00:00:00 GMT
  DOI: 10.1093/biomethods/bpab023
Desktop scanning electron microscopy in plant–insect interactions
research: a fast and effective way to capture electron micrographs with
minimal sample preparation
- Authors: Watts S; Kaur I, Singh S, et al.
  Abstract: AbstractThe ability to visualize cell and tissue morphology at a high magnification using scanning electron microscopy (SEM) has revolutionized plant sciences research. In plant–insect interactions studies, SEM-based imaging has been of immense assistance to understand plant surface morphology including trichomes [plant hairs; physical defense structures against herbivores], spines, waxes, and insect morphological characteristics such as mouth parts, antennae, and legs, that they interact with. While SEM provides finer details of samples, and the imaging process is simpler now with advanced image acquisition and processing, sample preparation methodology has lagged. The need to undergo elaborate sample preparation with cryogenic freezing, multiple alcohol washes, and sputter coating makes SEM imaging expensive, time consuming, and warrants skilled professionals, making it inaccessible to majority of scientists. Here, using a desktop version of SEM (SNE- 4500 Plus Tabletop), we show that the “plug and play” method can efficiently produce SEM images with sufficient details for most morphological studies in plant–insect interactions. We used leaf trichomes of Solanum genus as our primary model, and oviposition by tobacco hornworm (Manduca sexta; Lepidoptera: Sphingidae) and fall armyworm (Spodoptera frugiperda; Lepidoptera: Noctuidae), and leaf surface wax imaging as additional examples to show the effectiveness of this instrument and present a detailed methodology to produce the best results with this instrument. While traditional sample preparation can still produce better resolved images with less distortion, we show that even at a higher magnification, the desktop SEM can deliver quality images. Overall, this study provides detailed methodology with a simpler “no sample preparation” technique for scanning fresh biological samples without the use of any additional chemicals and machinery.
  PubDate: Wed, 13 Oct 2021 00:00:00 GMT
  DOI: 10.1093/biomethods/bpab020