Early-life caffeine exposure induces morphological changes and altered physiology in Caenorhabditiselegans.

Caffeine, a widely consumed stimulant, is known for its effects on alertness and fatigue reduction by blockade of adenosine receptors. While it holds therapeutic potential, its diverse impacts pose risks, particularly in early development. This study explores the developmental effects of caffeine exposure using Caenorhabditis elegans (C. elegans) as a model organism. We investigated morphological and behavioral changes induced by caffeine exposure at the L1 stage and assessed their impact at the L4 stage, which roughly corresponds to human infancy and adolescence, respectively. Caffeine-exposed worms displayed increased body length, body bends, and pharyngeal pumping rates compared to control worms. These findings indicate heightened food-seeking behavior and greater food intake, leading to the observed morphological changes. While caffeine did not affect other locomotor behaviors, its stimulatory effect on growth and development highlights its significance. This study provides insights into the potential impact of early-life caffeine exposure on long-term health and development, offering a foundation for future research in vertebrates to uncover its implications on metabolism and other metrics of health.

COSMIC Database and Structural Modeling Analysis of CYP2D6 Mutations in Human Cancers.

Single nucleotide polymorphisms (SNPs) in cytochrome P450 (CYP450) enzymes alter the metabolism of a variety of drugs. Numerous medications, including chemotherapies, are metabolized by CYP450 enzymes, making the expression of this suite of enzymes in tumor cells relevant to prescription regimens for cancer patients. We analyzed the characteristics of mutations of the CYP2D6 enzymes in cancer patients obtained from the Catalogue of Somatic Mutations in Cancer (COSMIC), including mutation type, age of the patient, tissue type, and histology. Mutations were analyzed through the Cancer-Related Analysis of Variants Toolkit (CRAVAT) software along with CHASM and VEST4 algorithms to determine the likelihood of being a driver and/or pathogenic mutation. For mutations with significant CHASM and VEST4 scores, structural analysis of each corresponding mutant protein was performed. The effect of each mutation was evaluated for its impact on the overall protein stability and ligand binding using Foldit Standalone and SwissDock, respectively. Structural analysis revealed that several missense mutations in CYP2D6 resulted in altered stability after energy minimization. Three missense mutations of CYP2D6 significantly altered docking stability and those located on alpha-helices near the docking site had a more significant impact than those not found in secondary protein structures. In conclusion, we have identified a series of mutations to CYP2D6 enzymes with possible relevance to cancer pathologies. Significance Statement CYP2D6 is responsible for the metabolism of many anti-cancer drugs. This study identified and characterized a series of mutations in the CYP2D6 enzyme that occurred in tumors. We found it likely that many of these mutations would alter enzyme function, leading to changes in drug metabolism in the tumor. We provide a basis for predicting the likelihood of a patient carrying these mutations to identify patients who may benefit from a precision medicine approach to drug selection and dosing.

Detection of driver mutations and genomic signatures in endometrial cancers using artificial intelligence algorithms.

Analyzed endometrial cancer (EC) genomes have allowed for the identification of molecular signatures, which enable the classification, and sometimes prognostication, of these cancers. Artificial intelligence algorithms have facilitated the partitioning of mutations into driver and passenger based on a variety of parameters, including gene function and frequency of mutation. Here, we undertook an evaluation of EC cancer genomes deposited on the Catalogue of Somatic Mutations in Cancers (COSMIC), with the goal to classify all mutations as either driver or passenger. Our analysis showed that approximately 2.5% of all mutations are driver and cause cellular transformation and immortalization. We also characterized nucleotide level mutation signatures, gross chromosomal re-arrangements, and gene expression profiles. We observed that endometrial cancers show distinct nucleotide substitution and chromosomal re-arrangement signatures compared to other cancers. We also identified high expression levels of the CLDN18 claudin gene, which is involved in growth, survival, metastasis and proliferation. We then used in silico protein structure analysis to examine the effect of certain previously uncharacterized driver mutations on protein structure. We found that certain mutations in CTNNB1 and TP53 increase protein stability, which may contribute to cellular transformation. While our analysis retrieved previously classified mutations and genomic alterations, which is to be expected, this study also identified new signatures. Additionally, we show that artificial intelligence algorithms can be effectively leveraged to accurately predict key drivers of cancer. This analysis will expand our understanding of ECs and improve the molecular toolbox for classification, diagnosis, or potential treatment of these cancers.

Quality Control Method and Device for Producing Agarose Micropads.

In brightfield and fluorescence microscopy, capturing images that show well-focused and immobile microorganisms can be challenging. An agarose-based gel pad reduces the variability of results, especially in conditions like uneven specimen staging, variable fluid dynamics, and Brownian motion that plague conventional wet mount setups. To correct these discrepancies during image acquisition, we analyzed three micropad preparation setups. We tested the quality and consistency of pads and images resulting from each setup. Our examination reveals that improved gel pad flatness is associated with better image quality. Moreover, we observe increased consistency in gel pad construction connected to the use of a 3D-printed setup. These findings highlight the technical benefits arising from incorporating micropad-generating platforms that increase the consistency of results in imaging pipelines. Additionally, our use of a quantitative approach to examine pad flatness suggests its inclusion in quality control pipelines to reduce variation in gel pad construction and image quality over time and between investigators. Finally, our use of a 3D-printed setup coupled with a quantitative downstream routine suggests their application in microscopy experiments that involve model organisms relevant to human health and disease.

The Fourth Annual Symposium of the Midwest Aging Consortium.

The Midwest Aging Consortium (MAC) has emerged as a critical collaborative initiative aimed at advancing our understanding of aging and developing strategies to combat the rising prevalence of age-related diseases. Founded in 2019, MAC brings together researchers from various disciplines and institutions across the Midwestern United States to foster interdisciplinary geroscience research. This report summarizes the highlights of the Fourth Annual Symposium of MAC, which was held at Iowa State University in May 2023. The symposium featured presentations on a wide array of topics, including studies on slow-aging animals, cellular senescence and senotherapeutics, the role of the immune system in aging, metabolic changes in aging, neuronal health in aging, and biomarkers for measuring the aging process. Speakers shared findings from studies involving a variety of animals, ranging from commonly used species such as mice, rats, worms, yeast, and fruit flies, to less-common ones like naked mole-rats, painted turtles, and rotifers. MAC continues to emphasize the importance of supporting emerging researchers and fostering a collaborative environment, positioning itself as a leader in aging research. This symposium not only showcased the current state of aging biology research but also highlighted the consortium's role in training the next generation of scientists dedicated to improving the healthspan and well-being of the aging population.

Papaya and pineapple juices facilitate rehydration of mummified dermal tissue for fingerprint capture.

Mummified remains pose an issue for forensic scientists as identification of the deceased can be difficult due to extreme shriveling of dermal tissue and a resulting lack of quality fingerprint features. The typical protocols used to address this problem include corrosive chemicals that may further damage the already susceptible tissues. An alternative approach is found in the juice of two fruit species known to contain proteolytically active enzymes that tenderize soft tissues, thereby promoting water uptake. In this study, we saturated mummified fingers in papaya and pineapple juice treatments, followed by syringe-facilitated finger volume distension. After juice saturation, the data showed statistically significant increases in mass and volume of the samples, (papaya: relative mass p < 0.02833, relative volume p < 0.008466; pineapple: relative mass p < 0.01426, relative volume p < 0.04182). The post-treatment tissues were then rehydrated through a hydraulic mechanism that exerted the required turgor for effective fingerprint capture. This novel protocol utilizes fruit-based reagents to rehydrate mummified fingers without risk of corrosive damage, allowing for the restoration of accurate fingerprints and the positive identification of decedents. The value of this protocol lies in its simple implementation, affordability, instrument availability, and time effectiveness.

Photo Phenosizer, a rapid machine learning-based method to measure cell dimensions in fission yeast.

Cell metrics such as area, length, and width provide informative data about cell cycle dynamics. Factors that affect these dimensions include environmental conditions and genotypic differences. Fission yeast ( Schizosaccharomyces pombe ) is a rod-shaped ascomycete fungus in which cell cycle progression is linked to changes in cell length. Microscopy work to obtain these metrics places considerable burdens on time and effort. We now report on Photo Phenosizer (PP), a machine learning-based methodology that measures cell dimensions in fission yeast. It does this in an unbiased, automated manner and streamlines workflow from image acquisition to statistical analysis. Using this new approach, we constructed an efficient and flexible pipeline for experiments involving different growth media (YES and EMM) and treatments (Untreated and MMS) as well as different genotypes ( cut6-621 versus wildtype). This methodology allows for the analysis of larger sample sizes and faster image processing relative to manual segmentation. Our findings suggest that researchers using PP can quickly and efficiently determine cell size differences under various conditions that highlight genetic or environmental disruptions.

Maf1 regulates intracellular lipid homeostasis in response to DNA damage response activation.

Surveillance of DNA damage and maintenance of lipid metabolism are critical factors for general cellular homeostasis. We discovered that in response to DNA damage-inducing UV light exposure, intact Caenorhabditis elegans accumulate intracellular lipids in a dose-dependent manner. The increase in intracellular lipids in response to exposure to UV light utilizes mafr-1, a negative regulator of RNA polymerase III and the apical kinases atm-1 and atl-1 of the DNA damage response (DDR) pathway. In the absence of exposure to UV light, the genetic ablation of mafr-1 results in the activation of the DDR, including increased intracellular lipid accumulation, phosphorylation of ATM/ATR target proteins, and expression of the Bcl-2 homology region genes, egl-1 and ced-13. Taken together, our results reveal mafr-1 as a component the DDR pathway response to regulating lipid homeostasis following exposure to UV genotoxic stress.

A visual atlas of meiotic protein dynamics in living fission yeast.

Meiosis is a carefully choreographed dynamic process that re-purposes proteins from somatic/vegetative cell division, as well as meiosis-specific factors, to carry out the differentiation and recombination pathway common to sexually reproducing eukaryotes. Studies of individual proteins from a variety of different experimental protocols can make it difficult to compare details between them. Using a consistent protocol in otherwise wild-type fission yeast cells, this report provides an atlas of dynamic protein behaviour of representative proteins at different stages during normal zygotic meiosis in fission yeast. This establishes common landmarks to facilitate comparison of different proteins and shows that initiation of S phase likely occurs prior to nuclear fusion/karyogamy.

Comprehensive Genetic Analysis of DGAT2 Mutations and Gene Expression Patterns in Human Cancers.

DGAT2 is a transmembrane protein encoded by the DGAT2 gene that functions in lipid metabolism, triacylglycerol synthesis, and lipid droplet regulation. Cancer cells exhibit altered lipid metabolism and mutations in DGAT2 may contribute to this state. Using data from the Catalogue of Somatic Mutations in Cancer (COSMIC), we analyzed all cancer genetic DGAT2 alterations, including mutations, copy number variations and gene expression. We find that several DGAT2 mutations fall within the catalytic site of the enzyme. Using the Variant Effect Scoring Tool (VEST), we identify multiple mutations with a high likelihood of contributing to cellular transformation. We also found that D222V is a mutation hotspot neighboring a previously discovered Y223H mutation that causes Axonal Charcot-Marie-Tooth disease. Remarkably, Y223H has not been detected in cancers, suggesting that it is inhibitory to cancer progression. We also identify several single nucleotide polymorphisms (SNP) with high VEST scores, indicating that certain alleles in human populations have a pathogenic predisposition. Most mutations do not correlate with a change in gene expression, nor is gene expression dependent on high allele copy number. However, we did identify eight alleles with high expression levels, suggesting that at least in certain cases, the excess DGAT2 gene product is not inhibitory to cellular proliferation. This work uncovers unknown functions of DGAT2 in cancers and suggests that its role may be more complex than previously appreciated.

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Tetrad Dissection in Fission Yeast.

Tetrad dissection is a powerful tool in yeast genetics that allows the analysis of products of a single meiosis. With just a few tetrads, it is possible to determine linkage, identify unique phenotypes associated with double mutants, or assess specific meiotic defects. Strains are crossed on nitrogen-limiting medium for 3 days. With the help of a micromanipulator, ripe asci are isolated to spots 5 mm apart on a YES plate. Incubation at 36 °C for about 3-5 h is necessary for the ascus walls to break down. Once the spores are released, they are individually placed in a row containing four tetrad products, separated by 5 mm. The spores are then put in the appropriate temperature for the cross until colonies form, and phenotypes are assessed by replica plating or microscopic analysis.

Random Spore Analysis in Fission Yeast.

Random spore analysis (RSA) is a tool that allows for the screening of a large number of meiotic products. It requires only a limited effort, and is often the method of choice for constructing strains with unambiguous genotypes. It is also useful to identify the frequency of rare events. Strains are crossed on a nitrogen-limiting medium for three days. Mated cells are observed under the microscope to check for the presence of ripe asci. To release spores from their ascus, a sample of the cross is taken from the mating plate and resuspended in an enzyme solution overnight at 25-29 °C. Spores are then counted using a hemocytometer before plating an appropriate number. Incubation at the appropriate temperature follows until colonies form.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Destabilization of the replication fork protection complex disrupts meiotic chromosome segregation.

The replication fork protection complex (FPC) coordinates multiple processes that are crucial for unimpeded passage of the replisome through various barriers and difficult to replicate areas of the genome. We examine the function of Swi1 and Swi3, fission yeast's primary FPC components, to elucidate how replication fork stability contributes to DNA integrity in meiosis. We report that destabilization of the FPC results in reduced spore viability, delayed replication, changes in recombination, and chromosome missegregation in meiosis I and meiosis II. These phenotypes are linked to accumulation and persistence of DNA damage markers in meiosis and to problems with cohesion stability at the centromere. These findings reveal an important connection between meiotic replication fork stability and chromosome segregation, two processes with major implications to human reproductive health.