A Normalization Protocol Reduces Edge Effect in High-Throughput Analyses of Hydroxyurea Hypersensitivity in Fission Yeast.

Edge effect denotes better growth of microbial organisms situated at the edge of the solid agar media. Although the precise reason underlying edge effect is unresolved, it is generally attributed to greater nutrient availability with less competing neighbors at the edge. Nonetheless, edge effect constitutes an unavoidable confounding factor that results in misinterpretation of cell fitness, especially in high-throughput screening experiments widely employed for genome-wide investigation using microbial gene knockout or mutant libraries. Here, we visualize edge effect in high-throughput high-density pinning arrays and report a normalization approach based on colony growth rate to quantify drug (hydroxyurea)-hypersensitivity in fission yeast strains. This normalization procedure improved the accuracy of fitness measurement by compensating cell growth rate discrepancy at different locations on the plate and reducing false-positive and -negative frequencies. Our work thus provides a simple and coding-free solution for a struggling problem in robotics-based high-throughput screening experiments.

Usneaceratins A and B, two new secondary metabolites from the lichen Usnea ceratina.

Two new compounds, comprising one dibenzofuran, named usneaceratin A (1), and one phenolic acid, named usneaceratin B (2), together with one known dibenzofuran, isousnic acid (3), and two known phenolics, orsellinic acid (4) and methyl orsellinate (5) were clarified from the lichen Usnea ceratina using variously chromatographic methods. Their structures were testified by comprehensive HR-ESI-MS, and NMR spectroscopic analysis, and comparison with published data. Their α-glucosidase inhibitory activity of all compounds was measured. Usneaceratin B (2) possessed better inhibition against α-glucosidase enzyme (IC50 value of 41.8 µM) than the standard drug acarbose (IC50 value of 214.50 µM).

Preclinical Immune Response and Safety Evaluation of the Protein Subunit Vaccine Nanocovax for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health concern. The development of vaccines with high immunogenicity and safety is crucial for controlling the global COVID-19 pandemic and preventing further illness and fatalities. Here, we report the development of a SARS-CoV-2 vaccine candidate, Nanocovax, based on recombinant protein production of the extracellular (soluble) portion of the spike (S) protein of SARS-CoV-2. The results showed that Nanocovax induced high levels of S protein-specific IgG and neutralizing antibodies in three animal models: BALB/c mouse, Syrian hamster, and a non-human primate (Macaca leonina). In addition, a viral challenge study using the hamster model showed that Nanocovax protected the upper respiratory tract from SARS-CoV-2 infection. Nanocovax did not induce any adverse effects in mice (Mus musculus var. albino) and rats (Rattus norvegicus). These preclinical results indicate that Nanocovax is safe and effective.

SAHA and cisplatin sensitize gastric cancer cells to doxorubicin by induction of DNA damage, apoptosis and perturbation of AMPK-mTOR signalling.

Chemotherapy remains the most prescribed anti-cancer therapy, despite patients suffering severe side effects and frequently developing chemoresistance. These complications can be partially overcome by combining different chemotherapeutic agents that target multiple biological pathways. However, selecting efficacious drug combinations remains challenging. We previously used fission yeast Schizosaccharomycespombe as a surrogate model to predict drug combinations, and showed that suberoylanilide hydroxamic acid (SAHA) and cisplatin can sensitise gastric adenocarcinoma cells toward the cytotoxic effects of doxorubicin. Yet, how this combination undermines cell viability is unknown. Here, we show that SAHA and doxorubicin markedly enhance the cleavage of two apoptosis markers, caspase 3 and poly-ADP ribose polymerase (PARP-1), and increase the phosphorylation of γH2AX, a marker of DNA damage. Further, we found a prominent reduction in Ser485 phosphorylation of AMP-dependent protein kinase (AMPK), and reductions in its target mTOR and downstream ribosomal protein S6 phosphorylation. We show that SAHA contributes most of the effect, as confirmed using another histone deacetylase inhibitor, trichostatin A. Overall, our results show that the combination of SAHA and doxorubicin can induce apoptosis in gastric adenocarcinoma in a synthetically lethal manner, and that fission yeast offers an efficient tool for identifying potent drug combinations against human cancer cells.

Diagnostic value of 3DFLAIR in clinical practice for the detection of infratentorial lesions in multiple sclerosis in regard to dual echo T2 sequences.

BACKGROUND AND PURPOSE: The aim of this prospective study is to investigate and evaluate in clinical practice the diagnostic impact of 3DFLAIR in regards to 2DT2/PD in terms of infratentorial lesions detection in multiple sclerosis (MS). MATERIAL AND METHODS: 164 MS patients from the OFSEP database were reviewed retrospectively. MR examinations were performed on 1.5T or 3T systems from four different centers. Infratentorial lesions were counted and allocated to different regions of the posterior fossa by three raters independently (junior resident, resident with an expertise in neuroradiology, and senior neuro-radiologist) on the 3DFLAIR and 2DT2/PD. Both sequences do not have the same spatial resolution but reflect what is recommended by most of the consensus and done in clinical practice. RESULTS: With an overall number of 528 for Rater-1 and 798 for Rater-2 infratentorial lesions, 3DFLAIR had a significantly higher number of lesions detected than 2DT2/PD (303 for Rater-1 and 370 for Rater-2). The prevalence of trigeminal lesions detected by using 3DFLAIR was also significantly higher than 2DT2/PD. ROC analysis showed 3DFLAIR to be more specific and sensitive than 2DT2/PD. An overall difference between all three Raters has been observed. The more the Rater is experienced the more lesions he detects. CONCLUSION: Along with the radiologist ability to detect lesions based on his level of experience, the OFSEP optimized 3DFLAIR can significantly improve infratentorial lesion detection in MS compared to 2DT2/PD. This is important in MS follow-up that takes into account new lesions number to adapt patients' treatment.

Histone H3 lysine 36 methyltransferase mobilizes NER factors to regulate tolerance against alkylation damage in fission yeast.

The Set2 methyltransferase and its target, histone H3 lysine 36 (H3K36), affect chromatin architecture during the transcription and repair of DNA double-stranded breaks. Set2 also confers resistance against the alkylating agent, methyl methanesulfonate (MMS), through an unknown mechanism. Here, we show that Schizosaccharomyces pombe (S. pombe) exhibit MMS hypersensitivity when expressing a set2 mutant lacking the catalytic histone methyltransferase domain or a H3K36R mutant (reminiscent of a set2-null mutant). Set2 acts synergistically with base excision repair factors but epistatically with nucleotide excision repair (NER) factors, and determines the timely nuclear accumulation of the NER initiator, Rhp23, in response to MMS. Set2 facilitates Rhp23 recruitment to chromatin at the brc1 locus, presumably to repair alkylating damage and regulate the expression of brc1+ in response to MMS. Set2 also show epistasis with DNA damage checkpoint proteins; regulates the activation of Chk1, a DNA damage response effector kinase; and acts in a similar functional group as proteins involved in homologous recombination. Consistently, Set2 and H3K36 ensure the dynamicity of Rhp54 in DNA repair foci formation after MMS treatment. Overall, our results indicate a novel role for Set2/H3K36me in coordinating the recruitment of DNA repair machineries to timely manage alkylating damage.

Regulation of transcriptional silencing and chromodomain protein localization at centromeric heterochromatin by histone H3 tyrosine 41 phosphorylation in fission yeast.

Heterochromatin silencing is critical for genomic integrity and cell survival. It is orchestrated by chromodomain (CD)-containing proteins that bind to methylated histone H3 lysine 9 (H3K9me), a hallmark of heterochromatin. Here, we show that phosphorylation of tyrosine 41 (H3Y41p)-a novel histone H3 modification-participates in the regulation of heterochromatin in fission yeast. We show that a loss-of-function mutant of H3Y41 can suppress heterochromatin de-silencing in the centromere and subtelomere repeat regions, suggesting a de-silencing role for H3Y41p on heterochromatin. Furthermore, we show both in vitro and in vivo that H3Y41p differentially regulates two CD-containing proteins without the change in the level of H3K9 methylation: it promotes the binding of Chp1 to histone H3 and the exclusion of Swi6. H3Y41p is preferentially enriched on centromeric heterochromatin during M- to early S phase, which coincides with the localization switch of Swi6/Chp1. The loss-of-function H3Y41 mutant could suppress the hypersensitivity of the RNAi mutants towards hydroxyurea (HU), which arrests replication in S phase. Overall, we describe H3Y41p as a novel histone modification that differentially regulates heterochromatin silencing in fission yeast via the binding of CD-containing proteins.

Nanostructured lipid carriers to enhance transdermal delivery and efficacy of diclofenac.

Lipid carrier-mediated transdermal drug delivery offers several advantages because it is non-irritating and non-toxic, provides effective control of drug release, and forms an adhesive film that hydrates the outer skin layers. However, to penetrate the deeper skin layers, these formulations need to overcome several barriers in the stratum corneum. This study evaluates factors influencing particle size and drug-loading capacity, which play a key role in drug permeation and efficacy. Diclofenac sodium was chosen as the model drug. The fabrication of diclofenac sodium-loaded lipid nanoparticles was optimized by modulating several parameters, including the lipids and surfactants employed, the drug/lipid ratio, and the pH of the aqueous phase. The physical properties and loading efficiencies of the nanoparticles were characterized. The optimized formulation was then dispersed into a polymer solution to form a gel, which demonstrated a sustained ex vivo permeation of diclofenac sodium over 24 h through excised rat skin and a higher drug penetrating capacity than that of a commercially available gel. In vivo anti-inflammatory activity was assessed in a rat carrageenan-induced paw edema model; the anti-edema effects of the prepared gel and commercially available gel over 24 h were comparable. The present findings indicate the effects of particle size and drug loading on the ability of nanostructured lipid carrier preparations to provide transdermal drug delivery.

Calcium modulation of doxorubicin cytotoxicity in yeast and human cells.

Doxorubicin is a widely used chemotherapeutic agent, but its utility is limited by cellular resistance and off-target effects. To understand the molecular mechanisms regulating chemotherapeutic responses to doxorubicin, we previously carried out a genomewide search of doxorubicin-resistance genes in Schizosaccharomyces pombe fission yeast and showed that these genes are organized into networks that counteract doxorubicin cytotoxicity. Here, we describe the identification of a subgroup of doxorubicin-resistance genes that, when disrupted, leads to reduced tolerance to exogenous calcium. Unexpectedly, we observed a suppressive effect of calcium on doxorubicin cytotoxicity, where concurrent calcium and doxorubicin treatment resulted in significantly higher cell survival compared with cells treated with doxorubicin alone. Conversely, inhibitors of voltage-gated calcium channels enhanced doxorubicin cytotoxicity in the mutants. Consistent with these observations in fission yeast, calcium also suppressed doxorubicin cytotoxicity in human breast cancer cells. Further epistasis analyses in yeast showed that this suppression of doxorubicin toxicity by calcium was synergistically dependent on Rav1 and Vph2, two regulators of vacuolar-ATPase assembly; this suggests potential modulation of the calcium-doxorubicin interaction by fluctuating proton concentrations within the cellular environment. Thus, the modulatory effects of drugs or diet on calcium concentrations should be considered in doxorubicin treatment regimes.

Predicting chemotherapeutic drug combinations through gene network profiling.

Contemporary chemotherapeutic treatments incorporate the use of several agents in combination. However, selecting the most appropriate drugs for such therapy is not necessarily an easy or straightforward task. Here, we describe a targeted approach that can facilitate the reliable selection of chemotherapeutic drug combinations through the interrogation of drug-resistance gene networks. Our method employed single-cell eukaryote fission yeast (Schizosaccharomyces pombe) as a model of proliferating cells to delineate a drug resistance gene network using a synthetic lethality workflow. Using the results of a previous unbiased screen, we assessed the genetic overlap of doxorubicin with six other drugs harboring varied mechanisms of action. Using this fission yeast model, drug-specific ontological sub-classifications were identified through the computation of relative hypersensitivities. We found that human gastric adenocarcinoma cells can be sensitized to doxorubicin by concomitant treatment with cisplatin, an intra-DNA strand crosslinking agent, and suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. Our findings point to the utility of fission yeast as a model and the differential targeting of a conserved gene interaction network when screening for successful chemotherapeutic drug combinations for human cells.

Fitness profiling links topoisomerase II regulation of centromeric integrity to doxorubicin resistance in fission yeast.

Doxorubicin, a chemotherapeutic agent, inhibits the religation step of topoisomerase II (Top2). However, the downstream ramifications of this action are unknown. Here we performed epistasis analyses of top2 with 63 genes representing doxorubicin resistance (DXR) genes in fission yeast and revealed a subset that synergistically collaborate with Top2 to confer DXR. Our findings show that the chromatin-regulating RSC and SAGA complexes act with Top2 in a cluster that is functionally distinct from the Ino80 complex. In various DXR mutants, doxorubicin hypersensitivity was unexpectedly suppressed by a concomitant top2 mutation. Several DXR proteins showed centromeric localization, and their disruption resulted in centromeric defects and chromosome missegregation. An additional top2 mutation could restore centromeric chromatin integrity, suggesting a counterbalance between Top2 and these DXR factors in conferring doxorubicin resistance. Overall, this molecular basis for mitotic catastrophe associated with doxorubicin treatment will help to facilitate drug combinatorial usage in doxorubicin-related chemotherapeutic regimens.

Two fission yeast high mobility group box proteins in the maintenance of genomic integrity following doxorubicin insult.

Drug resistance is a challenge in chemotherapy, and, to date, there has been little resolution as to how it is induced. We previously isolated a host of doxorubicin resistance (DXR) genes in fission yeast and here we investigate the regulation of this resistance through two high mobility group (HMG) motif-containing DXR proteins, Nht1 and Hap2. The concurrent deletion of nht1 and hap2 did not confer cumulative sensitivity to doxorubicin, indicating that these factors cooperate closely in similar epistatic groups. We show that doxorubicin treatment resulted in the subcellular reorganization of Rhp54, a homologous recombination-dependent DNA damage repair protein. The disruption of either nht1 or hap2 attenuated Rhp54-foci formation, suggesting that these factors modulate the repair of doxorubicin-induced DNA lesions via the recruitment of homologous recombination machinery. Epistatic analyses further confirmed that Nht1 and Hap2 act in similar functional groups with complexes related to DSB repair but act synergistically with factors that regulate transcription and chromosome segregation. Overall, this work shows the molecular crosstalk coordinated by HMG proteins in conferring doxorubicin resistance in fission yeast.