MethodsX

In in-vitro or in-vivo bioassays, the no observed effect concentration (NOEC) is often determined. This simple procedure has several disadvantages, including the limitation of being able to estimate only experimental doses. Linear interpolation between adjacent doses overcomes this drawback while maintaining the level of a familywise error rate (FWER) using multiple contrast tests.

The sperm selection assay (SSA) is a method based on chemotaxis to obtain spermatozoa at the optimum physiological state to successfully fertilize the egg. It consists of a device made of acrylic and an attractant solution which includes progesterone. We evaluate potential cytotoxicity interactions by means of Neutral Red uptake, the MTT and colony formation assays, according to ISO normative. Here we showed that even stressing the conditions of the assays, the SSA device alone or together with the progesterone solutions employed, showed to be innocuous for the cells. Suggesting that SSA could be incorporated into the ART procedures.

Calcium signaling data analysis has become increasing complex as the size of acquired datasets increases. In this paper we present a Ca2+ signaling data analysis method that employs custom written software scripts deployed in a collection of Jupyter-Lab "notebooks" which were designed to cope with this complexity. The notebook contents are organized to optimize data analysis workflow and efficiency. The method is demonstrated through application to several different Ca2+ signaling experiment types.

Reduction oxidation (redox) reactions are central in life and altered redox state is associated with a spectrum of human diseases. Glutathione (GSH) is the most abundant antioxidant in eukaryotic cells and plays critical roles in maintaining redox homeostasis. Thus, measuring intracellular GSH level is an important method to assess the redox state of organism. The currently available GSH probes are based on irreversible chemical reactions with glutathione and cant monitor the real-time glutathione dynamics. Our group developed the first reversible reaction based fluorescent probe for glutathione, which can measure glutathione levels at high resolution using a confocal microscope and in the bulk scale with a flow cytometry. Most importantly it can quantitatively monitor the real-time GSH dynamics in living cells. Using the 2nd generation of GSH probe, RealThiol (RT), this study measured the GSH level in living Hela cells after treatment with varying concentrations of DL-Buthionine sulfoximine (BSO) which inhibits GSH synthesis, using a high throughput imaging system, Cytation 5 cell imaging reader. The results revealed that GSH probe RT at the concentration of 2.0 {micro}M accurately monitored the BSO treatment effect on GSH level in the Hela cells. The present results demonstrated that the GSH probe RT is sensitive and precise in GSH measurement in living cells at a high throughput imaging platform and has the potential to be applied to any cell lines.

Epithelial tissues form barriers to the flow of ions, nutrients, waste products, bacteria, and viruses. The conventional electrophysiology measurement of transepithelial resistance (TER) can quantify epithelial barrier integrity, but does not capture all the electrical behavior of the tissue or provide insight into membrane specific properties. Electrochemical impedance spectroscopy, in addition to measurement of TER, enables measurement of transepithelial capacitance (TEC) and a ratio of electrical time constants for the tissue, which we term membrane ratio. This protocol describes how to perform galvanostatic electrochemical impedance spectroscopy on epithelia using commercially available cell culture inserts and chambers, detailing the apparatus, electrical signal, fitting techniques, and error quantification. The measurement can be performed in approximately one minute using instrumentation capable of galvanostatic sinusoidal signal processing (4 A amplitude, 2 Hz-50 kHz). All fits to the model have less than 10 {Omega} mean absolute error, revealing repeatable values distinct for each cell type. On representative retinal pigment (n=3) and bronchiolar epithelial samples (n=4), we measured TER 500-667 {Omega}.cm2 and 955-1034 {Omega}.cm2, within the expected range, TEC 3.65-4.10 F/cm2 and 1.07-1.10 F/cm2, and membrane ratios 18-22 and 1.9-2.2, respectively.

The range of the high-sensitivity cardiac troponin (HSCTN) values in the gen-eral population increases progressively with age, being several times higher in older patients, beyond 70 years old, than in younger ones, below 40. More accurate normal limits for HSCTN, taking into account age and gender, are needed for the differential diagnosis and evaluation of the prognostic signif-icance of increases that do not reach the vendor-supplied upper reference limits (URL). We performed an analysis of the high sensitivity cardiac troponin (HSCTN) of 21743 individuals, representative for the general US population, that were studied in the NHANES survey performed by the Center for Disease Control of the USA. The vendor supplied URL values are typically several times higher than the actual upper limit in subjects under 40 and correspond to the URL at ages between 51 and 84 for specific vendors and genders. For each HSCTN test variant, we considered each one year age group between 1 and 85 and either gender. The distribution of the logarithmed HSCTN for a given test variant and gender, in each subsample i, is relatively close to Gaussian (N ({micro}i, {sigma}i)). Two quadratic models, for the 1-15 and the 16-85 age ranges, were found to fit well with the {micro}i, while the{sigma} i follow a linear model. These theoretical distributions can be used used to estimate any quantiles of the HSCTN distribution as functions of age and gender, including the upper reference limit (URL) for the HSCTN I and T in the general population. Consequently, it is possible to construct a calculated HSCTN indicator that represents the estimated age and gender specific centile in which a measurement with a specific test kit falls, or the percentual distance above the 99th centile. This indicator would be independent of age, gender and kit-vendor, and thus more accurate and easier to employ in clinical practice.

In different cellular activities like signal transduction, cell division, and intracellular transportation, small GTPases take on a vital role. Their functioning involves hydrolysing guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In this article we explain the application of a commercially accessible GTPase assay, known as the GTPase Glo assay by Promega, for the quantitative investigation of GTPase - effector interactions and the interplay between effectors. Basic ProtocolConducting GTPase assays with GTPase : effector protein mixtures using the GTPase Glo assay (Promega). Supporting Protocol 1Analysing GTPase assays to correlate the assay readout (luminescence) to amount of remaining GTP. Supporting Protocol 2Fitting GTPase assay data to obtain GTPase cycling rates.

An often-overlooked aspect of mitochondrial biology is the mitochondrial DNA (mtDNA). The multi-copy mtDNA is highly dynamic, with changes in supercoiling, synthesis rates, and turnover rates that are tightly associated with mitochondrial and cellular functions. To better understand the state of the mtDNA, here we describe a protocol that selectively incorporates bromodeoxyuridine into the mtDNA for subsequent measurement via an adapted Southern blot followed by immunoblotting (a.k.a. Southwestern blot). This basic protocol can be applied with slight modifications for the measurement of mtDNA synthesis, turnover or supercoiling to understand mtDNA changes.

Few protocols are available for retrieving male germ cell-specific information on DNA damage dynamics or assessing the potential harmful effects of environmental contaminants, drugs, or lifestyle factors related to genotoxicity. Here, we present a protocol for evaluating testicular germ cell genotoxicity using a modified version of the alkaline comet assay in rodent testicular germ cells. The protocol includes experimental design, preparation of testicular cell suspensions, comet analysis, germ cell-specific scoring, and data curation methods to collect information on testicular male germ cells, specifically spermatids and primary spermatocytes. For complete details on the use of this protocol please refer to Olsen et al., in preparation HighlightsO_LIDNA damage levels can be specifically measured in testicular germ cells using the developed revised version of the alkaline comet assay C_LIO_LI1C spermatids as well as 4C primary spermatocytes can be assessed C_LIO_LIBoth manual- and modeling-based approaches were developed that facilitate user-friendly protocols to select 1C spermatid comets. C_LIO_LIThis protocol expands the limited methodologies available to study germ cell DNA damage dynamics C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/624648v7_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c18b1eorg.highwire.dtl.DTLVardef@1949f12org.highwire.dtl.DTLVardef@576e1eorg.highwire.dtl.DTLVardef@1feb14b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

Assessing and understanding the Size of the human fetus provides essential information for the management of the health of the newborn and its mother, a quality that is commonly measured by Fetal Ultrasound. Many equations for making such calculations have been considered, none of which have been entirely satisfactory. As we have shown in the previous paper in this series, a consideration of the formation of the body, in units of numbers of cells, N, an approach we call Binary Cellular Analysis, provides new quantitative tools for estimating the Size and Age of the fetus. These tools include a new mathematical basis for creating useful expressions, Binary Cellular Estimated Fetal Weight Equations, for calculating human Fetal Weight from Ultrasound Measurements. As we show here, the most promising of these expressions, the Abdominal Circumference Binary Cellular Estimated Fetal Weight Equation, performs better than other currently used Fetal Weight Equations, while also yielding up new methods for improving ultrasound size assessment. Web-based calculators (https://kidzgrowth.com) make these mathematical manipulations available for obstetric care.

1In vitro viability assays are widely used in drug discovery, development, and pharmacovigilance. Traditional methods for evaluating cell viability often involve destructive processes, rendering the culture non-viable. As such, these methods are suitable only as endpoint solutions, providing a single measurement per cell culture and precluding further analyses of the cells. In this study, we introduce Neural Viability Regression (NViR), a real-time, deep learning-based method that enables non-invasive quantification of culture viability using microscopy images. The non-intrusive nature of NViR allows for frequent viability evaluations throughout experiments, capturing subtle changes while maintaining the structural integrity of the culture and significantly reducing both culture and labor costs. We demonstrate NViRs applicability by using it to predict Drug-Induced Liver Injury (DILI) in known drugs. By exposing human liver spheroids to 108 FDA-approved drugs and capturing microscopy images over time, NViRs viability assessments accurately predict whether a drug induces DILI in humans, playing a critical role in enhancing liver safety protocols. The cost-effectiveness and non-invasive characteristics of NViR enable high-frequency, high-throughput viability assessments. Consequently, NViR is poised to reduce both the costs and incidences of failures in drug discovery and development.

This paper describes an objective statistical approach that can be used to decide between two alternate kinetic mechanisms of covalent enzyme inhibition from kinetic experiments based on the standard "kobs" method. The two alternatives are either a two-step kinetic mechanism, which involves a reversibly formed noncovalent intermediate, or a one-step kinetic mechanism, proceeding in a single bimolecular step. Recently published experimental data [Hopper et al. (2020) J. Pharm. Exp. Therap. 372, 331-338] on the irreversible inhibition of Bruton tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) by ibrutinib (PCI-32765) and acalabrutinib are used as an illustrative example. The results show that the kinetic mechanism of inhibition was misdiagnosed in the original publication for at least one of the four enzyme/inhibitor combinations. In particular, based on the available kobs data, ibrutinib behaves effectively as a one-step inhibitor of the TEC enzyme, which means that it is not possible to reliably determine either the inhibition constant Ki or the inactivation rate constant kinact, but only the covalent efficiency constant keff = kinact/Ki. Thus, the published values of Ki and kinact for this system are not statistically valid.

BackgroundTranscutaneous electrical nerve stimulation (TENS) has been a commonly used modality to relieve aches and pain for over 40 years. Commercially available devices provide multiple therapy modes involving a different combination of frequency and pulse width with intensity. While frequency sets sensation, intensity helps determine tolerability, longer pulse width is reported to induce a feeling of deeper stimulation. In fact, longer pulse width has been empirically shown to deliver current into deeper tissues, but in context of other electrical stimulation modalities. The goal of this study was to unpack the relationship between pulse width and activation depth in TENS. MethodsA highly realistic, anatomically-based, 3D finite element model of the forearm was used to simulate the electric field (E-field) distribution, as the pulse width is varied. A typical titration-guided mechanism was used to obtain the strength-duration (S-D) curves of a sensory McIntyre-Richardson-Grill (MRG) axonal model simulating the pain-transmitting A-delta fibers. The pulse widths tested ranged from 30 s to 495 s. ResultsAs expected, shorter pulse widths required more current to achieve activation, resulting in a larger E- field. The S-D curve of the target median nerve indicates a rheobase of 1.75 mA and a chronaxie of 232 {micro}sec. When the applied currents are the same, shorter pulse widths result in a smaller volume of tissue activated (VTA) compared to the longer pulse widths. A 21 fold difference in VTA was found between the longest and shortest pulse widths considered. We observed a linear relationship between pulse width and activation depth for the conditions tested in the study. ConclusionOur findings highlight the impact of pulse width on activation depth. While choice of a given therapy mode is usually based on an ad-hoc desirable sensation basis, medical professionals may consider advocating a certain therapy mode based on the depth of the intended target nerve.

In regulatory toxicology an outcome is claimed positive when both a trend is significant and any pairwise test against control. Two statistical approaches are proposed: a joint Dunnett and Williams test (assuming the dose as a qualitative factor) and a joint test of the Tukey regression test and Dunnett test (assuming the dose as a quantitative covariate). Related R software is available.

In (eco-)toxicological hazard characterization, the No Observed Adverse Effect Concentration or Level (NOAEC or NOAEL) approach is used and often required despite of its known limitations. For count data, statistical testing can be challenging, due to several confounding factors, such as zero inflation, low observation numbers, variance heterogeneity, over- or under-dispersion when applying the Poisson model or hierarchical experimental designs. As several tests are available for count data, we selected sixteen tests suitable for overdispersed counts and compared them in a simulation study. We assessed their performance considering data sets containing mixing distribution and over-dispersion with different observation numbers. It shows that there is no uniformly best approach because the assumed data conditions and assumptions are very different. However, the Dunnett-type procedure based on most likely transformation can be recommended, because of its size and power behavior, which is relatively better over most data conditions as compared to the available alternative test methods, and because it allows flexible modeling and effect sizes can be estimated by confidence intervals. Related R-code is provided for real data examples.

BackgroundSirolimus, an immunosuppressive drug widely used in organ transplantation, inhibits the mechanistic target of rapamycin (mTOR), decreases cellular respiration, stops cell cycle progression and causes apoptosis. It has been linked to cytotoxicity in the gonads, but the cause is unknown. AimsTo understand the effects of sirolimus on the gonads by measuring cellular respiration and ATP as indicators of cellular damage, and examining histological changes. Material and MethodsWe used 6-8 week old male and female BALB/c mice. They received 5 {micro}g/g sirolimus via intraperitoneal injections for 5 consecutive days per week for up to 3 weeks (treated group) or DMSO (control group). Upon euthanasia, the gonads were promptly removed while the heart was still beating, weighed, and processed for cellular respiration, ATP measurement, and histological studies. Cellular respiration was measured using a phosphorescence oxygen analyzer and ATP using a bioluminescent assay system. Histology was performed using processed tissue fragments stained with hematoxylin and eosin (H&E). ResultsCellular ATP levels in testicular tissue were higher than ovarian tissue in both groups, but this was not significant (p=0.4 and 0.2). Although the sirolimus group had higher cellular ATP levels, no significant difference was observed in either testicular (p=0.6) or ovarian (p=0.9) tissue. Both DMSO (p=0.01) and sirolimus groups (p=0.008) showed lower cellular respiration in ovarian than testicular tissue. Cellular respiration was lower in sirolimus group than DMSO group in both testicular (p=0.6) and ovarian (p=0.2) tissue. Testicular cellular respiration remained unchanged up to day 11 before declining, while ovarian respiration reduced up to day 11 and remained unchanged. Testicular histopathology showed normal sperm production and normal follicular development in ovarian tissue. ConclusionThe results suggest that the 5 {micro}g/g dose of sirolimus does not cause significant short-term changes in the cellular bioenergetics of the gonads or produce noticeable histopathological changes.

Image analysis is a rapidly developing field that provides unique opportunities to characterize and quantify spatial information in images. We study cis-regulatory modules (CRMs), non-coding DNA regions that regulate gene expression, during development using fluorescent reporters in vivo. Characterizing CRM activity during development presents challenges including image segmentation into biologically relevant regions of interest that are not easily distinguishable via common segmentation methods, fluorophore band passing, and variable transfection undermine standardized analysis. To quantify and compare CRM activity levels, we compiled an open-source computer vision tool stack in the form of a Python-based Jupyter notebook and tested four analysis methods to assess their efficacy in quantifying CRM expression in limb development. This Jupyter notebook provides a reproducible, standardized workflow that can be adapted to numerous image analysis applications.

In recent years, the structural analysis of tissue elements has gained significant importance in biomedical research. Advancements in imaging technologies have created a pressing need to quantify tissue and cell properties accurately. This paper introduces a MATLAB-based analytical tool designed to measure a spectrum of properties from 2D images of tissues and cells. Our software efficiently computes parameters such as eccentricity, orientation, density, co-localization, size, and perimeter. The algorithms precision in evaluating these characteristics has broad implications for enhancing the understanding of various biological processes and diseases. The codes flexibility allows for application across different tissue types and experimental conditions, providing researchers with a robust method for quantitative analysis. This advancement in computational image analysis represents a pivotal step towards more detailed and objective assessment in tissue engineering and cellular biology.

Bioimage analysis is a powerful tool for investigating complex biological processes, but its robustness depends on technical precision and rigorous experimental design. In particular, the use of appropriate controls and experimental repetition is critical for drawing meaningful conclusions. However, both are often used inadequately or overlooked, with "statistical significance" often prioritised, frequently obtained through the misuse or misinterpretation of statistical tests. In this study, we reanalyse publicly available image datasets to highlight the crucial role of robust experimental design in interpreting results. Our findings underscore the importance of focusing on effect sizes and biological relevance over arbitrary statistical thresholds. We also discuss the diminishing returns of increased data collection once statistical stability has been achieved. By refining control usage and emphasising effect sizes, this work aims to enhance the reproducibility and robustness of research findings. We provide open-access code to allow researchers to engage with the dataset, promoting better practices in experimental design and data interpretation.

Cerebral autoregulation (CA) is a critical mechanism that maintains cerebral blood flow (CBF) relatively stable despite fluctuations in arterial blood pressure (ABP), ensuring protection against ischemia and hyperperfusion. Alterations in CA are linked to adverse outcomes in various conditions, highlighting the need for precise and accessible methods to analyse CA. CardioBrain is a freely available user-friendly software developed to assess dynamic CA by performing Transfer Function Analysis (TFA) on continuously recorded ABP and CBF data. The software utilizes the Welch method for spectral density and cross-spectral analysis. Following TFA, the software calculates different dCA metrics as gain, phase, and coherence across various frequency bands (VLF, LF, HF). CardioBrain allows users to easily manipulate signal processing parameters, visualize data, and export results, making it suitable for both research and clinical settings without requiring advanced programming skills. Additionally, the software includes tools to address signal misalignments, such as a temporal shift feature, and ensures adherence to recommended standards for TFA analysis. Future development will focus on incorporating user feedback, validating the software against simulated and real-world data, and expanding its functionality for a broader range of regulatory analyses.