Developing centrifugal force real-time digital PCR for detecting extremely low DNA concentration.

Digital PCR (dPCR) is a technique for absolute quantification of nucleic acid molecules. To develop a dPCR technique that enables more accurate nucleic acid detection and quantification, we established a novel dPCR apparatus known as centrifugal force real-time dPCR (crdPCR). This system is efficient than other systems with only 2.14% liquid loss by dispensing samples using centrifugal force. Moreover, we applied a technique for analyzing the real-time graph of the each micro-wells and distinguishing true/false positives using artificial intelligence to mitigate the rain, a persistent issue with dPCR. The limits of detection and quantification were 1.38 and 4.19 copies/µL, respectively, showing a two-fold higher sensitivity than that of other comparable devices. With the integration of this new technology, crdPCR will significantly contribute to research on next-generation PCR targeting absolute micro-analysis.

Analysis of Self-Assembled Micelle Inhibitory RNA (SAMiRNA) Drug Using Ion-Pairing Reversed-Phase Liquid Chromatography Combined with Mass Spectrometry.

Small interfering RNA (siRNA) is known for its ability to silence the expression of specific genes, demonstrating its promising potential as a therapeutic approach. Self-assembled micelle inhibitory RNA (SAMiRNA) is an oligonucleotide duplex developed to overcome the in vivo delivery limitations of siRNA. SAMiRNA has hydrophilic and hydrophobic groups at both ends of a sense strand, forming a spherical nanostructure that enhances the in vivo delivery efficiency. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the most commonly used method for the analysis of oligonucleotides. Since SAMiRNA is heavily chemically modified, the behavior of SAMiRNA in IP-RPLC combined with mass spectrometry (MS) is anticipated to differ from that of the conventional siRNA drug. The current investigation using IP-RPLC-MS revealed that a distinct duplex peak along with two minor separate strands of antisense and sense was observed at column temperatures below 35 °C in the IP-RPLC system with a 100 mM ammonium bicarbonate buffer system. At column temperatures higher than 35 °C, however, two fully denatured single strands were observed. The mass spectrum from the chromatographic peak of the SAMiRNA duplex contained signals from the duplex, the antisense, and the sense, probably due to duplex denaturation during the MS ionization process. The current comprehensive analysis results will make a substantial contribution to the future application of IP-RPLC-MS in the analysis of SAMiRNA.

Anti-Melanogenic and Antioxidant Effects of Cell-Free Supernatant from Lactobacillus gasseri BNR17.

In recent years, there has been considerable interest in the use of cell-free supernatant of probiotics culture for nutritional and functional applications. In this study, we investigated the effect of the cell-free supernatant from Lactobacillus gasseri BNR17 (CFS) on anti-melanogenesis and reducing oxidative stress in B16-F10 murine melanoma cells and HaCaT human keratinocytes. Treatment with CFS significantly inhibited the production of extracellular and intracellular melanin without cytotoxicity during melanogenesis induced by the α-MSH in B16-F10 cells. The CFS dramatically reduced tyrosinase activity and the melanogenesis-related gene expression. Further, it showed antioxidative effects in a dose-dependent manner in DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assays and significantly increased the mRNA levels of HO-1 and CAT in HaCaT cells. Furthermore, the CFS increased HO-1 and anti-oxidative-related gene expression during H2O2-induced oxidative stress in HaCaT cells. Together, this study suggests that the CFS reduces hyperpigmentation and inhibits oxidative stress, and thus can be used as a potential skincare product in the future.

Evaluation of TBMDR® and XDRA® for the detection of multidrug resistant and pre-extensively drug resistant tuberculosis.

This study evaluated the diagnostic performance of the AccuPower® TB&MDR Real-Time PCR (TBMDR®) and AccuPower® XDR-TB Real-Time PCR Kit-A (XDRA®) to detect multidrug-resistant (MDR-TB) and pre-extensively drug-resistant tuberculosis (pre-XDR-TB) in comparison with phenotypic drug susceptibility testing (DST) using MGIT 960 on 234 clinical Mycobacterium tuberculosis isolates. Discrepant results were confirmed by direct-sequencing. Sensitivity and specificity of TBMDR and XDRA for cultured isolates were 81.2% and 95.8% for isoniazid (INH) resistance, 95.7% and 95.7% for rifampicin (RIF) resistance, 84.1% and 99.1% for fluoroquinolone (FQ) resistance, and 67.4% and 100% for second-line injectables resistance. The sensitivities of each drug were equivalent to other molecular DST methods. High concordance was observed when compared to direct-sequencing. We also found that TBMDR and XDRA assays can detect INH, RIF and FQ resistance in isolates with low level resistance-associated mutations which were missed by phenotypic DST. Our study showed TBMDR and XDRA assays could be the useful tools to detect MDR-TB and pre-XDR-TB.

SAMiRNA Targeting Amphiregulin Alleviate Total-Body-Irradiation-Induced Renal Fibrosis.

Fibrosis is a serious unintended side effect of radiation therapy. In this study, we aimed to investigate whether amphiregulin (AREG) plays a critical role in fibrosis development after total-body irradiation (TBI). We found that the expression of AREG and fibrotic markers, such as α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1α1), was elevated in the kidneys of 6 Gy TBI mice. Expression of AREG and α-SMA was mainly elevated in the proximal and distal tubules of the kidney in response to TBI, which was confirmed by immunofluorescence staining. Knockdown of Areg mRNA using self-assembled-micelle inhibitory RNA (SAMiRNA) significantly reduced the expression of fibrotic markers, including α-SMA and COL1α1, and inflammatory regulators. Finally, intravenous injections of SAMiRNA targeting mouse Areg mRNA (SAMiRNA-mAREG) diminished radiation-induced collagen accumulation in the renal cortex and medulla. Taken together, the results of the present study suggest that blocking of AREG signaling via SAMiRNA-mAREG treatment could be a promising therapeutic approach to alleviate radiation-induced kidney fibrosis.

Weekly treatment with SAMiRNA targeting the androgen receptor ameliorates androgenetic alopecia.

Androgenetic alopecia (AGA) is the most common type of hair loss in men and women. Dihydrotestosterone (DHT) and androgen receptor (AR) levels are increased in patients with AGA, and DHT-AR signaling correlates strongly with AGA pathogenesis. In this study, treatment with self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticle-type siRNA selectively suppressed AR expression in vitro. Clinical studies with application of SAMiRNA to the scalp and massaging to deliver it to the hair follicle confirmed its efficacy in AGA. For identification of a potent SAMiRNA for AR silencing, 547 SAMiRNA candidates were synthesized and screened. SAMiRNA-AR68 (AR68) was the most potent and could be efficiently delivered to human follicle dermal papilla cells (HFDPCs) and hair follicles, and this treatment decreased the AR mRNA and protein levels. We confirmed that 10 µM AR68 elicits no innate immune response in human PBMCs and no cytotoxicity up to 20 µM with HFDP and HaCaT cells. Clinical studies were performed in a randomized and double-blind manner with two different doses and frequencies. In the low-dose (0.5 mg/ml) clinical study, AR68 was applied three times per week for 24 weeks, and through quantitative analysis using a phototrichogram, we confirmed increases in total hair counts. In the high-dose (5 mg/ml) clinical study, AR68 was given once per week for 24 weeks and showed 83% efficacy in increasing hair counts compared with finasteride. No side effects were observed. Therefore, SAMiRNA targeting AR mRNA is a potential novel topical treatment for AGA.

Hydrochloric acid-treated Bacillus subtilis ghosts induce IL-1 beta, IL-6, and TNF-alpha in murine macrophage.

Background: Bacterial ghosts (BGs) are empty cell envelopes commonly generated using Gram-negative bacteria; they represent a potential platform for efficient adjuvant and vaccine delivery systems. However, the efficient production of BGs from bacteria in a short period of time is challenging. Objective: The purpose of this study was to investigate the possibility of producing BGs in the Gram-positive Bacillus subtilis using various chemicals, and the potential application of BGs as a novel immunomodulatory agent. Results: In this study, Bacillus subtilis ghosts (BSGs) were generated, for the first time to the best of our knowledge, using the minimum inhibitory concentration (MIC) of hydrochloric acid (HCl; 6.25 mg/mL), sulfuric acid (H2SO4; 3.125 mg/mL), and nitric acid (HNO3; 6.25 mg/mL). Among the BSGs generated using these chemicals, HCl-induced BSGs were completely DNA-free as confirmed by real-time polymerase chain reaction. Scanning electron microscopy showed the formation of transmembrane lysis tunnel structures in HCl-induced BSGs. Murine macrophages exposed to the HCl-induced BSGs at a concentration of 1 × 105 CFU/mL showed a cell viability of 97.8%. Additionally, HCl-induced BSGs upregulated the expression of pro-inflammatory cytokines including interleukin (IL)-1ß, tumor necrosis factor alpha, and IL-6. Furthermore, we found differences in the protein expression profiles between intact live bacteria and BSGs using two-dimensional electrophoresis coupled with peptide mass fingerprinting/matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Conclusion: These data suggest that the HCl-induced BSGs may be potentially safe and effective candidates for inactivated bacterial vaccines and/or immunostimulants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13273-022-00221-5.

Genotoxicity evaluation of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG), a novel siRNA nanoparticle for the treatment of fibrotic disease.

The self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG) is a novel small-interfering RNA (siRNA) nanoparticle that is used for treatment of pulmonary fibrosis. We investigated the potential genotoxicity of SAMiRNA-AREG based on the guidelines published by the Organization for Economic Cooperation and Development. In the bacterial reverse mutation assay (Ames test), SAMiRNA-AREG did not induce mutations in Salmonella typhimurium TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA at concentrations of up to 3000 µg/plate with or without metabolic activation. The SAMiRNA-AREG (concentrations up to 500 µg/mL) did not induce chromosomal aberrations in cultured Chinese hamster lung cells with or without metabolic activation. In the in vivo mouse bone marrow micronucleus assay, the SAMiRNA-AREG (concentrations up to 300 mg/kg body weight) did not affect the proportions of polychromatic erythrocytes and total erythrocytes, nor did it increase the number of micronucleated polychromatic erythrocytes in ICR mice. Collectively, these results suggest that SAMiRNA-AREG is safe with regard to genotoxicity such as mutagenesis or clastogenesis under the present experimental conditions. These results might support the safety of SAMiRNA-AREG as a potential therapeutic agent for pharmaceutical development.

Four-Week Repeated Intravenous Dose Toxicity of Self-Assembled-Micelle Inhibitory RNA-Targeting Amphiregulin in Mice.

The present study investigated the potential subchronic toxicity of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG) in mice. The test reagent was administered once-daily by intravenous injection for 4 weeks at 0, 100, 200, or 300 mg/kg/day doses. Additional recovery groups (vehicle control and high dose groups) were observed for a 2-week recovery period. During the test period, mortality, clinical signs, body weight, food consumption, ophthalmology, urinalysis, hematology, serum biochemistry, gross pathology, organ weight, and histopathology were examined. An increase in the percentages of basophil and large unstained cells was observed in the 200 and 300 mg/kg/day groups of both sexes. In addition, the absolute and relative weights of the spleen were higher in males given 300 mg/kg/day relative to the concurrent controls. However, these findings were considered of no toxicological significance because the changes were minimal, were not accompanied by other relevant results (eg, correlating microscopic changes), and were not observed at the end of the 2-week recovery period indicating recovery of the findings. Based on the results, SAMiRNA-AREG did not cause treatment-related adverse effects at dose levels of up to 300 mg/kg/day in mice after 4-week repeated intravenous doses. Under these conditions, the no-observed-adverse-effect level of the SAMiRNA-AREG was ≥300 mg/kg/day in both sexes and no target organs were identified.

The Effect of Lactobacillus gasseri BNR17 on Postmenopausal Symptoms in Ovariectomized Rats.

Clinical and preclinical studies have reported that Lactobacillus gasseri BNR17, a probiotic bacterial strain isolated from human breast milk, reduces body weight and white adipose tissue volume. In order to further explore the actions of L. gasseri BNR17, we investigated the anti-menopausal effects of L. gasseri BNR17 in an ovariectomized (OVX) rat model. The serum alanine aminotransferase levels of the rats in the OVX-BNR17 group were lower than those of the rats in the OVX-vehicle only (OVX-Veh) group. Upon administration of L. gasseri BNR17 after ovariectomy, calcitonin and Serotonin 2A levels increased significantly, whereas serum osteocalcin levels showed a decreasing tendency. Compared to the rats in the OVX-Veh group, those in the OVX-BNR17 group showed lower urine deoxypyridinoline levels, lower pain sensitivity, and improved vaginal cornification. Furthermore, L. gasseri BNR17 administration increased bone mineral density in the rats with OVX-induced femoral bone loss. These results suggest that L. gasseri BNR17 administration could alleviate menopausal symptoms, indicating that this bacterium could be a good functional probiotic for managing the health of older women.

Safety pharmacology of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG), a novel siRNA nanoparticle platform.

The present safety pharmacology core battery studies (neurobehavior, respiratory, cardiovascular system, and human ether a-go-go (hERG) channel current) investigated the potential harmful effects of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG). The SAMiRNA-AREG was administered by single intravenous injection at up to 300 mg/kg and 100 mg/kg in mice and monkeys, respectively. The hERG assay was performed in Chinese hamster ovary (CHO) cells at SAMiRNA-AREG concentrations of up to 200 µg/mL. In the evaluation on neurobehavior, a transient decrease in body temperature was found at 0.5 h (30 min) post-dose at both sexes in mice, with a single 300 mg/kg dose of SAMiRNA-AREG. However, these effects had returned to normal at 1 h post-dose. In the evaluation on hERG channel current, there were statistically significant differences in the inhibition of peak hERG potassium channel current between the 20, 100, and 200 µg/mL SAMiRNA-AREG treatment groups and the vehicle control group. However, these effects were less potent than that of E-4031, a positive control article. For the respiratory and cardiovascular systems, no treatment-related changes were observed in mice or monkeys. Thus, under these experimental conditions, these studies suggest that SAMiRNA-AREG showed no adverse effects on the neurobehavior, respiratory, and cardiovascular function.

In vivo silencing of amphiregulin by a novel effective Self-Assembled-Micelle inhibitory RNA ameliorates renal fibrosis via inhibition of EGFR signals.

Amphiregulin (AREG) is a transmembrane glycoprotein recently implicated in kidney fibrosis. Previously, we reported that the AREG-targeting Self-Assembled-Micelle inhibitory RNA (SAMiRNA-AREG) alleviated fibrosis by stably silencing the AREG gene, and reduced the side effects of conventional siRNA treatment of pulmonary fibrosis. However, the therapeutic effect of SAMiRNA-AREG in renal fibrosis has not been studied until now. We used two animal models of renal fibrosis generated by a unilateral ureteral obstruction (UUO) and an adenine diet (AD) to investigate whether SAMiRNA-AREG inhibited renal fibrosis. To investigate the delivery of SAMiRNA-AREG to the kidney, Cy5-labeled SAMiRNA-AREG was injected into UUO- and AD-induced renal fibrosis models. In both kidney disease models, SAMiRNA-AREG was delivered primarily to the damaged kidney. We also confirmed the protective effect of SAMiRNA-AREG in renal fibrosis models. SAMiRNA-AREG markedly decreased the UUO- and AD-induced AREG mRNA expression. Furthermore, the mRNA expression of fibrosis markers, including α-smooth muscle actin, fibronectin, α1(I) collagen, and α1(III) collagen in the UUO and AD-induced kidneys, was diminished in the SAMiRNA-AREG-treated mice. The transcription of inflammatory markers (tumor necrosis factor-α and monocyte chemoattractant protein-1) and adhesion markers (vascular cell adhesion molecule 1 and intercellular adhesion molecule 1) was attenuated. The hematoxylin and eosin, Masson's trichrome, and immunohistochemical staining results showed that SAMiRNA-AREG decreased renal fibrosis, AREG expression, and epidermal growth factor receptor (EGFR) phosphorylation in the UUO- and AD-induced models. Moreover, we studied the effects of SAMiRNA-AREG in response to TGF-ß1 in mouse and human proximal tubule cells, and mouse fibroblasts. TGF-ß1-induced extracellular matrix production and myofibroblast differentiation were attenuated by SAMiRNA-AREG. Finally, we confirmed that upregulated AREG in the UUO or AD models was mainly localized in the distal tubules. In conclusion, SAMiRNA-AREG represents a novel siRNA therapeutic for renal fibrosis by suppressing EGFR signals.

A Salmonella typhimurium ghost vaccine induces cytokine expression in vitro and immune responses in vivo and protects rats against homologous and heterologous challenges.

Salmonella enteritidis and Salmonella typhimurium are important food-borne bacterial pathogens, which are responsible for diarrhea and gastroenteritis in humans and animals. In this study, S. typhimurium bacterial ghost (STG) was generated based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH). Experimental studies performed using in vitro and in vivo experimental model systems to characterize effects of STG as a vaccine candidate. When compared with murine macrophages (RAW 264.7) exposed to PBS buffer (98.1%), the macrophages exposed to formalin-killed inactivated cells (FKC), live wild-type bacterial cells and NaOH-induced STG at 1 × 108 CFU/mL showed 85.6%, 66.5% and 84.6% cell viability, respectively. It suggests that STG significantly reduces the cytotoxic effect of wild-type bacterial cells. Furthermore, STG is an excellent inducer for mRNAs of pro-inflammatory cytokine (TNF-α, IL-1ß) and factor (iNOS), anti-inflammatory cytokine (IL-10) and dual activities (IL-6) in the stimulated macrophage cells. In vivo, STG vaccine induced humoral and cellular immune responses and protection against homologous and heterologous challenges in rats. Furthermore, the immunogenicity and protective efficacy of STG vaccine were compared with those of FKC and non-vaccinated PBS control groups. The vaccinated rats from STG group exhibited higher levels of serum IgG antibody responses, serum bactericidal antibodies, and CD4+ and CD8+ T-cell populations than those of the FKC and PBS control groups. Most importantly, after challenge with homologous and heterologous strains, the bacterial loads in the STG group were markedly lower than the FKC and PBS control groups. In conclusion, these findings suggest that the STG vaccine induces protective immunity against homologous and heterologous challenges.

A nanofilter composed of carbon nanotube-silver composites for virus removal and antibacterial activity improvement.

We have developed a new nanofilter using a carbon nanotube-silver composite material that is capable of efficiently removing waterborne viruses and bacteria. The nanofilter was subjected to plasma surface treatment to enhance its flow rate, which was improved by approximately 62%. Nanoscale pores were obtained by fabricating a carbon nanotube network and using nanoparticle fixation technology for the removal of viruses. The pore size of the nanofilter was approximately 38 nm and the measured flow rate ranged from 21.0 to 97.2L/(min·m(2)) under a pressure of 1-6 kgf/cm(2) when the amount of loaded carbon nanotube-silver composite was 1.0 mg/cm(2). The nanofilter was tested against Polio-, Noro-, and Coxsackie viruses using a sensitive real-time polymerase chain reaction assay to detect the presence of viral particles within the outflow. No trace of viruses was found to flow through the nanofilter with carbon nanotube-silver composite loaded above 0.8 mg/cm(2). Moreover, the surface of the filter has antibacterial properties to prevent bacterial clogging due to the presence of 20-nm silver nanoparticles, which were synthesized on the carbon nanotube surface.

Self-assembled Micelle Interfering RNA for Effective and Safe Targeting of Dysregulated Genes in Pulmonary Fibrosis.

The siRNA silencing approach has long been used as a method to regulate the expression of specific target genes in vitro and in vivo. However, the effectiveness of delivery and the nonspecific immune-stimulatory function of siRNA are the limiting factors for therapeutic applications of siRNAs. To overcome these limitations, we developed self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticles made of individually biconjugated siRNAs with a hydrophilic polymer and lipid on their ends and characterized their stability, immune-stimulatory function, and in vivo silencing efficacy. SAMiRNAs form very stable nanoparticles with no significant degradation in size distribution and polydispersity index over 1 year. Overnight incubation of SAMiRNAs (3 µm) on murine peripheral blood mononuclear cells did not cause any significant elaboration of innate immune cytokines such as TNF-α, IL-12, or IL-6, whereas unmodified siRNAs or liposomes or liposome complexes significantly stimulated the expression of these cytokines. Last, the in vivo silencing efficacy of SAMiRNAs was evaluated by targeting amphiregulin and connective tissue growth factor in bleomycin or TGF-ß transgenic animal models of pulmonary fibrosis. Intratracheal or intravenous delivery two or three times of amphiregulin or connective tissue growth factor SAMiRNAs significantly reduced the bleomycin- or TGF-ß-stimulated collagen accumulation in the lung and substantially restored the lung function of TGF-ß transgenic mice. This study demonstrates that SAMiRNA nanoparticle is a less toxic, stable siRNA silencing platform for efficient in vivo targeting of genes implicated in the pathogenesis of pulmonary fibrosis.

Identification of a Mitochondrial DNA Polymerase Affecting Cardiotoxicity of Sunitinib Using a Genome-Wide Screening on S. pombe Deletion Library.

Drug toxicity is a key issue for drug R&D, a fundamental challenge of which is to screen for the targets genome-wide. The anticancer tyrosine kinase inhibitor sunitinib is known to induce cardiotoxicity. Here, to understand the molecular insights of cardiotoxicity by sunitinib at the genome level, we used a genome-wide drug target screening technology (GPScreen) that measures drug-induced haploinsufficiency (DIH) in the fission yeast Schizosaccharomyces pombe genome-wide deletion library and found a mitochondrial DNA polymerase (POG1). In the results, sunitinib induced more severe cytotoxicity and mitochondrial damage in POG1-deleted heterozygous mutants compared to wild type (WT) of S. pombe. Furthermore, knockdown of the human ortholog POLG of S. pombe POG1 in human cells significantly increased the cytotoxicity of sunitinib. Notably, sunitinib dramatically decreased the levels of POLG mRNAs and proteins, of which downregulation was already known to induce mitochondrial damage of cardiomyocytes, causing cardiotoxicity. These results indicate that POLG might play a crucial role in mitochondrial damage as a gene of which expressional pathway is targeted by sunitinib for cardiotoxicity, and that genome-wide drug target screening with GPScreen can be applied to drug toxicity target discovery to understand the molecular insights regarding drug toxicity.

Systematic targeted gene deletion using the gene-synthesis method in fission yeast.

Genome-wide targeted gene deletion, a systematic method to study gene function by replacing target genes with deletion cassettes, using serial-PCR or block-PCR requires elaborate skill. We developed a novel gene-synthesis method to systematically prepare deletion cassettes on a 96-well basis in fission yeast. We designed the 2129-bp deletion cassette as three modules: a central 1397-bp KanMX4 selection marker module and two flanking 366-bp gene-specific artificial linker modules. The central KanMX4 module can be used in multiple deletion cassettes in combination with different sets of flanking modules. The deletion cassettes consisted of 147 oligonucleotides (93 for the central module+25 for each of the flanking modules+4 for the joints) and the oligonucleotides were designed as ~29mers using an in-house program. Oligonucleotides were synthesized on a 96-well basis and ligated into deletion cassettes without gaps by ligase chain reaction, which was followed by two rounds of nested PCR to amplify trace amounts of the ligated cassettes. After the artificial linkers were removed from the deletion cassettes, the cassettes were transformed into wild-type diploid fission yeast strain SP286. We validated the transformed colonies via check PCR and subjected them to tetrad analysis to confirm functional integrity. Using this method, we systematically deleted 563 genes in the fission yeast Schizosaccharomyces pombe with a >90% success rate and a point-mutation rate of ~0.4 mutations per kb. Our method can be used to create systematic gene deletions in a variety of yeasts especially when it included a bar-code system for parallel analyses.

Construction of the first compendium of chemical-genetic profiles in the fission yeast Schizosaccharomyces pombe and comparative compendium approach.

Genome-wide chemical genetic profiles in Saccharomyces cerevisiae since the budding yeast deletion library construction have been successfully used to reveal unknown mode-of-actions of drugs. Here, we introduce comparative approach to infer drug target proteins more accurately using two compendiums of chemical-genetic profiles from the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe. For the first time, we established DNA-chip based growth defect measurement of genome-wide deletion strains of S. pombe, and then applied 47 drugs to the pooled heterozygous deletion strains to generate chemical-genetic profiles in S. pombe. In our approach, putative drug targets were inferred from strains hypersensitive to given drugs by analyzing S. pombe and S. cerevisiae compendiums. Notably, many evidences in the literature revealed that the inferred target genes of fungicide and bactericide identified by such comparative approach are in fact the direct targets. Furthermore, by filtering out the genes with no essentiality, the multi-drug sensitivity genes, and the genes with less eukaryotic conservation, we created a set of drug target gene candidates that are expected to be directly affected by a given drug in human cells. Our study demonstrated that it is highly beneficial to construct the multiple compendiums of chemical genetic profiles using many different species. The fission yeast chemical-genetic compendium is available at http://pombe.kaist.ac.kr/compendium.