Kim Kiat Lim

Optical density (OD)-based cell growth measurement is commonly used in high-throughput screening (HTS) during drug discovery or when deciphering the pharmaceutical mechanism of action. While resuspending the cells via a mixing step is often assumed to be necessary prior to OD measurement, its essentiality in HTS workflows has not been systematically verified. Here, through the measurement of the growth of several strains of the microbial yeast Schizosaccharomyces pombe cells, we compared the overall growth dynamics between samples that have been mixed and not mixed. Using statistical quantification by a two-tailed paired t-test followed by multiple comparison corrections, we concluded from the comparison of the doubling time of cells growing in the exponential phase that mixing did not significantly affect the biological interpretation compared to unmixed samples. Doubling time quantification between mixed and unmixed samples showed a difference of approximately 10% on average based on the assessment of the growth of eight strains. As such, if the experimental outcome can accommodate this level of variability, incorporating a mixing step before OD determination would not be necessary. These observations support the simplification of HTS processes, improving the cost efficacy and process efficiency of readouts, yet maintaining the accuracy of data acquisition.

Autism spectrum disorder (ASD) is a complex neurological and developmental condition that occurs in approximately 1 in 100 children. ASD is a lifelong condition defined by difficulties with social communication, restricted interests, and repetitive behaviors, among other symptoms. Currently, we understand that there is no cure and the disorder can only be managed with occupational therapy alongside limited medical treatments. Reasons underlying the pathogenesis of ASD are still not well understood, but recent studies point to the influence of epigenetic dysregulation in ASD development, which opens up avenues to novel diagnosis and treatment options. In this review, we summarize recent findings and emerging therapeutics for ASD, with a focus on implications of epigenetic regulatory pathways and factors. We expound the implications of these findings to enable preventive measurements for mothers to reduce the impact of ASD at birth, non-invasive diagnostic tests for early detection, and personalized medicine management. Finally, we discuss several critical issues to be addressed and future directions of this important research field.

5-Fluorouracil (5-FU) is a conventional chemotherapeutic drug widely used in clinics worldwide, but development of resistance that compromises responsiveness remains a major hurdle to its efficacy. The mechanism underlying 5-FU resistance is conventionally attributed to the disruption of nucleotide synthesis, even though research has implicated other pathways such as RNA processing and chromatin dysregulation. Aiming to clarify resistance mechanisms of 5-FU, we tested the response of a collection of fission yeast (Schizosaccharomyces pombe) null mutants, which confer multiple environmental factor responsiveness (MER). Our screen identified disruption of membrane transport, chromosome segregation and mitochondrial oxidative phosphorylation to increase cellular susceptibility towards 5-FU. Conversely, we revealed several null mutants of Ino80 complex factors exhibited resistance to 5-FU. Furthermore, attenuation of Ino80 function via deleting several subunit genes reversed loss of chromosome-segregation fidelity in 5-FU in the loss-of-function mutant of the Argonaute protein, which regulates RNA interference (RNAi)-dependent maintenance of pericentromeric heterochromatin. Our study thus uncovered a critical role played by chromatin remodeling Ino80 complex factors in 5-FU resistance, which may constitute a possible target to modulate in reversing 5-FU resistance.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate metabolic pathway, and its loss of function through polymorphisms is often associated with human conditions, including cancer, congenital heart disease, and Down syndrome. MTHFR is also required in the maintenance of heterochromatin, a crucial determinant of genomic stability and precise chromosomal segregation. Here, we characterize the function of a fission yeast gene met11⁺, which encodes a protein that is highly homologous to the mammalian MTHFR. We show that, although met11⁺ is not essential for viability, its disruption increases chromosome missegregation and destabilizes constitutive heterochromatic regions at pericentromeric, sub-telomeric and ribosomal DNA (rDNA) loci. Transcriptional silencing at these sites were disrupted, which is accompanied by the reduction in enrichment of histone H3 lysine 9 dimethylation (H3K9me2) and binding of the heterochromatin protein 1 (HP1)-like Swi6. The met11 null mutant also dominantly disrupts meiotic fidelity, as displayed by reduced sporulation efficiency and defects in proper partitioning of the genetic material during meiosis. Interestingly, the faithful execution of these meiotic processes is synergistically ensured by cooperation among Met11, Rec8, a meiosis-specific cohesin protein, and the shugoshin protein Sgo1, which protects Rec8 from untimely cleavage. Overall, our results suggest a key role for Met11 in maintaining pericentromeric heterochromatin for precise genetic inheritance during mitosis and meiosis.

Drug resistance is a major hurdle to the success of chemotherapy. The permeability glycoprotein (P-gp) is an important factor dictating drug access to the cells, as it controls the efflux of chemotherapeutic agents against the concentration gradient. Pmd1, a P-gp-like protein, was recently isolated as a doxorubicin resistance gene in fission yeast. Although the null mutant of pmd1 (Δpmd1) exhibited sensitivity to doxorubicin, it showed an unexpectedly high resistance to the drug at relatively high concentrations. The data presented here suggest that this is due to the presence of cooperative processes that can complement and counteract drug cytotoxicity in the absence of Pmd1. One such factor, Rav1, is an essential factor in controlling the assembly of the pH-regulating transporter vacuolar-ATPase (V-ATPase) in fission yeast. The simultaneous disruption of Pmd1 and Rav1 resulted in a prominent accumulation of doxorubicin in the cytoplasm of cells, accompanied by a decline in cell viability. With concurrent treatment of pharmacological inhibitors in human cervical cancer cells, P-gp and V-ATPase were further shown to act synergistically to sensitize cells to doxorubicin also in the human cells. Furthermore, a novel Cornichon-like protein SPAC2C4.05 (herein named as Cor1) was demonstrated for the first time to be involved in the interaction with P-gp and V-ATPase to counteract doxorubicin-dependent cytotoxicity. Therefore this study identified a molecular cooperation between multiple membrane transporter proteins that confers chemoresistance to cells against the chemical insult of doxorubicin. Interestingly, this network exhibited differential effects to doxorubicin as compared with its close epimeric analog epirubicin, suggestive of the intricacy of the drug response regulated by this synergistic interaction. A model is discussed on how the versatility of this network can differentiate closely related chemical drug structures yet allow for the robustness to counteract a vast range of drugs.