Exploratory study of cold hypersensitivity in Japanese women: genetic associations and somatic symptom burden.

Temperature perception is essential for humans to discern the environment and maintain homeostasis. However, some individuals experience cold hypersensitivity, characterized by a subjective feeling of coldness despite ambient environmental temperatures being normal, the underlying mechanisms of which are unknown. In this study, we aimed to investigate the relationship between subjective cold symptoms and somatic burden or single nucleotide polymorphisms to understand the causes of cold hypersensitivity. We conducted an online questionnaire survey [comprising 30 questions, including past medical history, subjective symptoms of cold hypersensitivity, and the Somatic Symptom Scale-8 (SSS-8)]. Respondents were 1200 Japanese adult female volunteers (age: 20-59 years), recruited between April 21 and May 25, 2022, who were customers of MYCODE, a personal genome service in Japan. Among the 1111 participants, 599 (54%) reported cold hypersensitivity. Higher cold hypersensitivity severity was positively associated with the SSS-8 scores. Additionally, a genome-wide association study for cold hypersensitivity was conducted using array-based genomic data obtained from genetic testing. We identified 11 lead variants showing suggestive associations (P < 1 × 10-5) with cold hypersensitivity, some of which showed a reasonable change in expression in specific tissues in the Genotype-Tissue Expression database. The study findings shed light on the underlying causes of cold hypersensitivity.

Newly developed preclinical models reveal broad-spectrum CDK inhibitors as potent drugs for CRPC exhibiting primary resistance to enzalutamide.

Androgen-deprivation therapy is a standard treatment for advanced prostate cancer. However, most patients eventually acquire resistance and progress to castration-resistant prostate cancer (CRPC). In this study, we established new CRPC cell lines, AILNCaP14 and AILNCaP15, from LNCaP cells under androgen-deprived conditions. Unlike most pre-existing CRPC cell lines, both cell lines expressed higher levels of androgen receptor (AR) and prostate-specific antigen (PSA) than parental LNCaP cells. Moreover, these cells exhibited primary resistance to enzalutamide. Since AR signaling plays a significant role in the development of CRPC, PSA promoter sequences fused with GFP were introduced into AILNCaP14 cells to conduct GFP fluorescence-based chemical screening. We identified flavopiridol, a broad-spectrum CDK inhibitor, as a candidate drug that could repress AR transactivation of CRPC cells, presumably through the inhibition of phosphorylation of AR on the serine 81 residue (pARSer81 ). Importantly, this broad-spectrum CDK inhibitor inhibited the proliferation of AILNCaP14 cells both in vitro and in vivo. Moreover, a newly developed liver metastatic model using AILNCaP15 cells revealed that the compound attenuated tumor growth of CRPC harboring highly metastatic properties. Finally, we developed a patient-derived xenograft (PDX) model of CRPC and DCaP CR from a patient presenting therapeutic resistance to enzalutamide, abiraterone, and docetaxel. Flavopiridol successfully suppressed the tumor growth of CRPC in this PDX model. Since ARSer81 was found to be phosphorylated in clinical CRPC samples, our data suggested that broad-spectrum CDK inhibitors might be a potent candidate drug for the treatment of CRPC, including those exhibiting primary resistance to enzalutamide.

Data of RNA-seq transcriptomes of liver, bone, heart, kidney and blood in klotho mice at a pre-symptomatic state and the effect of a traditional Japanese multi-herbal medicine, juzentaihoto.

We performed RNA-seq analyses of mRNA isolated from five organs, liver, bone, heart, kidney and blood at the pre-symptomatic state of klotho mice with/without administration of a Japanese traditional herbal medicine, juzentaihoto (JTT). Data of differentially expressed genes (DEG) with/without JTT was included. Intron retention (IR) is an important regulatory mechanism that affects gene expression and protein functions. We collected data in which retained-introns were accumulated in a particular set of genes of these organs, and showed that among these retained introns in the liver and bone a subset was recovered to the normal state by the medicine. All of the data present changes of molecular events on the levels of metabolites, proteins and gene expressions observed at the pre- symptomatic state of aging in klotho mice with/without JTT. The research article related to this Data in Brief is published in GENE entitled as "Intron retention as a new pre-symptomatic marker of aging and its recovery to the normal state by a traditional Japanese herbal medicine".

SNAIL2 contributes to tumorigenicity and chemotherapy resistance in pancreatic cancer by regulating IGFBP2.

Pancreatic cancer has an extremely poor prognosis because of its resistance to conventional therapies. Cancer stem cell (CSC)-targeted therapy is considered a promising approach for this disease. Epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) contribute to CSC properties in some solid tumors; however, this mechanism has not been fully elucidated in pancreatic cancer. Zinc finger protein, SNAIL2 (also known as SLUG), is a member of the SNAIL superfamily of EMT-TFs and is commonly overexpressed in pancreatic cancer. Patients exhibiting high SNAIL2 expression have a poor prognosis. In this study, we showed that the suppression of SNAIL2 expression using RNA interference decreased tumorigenicity in vitro (sphere formation assay) and in vivo (xenograft assay) in 2 pancreatic cancer cell lines, KLM1 and KMP5. In addition, SNAIL2 suppression resulted in increased sensitivity to gemcitabine and reduced the expression of CD44, a pancreatic CSC marker. Moreover, experiments on tumor spheroids established from surgically resected pancreatic cancer tissues yielded similar results. A microarray analysis revealed that the mechanism was mediated by insulin-like growth factor (IGF) binding protein 2. These results indicate that IGFBP2 regulated by SNAIL2 may represent an effective therapeutic target for pancreatic cancer.

Intron retention as a new pre-symptomatic marker of aging and its recovery to the normal state by a traditional Japanese multi-herbal medicine.

Intron retention (IR) is an important regulatory mechanism that affects gene expression and protein functions. Using klotho mice at the pre-symptomatic state, we discovered that retained-introns accumulated in several organs including the liver and that among these retained introns in the liver a subset was recovered to the normal state by a Japanese traditional herbal medicine. This is the first report of IR recovery by a medicine. IR-recovered genes fell into two categories: those involved in liver-specific metabolism and in splicing. Metabolome analysis of the liver showed that the klotho mice were under starvation stress. In addition, our differentially expressed gene analysis showed that liver metabolism was actually recovered by the herbal medicine at the transcriptional level. By analogy with the widespread accumulation of intron-retained pre-mRNAs induced by heat shock stress, we propose a model in which retained-introns in klotho mice were induced by an aging stress and in which this medicine-related IR recovery is indicative of the actual recovery of liver-specific metabolic function to the healthy state. Accumulation of retained-introns was also observed at the pre-symptomatic state of aging in wild-type mice and may be an excellent marker for this state in general.

Effects of maoto (ma-huang-tang) on host lipid mediator and transcriptome signature in influenza virus infection.

Maoto, a traditional kampo medicine, has been clinically prescribed for influenza infection and is reported to relieve symptoms and tissue damage. In this study, we evaluated the effects of maoto as an herbal multi-compound medicine on host responses in a mouse model of influenza infection. On the fifth day of oral administration to mice intranasally infected with influenza virus [A/PR/8/34 (H1N1)], maoto significantly improved survival rate, decreased viral titer, and ameliorated the infection-induced phenotype as compared with control mice. Analysis of the lung and plasma transcriptome and lipid mediator metabolite profile showed that maoto altered the profile of lipid mediators derived from ω-6 and ω-3 fatty acids to restore a normal state, and significantly up-regulated the expression of macrophage- and T-cell-related genes. Collectively, these results suggest that maoto regulates the host's inflammatory response by altering the lipid mediator profile and thereby ameliorating the symptoms of influenza.

Allelic composition of carotenoid metabolic genes in 13 founders influences carotenoid composition in juice sac tissues of fruits among Japanese citrus breeding population.

To enrich carotenoids, especially ß-cryptoxanthin, in juice sac tissues of fruits via molecular breeding in citrus, allele mining was utilized to dissect allelic variation of carotenoid metabolic genes and identify an optimum allele on the target loci characterized by expression quantitative trait (eQTL) analysis. SNPs of target carotenoid metabolic genes in 13 founders of the Japanese citrus breeding population were explored using the SureSelect target enrichment method. An independent allele was determined based on the presence or absence of reliable SNPs, using trio analysis to confirm inheritability between parent and offspring. Among the 13 founders, there were 7 PSY alleles, 7 HYb alleles, 11 ZEP alleles, 5 NCED alleles, and 4 alleles for the eQTL that control the transcription levels of PDS and ZDS among the ancestral species, indicating that some founders acquired those alleles from them. The carotenoid composition data of 263 breeding pedigrees in juice sac tissues revealed that the phenotypic variance of carotenoid composition was similar to that in the 13 founders, whereas the mean of total carotenoid content increased. This increase in total carotenoid content correlated with the increase in either or both ß-cryptoxanthin and violaxanthin in juice sac tissues. Bayesian statistical analysis between allelic composition of target genes and carotenoid composition in 263 breeding pedigrees indicated that PSY-a and ZEP-e alleles at PSY and ZEP loci had strong positive effects on increasing the total carotenoid content, including ß-cryptoxanthin and violaxanthin, in juice sac tissues. Moreover, the pyramiding of these alleles also increased the ß-cryptoxanthin content. Interestingly, the offset interaction between the alleles with increasing and decreasing effects on carotenoid content and the epistatic interaction among carotenoid metabolic genes were observed and these interactions complexed carotenoid profiles in breeding population. These results revealed that allele composition would highly influence the carotenoid composition in citrus fruits. The allelic genotype information for the examined carotenoid metabolic genes in major citrus varieties and the trio-tagged SNPs to discriminate the optimum alleles (PSY-a and ZEP-e) from the rest would promise citrus breeders carotenoid enrichment in fruit via molecular breeding.

SNAIL regulates gastric carcinogenesis through CCN3 and NEFL.

Among cancer cells, there are specific cell populations of whose activities are comparable to those of stem cells in normal tissues, and for whom the levels of cell dedifferentiation are reported to correlate with poor prognosis. Information concerning the mechanisms that modulate the stemness like traits of cancer cells is limited. Therefore, we examined five gastric cancer cell lines and isolated gastric oncospheres from three gastric cancer cell lines. The gastric cancer cells that expanded in the spheres expressed relatively elevated proportion of CD44, which is a marker of gastric cancer stem cells (CSCs), and displayed many properties of CSCs, for example: chemoresistance, tumorigenicity and epithelial-mesenchymal transition (EMT) acquisition. SNAIL, which is a key factor in EMT, was highly expressed in the gastric spheres. Microarray analysis in gastric cancer cell line HGC27 showed that CCN3 and NEFL displayed the greatest differential expression by knocking down of SNAIL; the former was upregulated and the latter downregulated, respectively. Downregulation of CCN3 and upregulation of NEFL gene expression impaired the SNAIL-dependent EMT activity: high tumorigenicity, and chemoresistance in gastric cancer cells. Thus, approach that disrupts SNAIL/CCN3/NEFL axis may be credible in inhibiting gastric cancer development.

Promoter-Level Transcriptome Identifies Stemness Associated With Relatively High Proliferation in Pancreatic Cancer Cells.

Both pancreatic intraepithelial neoplasia (PanIN), a frequent precursor of pancreatic cancer, and intraductal papillary mucinous neoplasm (IPMN), a less common precursor, undergo several phases of molecular conversions and finally develop into highly malignant solid tumors with negative effects on the quality of life. We approached this long-standing issue by examining the following PanIN/IPMN cell lines derived from mouse models of pancreatic cancer: Ptf1a-Cre; KrasG12D; p53f/+ and Ptf1a-Cre; KrasG12D; and Brg1f/f pancreatic ductal adenocarcinomas (PDAs). The mRNA from these cells was subjected to a cap analysis of gene expression (CAGE) to map the transcription starting sites and quantify the expression of promoters across the genome. Two RNA samples extracted from three individual subcutaneous tumors generated by the transplantation of PanIN or IPMN cancer cell lines were used to generate libraries and Illumina Seq, with four RNA samples in total, to depict discrete transcriptional network between IPMN and PanIN. Moreover, in IPMN cells, the transcriptome tended to be enriched for suppressive and inhibitory biological processes. In contrast, the transcriptome of PanIN cells exhibited properties of stemness. Notably, the proliferation capacity of the latter cells in culture was only minimally constrained by well-known chemotherapy drugs such as GSK690693 and gemcitabine. The various transcriptional factor network systems detected in PanIN and IPMN cells reflect the distinct molecular profiles of these cell types. Further, we hope that these findings will enhance our mechanistic understanding of the characteristic molecular alterations underlying pancreatic cancer precursors. These data may provide a promising direction for therapeutic research.

Clinical significance of TP53 variants as possible secondary findings in tumor-only next-generation sequencing.

In tumor-only next-generation sequencing (NGS), identified variants have the potential to be secondary findings (SFs), but they require verification through additional germline testing. In the present study, 194 patients with advanced cancer who underwent tumor-only NGS between April 2015 and March 2018 were enrolled, and the incidences of possible and true SFs were evaluated. Among them, 120 patients (61.9%) harbored at least one possible SF. TP53 was the most frequent gene in which 97 variants were found in 91 patients (49.5%). Nine patients provided informed consent to undergo additional germline testing, and a total of 14 variants (BRCA1, n = 1; BRCA2, n = 2; PTEN, n = 2; RB1, n = 1; SMAD4, n = 1; STK11, n = 1; TP53, n = 6) were analyzed. Three variants (BRCA1, n = 1; BRCA2, n = 2) were confirmed to be SFs, whereas TP53 variants were confirmed to be somatic variants. To confirm the low prevalence of SFs in TP53, we analyzed 24 patients with TP53 variants who underwent a paired tumor-normal NGS assay. As expected, all TP53 variants were confirmed to be somatic variants. A total of 30 patients were tested for germline variants in TP53, but none of them resulted in true SFs, suggesting the low prevalence of SFs in this gene. Therefore, the significance of additional germline testing for TP53 variants appears to be relatively low in daily clinical practice using a tumor-only NGS assay, unless patients have any relevant medical or family history.

Identification of modules and functional analysis in CRC subtypes by integrated bioinformatics analysis.

Colorectal cancer is one of the top three causes of cancer-related mortality globally, but no predictive molecular biomarkers are currently available for identifying the disease stage of colorectal cancer patients. Common molecular patterns in the disease, beyond superficial manifestations, can be significant in determining treatment choices. In this study, we used microarray data from colorectal cancer and adjacent normal tissue from the GEO database. These data were categorized into four consensus molecular subtypes based on distinct gene expression signatures. Weighted gene-based protein-protein interaction network analysis was performed for each subtype. NUSAP1, CD44, and COL4A1 modules were found to be statistically significant and present among all the subtypes and displayed though similar but not identical functional enrichment results. Reference of the characteristics of the subtypes to functional modules is necessary since the latter can stay resistant to platform changes and technique noise when compared with other analyses. The CMS4-mesenchymal group, which currently has a poor prognosis, was examined in the study. It is composed mainly of genes involved in immune and stromal expression, with modules focused on ECM dysregulation and chemokine biological processes. Hub genes detection and its' mapping into the protein-protein interaction network can be indicative of possible targets against specific modules. This approach identified subtypes using enrichment-oriented analysis in functional modules. Proper annotation of functional analysis of modules from different subtypes of CRC might be directive for finding extra options for treatment targets and guiding clinical routines.

TFE3 Xp11.2 Translocation Renal Cell Carcinoma Mouse Model Reveals Novel Therapeutic Targets and Identifies GPNMB as a Diagnostic Marker for Human Disease.

Renal cell carcinoma (RCC) associated with Xp11.2 translocation (TFE3-RCC) has been recently defined as a distinct subset of RCC classified by characteristic morphology and clinical presentation. The Xp11 translocations involve the TFE3 transcription factor and produce chimeric TFE3 proteins retaining the basic helix-loop-helix leucine zipper structure for dimerization and DNA binding suggesting that chimeric TFE3 proteins function as oncogenic transcription factors. Diagnostic biomarkers and effective forms of therapy for advanced cases of TFE3-RCC are as yet unavailable. To facilitate the development of molecular based diagnostic tools and targeted therapies for this aggressive kidney cancer, we generated a translocation RCC mouse model, in which the PRCC-TFE3 transgene is expressed specifically in kidneys leading to the development of RCC with characteristic histology. Expression of the receptor tyrosine kinase Ret was elevated in the kidneys of the TFE3-RCC mice, and treatment with RET inhibitor, vandetanib, significantly suppressed RCC growth. Moreover, we found that Gpnmb (Glycoprotein nonmetastatic B) expression was notably elevated in the TFE3-RCC mouse kidneys as seen in human TFE3-RCC tumors, and confirmed that GPNMB is the direct transcriptional target of TFE3 fusions. While GPNMB IHC staining was positive in 9/9 cases of TFE3-RCC, Cathepsin K, a conventional marker for TFE3-RCC, was positive in only 67% of cases. These data support RET as a potential target and GPNMB as a diagnostic marker for TFE3-RCC. The TFE3-RCC mouse provides a preclinical in vivo model for the development of new biomarkers and targeted therapeutics for patients affected with this aggressive form of RCC. IMPLICATIONS: Key findings from studies with this preclinical mouse model of TFE3-RCC underscore the potential for RET as a therapeutic target for treatment of patients with TFE3-RCC, and suggest that GPNMB may serve as diagnostic biomarker for TFE3 fusion RCC.

MITE insertion-dependent expression of CitRKD1 with a RWP-RK domain regulates somatic embryogenesis in citrus nucellar tissues.

BACKGROUND: Somatic embryogenesis in nucellar tissues is widely recognized to induce polyembryony in major citrus varieties such as sweet oranges, satsuma mandarins and lemons. This capability for apomixis is attractive in agricultural production systems using hybrid seeds, and many studies have been performed to elucidate the molecular mechanisms of various types of apomixis. To identify the gene responsible for somatic embryogenesis in citrus, a custom oligo-DNA microarray including predicted genes in the citrus polyembryonic locus was used to compare the expression profiles in reproductive tissues between monoembryonic and polyembryonic varieties. The full length of CitRKD1, which was identified as a candidate gene responsible for citrus somatic embryogenesis, was isolated from satsuma mandarin and its molecular function was investigated using transgenic 'Hamlin' sweet orange by antisense-overexpression. RESULTS: The candidate gene CitRKD1, predominantly transcribed in reproductive tissues of polyembryonic varieties, is a member of the plant RWP-RK domain-containing protein. CitRKD1 of satsuma mandarin comprised two alleles (CitRKD1-mg1 and CitRKD1-mg2) at the polyembryonic locus controlling embryonic type (mono/polyembryony) that were structurally divided into two types with or without a miniature inverted-repeat transposable element (MITE)-like insertion in the upstream region. CitRKD1-mg2 with the MITE insertion was the predominant transcript in flowers and young fruits where somatic embryogenesis of nucellar cells occurred. Loss of CitRKD1 function by antisense-overexpression abolished somatic embryogenesis in transgenic sweet orange and the transgenic T1 plants were confirmed to derive from zygotic embryos produced by self-pollination by DNA diagnosis. Genotyping PCR analysis of 95 citrus traditional and breeding varieties revealed that the CitRKD1 allele with the MITE insertion (polyembryonic allele) was dominant and major citrus varieties with the polyembryonic allele produced polyembryonic seeds. CONCLUSION: CitRKD1 at the polyembryonic locus plays a principal role in regulating citrus somatic embryogenesis. CitRKD1 comprised multiple alleles that were divided into two types, polyembryonic alleles with a MITE insertion in the upstream region and monoembryonic alleles without it. CitRKD1 was transcribed in reproductive tissues of polyembryonic varieties with the polyembryonic allele. The MITE insertion in the upstream region of CitRKD1 might be involved in regulating the transcription of CitRKD1.

Phospholipase D2 promotes disease progression of renal cell carcinoma through the induction of angiogenin.

A hallmark of clear cell renal cell carcinoma (ccRCC) is the presence of intracellular lipid droplets (LD) and it is assumed that phosphatidic acid (PA) produced by phospholipase D (PLD) plays some role in the LD formation. However, little is known about the significance of PLD in ccRCC. In this study, we examined the expression levels of PLD in ccRCC. The classical mammalian isoforms of PLD are PLD1 and PLD2, and the levels of both mRNA were higher at the primary tumor sites than in normal kidney tissues. Similarly, both PLD were significantly abundant in tumor cells as determined by analysis using immunohistochemical staining. Importantly, a higher level of PLD was significantly associated with a higher tumor stage and grade. Because PLD2 knockdown effectively suppressed the cell proliferation and invasion of ccRCC as compared with PLD1 in vitro, we examined the effect of PLD2 in vivo. Notably, shRNA-mediated knockdown of PLD2 suppressed the growth and invasion of tumors in nude mouse xenograft models. Moreover, the higher expression of PLD2 was significantly associated with poorer prognosis in 67 patients. As for genes relating to the tumor invasion of PLD2, we found that angiogenin (ANG) was positively regulated by PLD2. In fact, the expression levels of ANG were elevated in tumor tissues as compared with normal kidney and the inhibition of ANG activity with a neutralizing antibody significantly suppressed tumor invasion. Overall, we revealed for the first time that PLD2-produced PA promoted cell invasion through the expression of ANG in ccRCC cells.

Snail promotes ovarian cancer progression by recruiting myeloid-derived suppressor cells via CXCR2 ligand upregulation.

Snail is a major transcriptional factor that induces epithelial-mesenchymal transition (EMT). In this study, we explore the effect of Snail on tumor immunity. Snail knockdown in mouse ovarian cancer cells suppresses tumor growth in immunocompetent mice, associated with an increase of CD8+ tumor-infiltrating lymphocytes and a decrease of myeloid-derived suppressor cells (MDSCs). Snail knockdown reduces the expression of CXCR2 ligands (CXCL1 and CXCL2), chemokines that attract MDSCs to the tumor via CXCR2. Snail upregulates CXCR ligands through NF-kB pathway, and most likely, through direct binding to the promoters. A CXCR2 antagonist suppresses MDSC infiltration and delays tumor growth in Snail-expressing mouse tumors. Ovarian cancer patients show elevated serum CXCL1/2, which correlates with Snail expression, MDSC infiltration, and short overall survival. Thus, Snail induces cancer progression via upregulation of CXCR2 ligands and recruitment of MDSCs. Blocking CXCR2 represents an immunological therapeutic approach to inhibit progression of Snail-high tumors undergoing EMT.

Allelic diversity of phytoene synthase gene influences the transcription level in citrus fruit among a citrus F1 hybrid population.

Phytoene synthase (PSY) is one of the key regulatory enzyme on the biosynthesis and accumulation of carotenoid in citrus fruits. The transcriptional diversity of PSY is mainly attributed to the structural variation in promoter region among PSY alleles. In aim to clarify how this transcriptional diversity is regulated among them, PSY alleles responsible for carotenoid biosynthesis in the fruits are characterized and their promoter sequences were compared. Based on gene structure and expression pattern of PSY homologues on the clementine mandarin genome sequence, PSY alleles responsible for carotenoid biosynthesis are derived from a single locus in the scaffold 6. AG mapping population possessed four PSY alleles derived from parent lines of A255 and G434, and their F1 individuals with PSY-g2 allele tended to have low transcription level. From sequence comparison of their promoter regions, the cis-motif alternation from MYBPZM to RAV1AAT might be a candidate to influence the transcription level. Among the ancestral pedigree varieties of AG mapping population, the transcription level of PSY correlated with genotypes of MYBPZM and RAV1AAT motifs in the promoter region of PSY alleles, so that homozygous genotype of MYBPZM showed higher transcription level while heterozygous genotype of MYBPZM and RAV1AAT showed lower transcription level.

Clinical sequencing using a next-generation sequencing-based multiplex gene assay in patients with advanced solid tumors.

Advances in next-generation sequencing (NGS) technologies have enabled physicians to test for genomic alterations in multiple cancer-related genes at once in daily clinical practice. In April 2015, we introduced clinical sequencing using an NGS-based multiplex gene assay (OncoPrime) certified by the Clinical Laboratory Improvement Amendment. This assay covers the entire coding regions of 215 genes and the rearrangement of 17 frequently rearranged genes with clinical relevance in human cancers. The principal indications for the assay were cancers of unknown primary site, rare tumors, and any solid tumors that were refractory to standard chemotherapy. A total of 85 patients underwent testing with multiplex gene assay between April 2015 and July 2016. The most common solid tumor types tested were pancreatic (n = 19; 22.4%), followed by biliary tract (n = 14; 16.5%), and tumors of unknown primary site (n = 13; 15.3%). Samples from 80 patients (94.1%) were successfully sequenced. The median turnaround time was 40 days (range, 18-70 days). Potentially actionable mutations were identified in 69 of 80 patients (86.3%) and were most commonly found in TP53 (46.3%), KRAS (23.8%), APC (18.8%), STK11 (7.5%), and ATR (7.5%). Nine patients (13.0%) received a subsequent therapy based on the NGS assay results. Implementation of clinical sequencing using an NGS-based multiplex gene assay was feasible in the clinical setting and identified potentially actionable mutations in more than 80% of patients. Current challenges are to incorporate this genomic information into better therapeutic decision making.

Overexpression of a citrus basic helix-loop-helix transcription factor (CubHLH1), which is homologous to Arabidopsis activation-tagged bri1 suppressor 1 interacting factor genes, modulates carotenoid metabolism in transgenic tomato.

To explore the transcription factors associated with carotenoid metabolism in citrus fruit, one transcription factor (CubHLH1) was selected through microarray screening in Satsuma mandarin (Citrus unshiu Marc.) fruit, which was treated with exogenous ethylene or gibberellin (GA), accelerating or retarding carotenoid accumulation in peel, respectively. The amino acid sequence of CubHLH1 has homology to Arabidopsis activation-tagged bri1 suppressor 1 (ATBS1) interacting factor (AIF), which is functionally characterized as a negative regulator of the brassinolide (BR) signalling pathway. Yeast two-hybrid analysis revealed that protein for CubHLH1 could interact with Arabidopsis and tomato ATBS1. Overexpression of CubHLH1 caused a dwarf phenotype in transgenic tomato (Solanum lycopersicum L.), suggesting that CubHLH1 has a similar function to Arabidopsis AIF. In the transgenic tomato fruit at ripening stage, the lycopene content was reduced along with the changes in carotenoid biosynthetic gene expression. The abscisic acid (ABA) content of all the transgenic tomato fruit was higher than that of the wild type. These results implied that CubHLH1 is considered to have a similar function to Arabidopsis AIFs and might be directly involved in carotenoid metabolism in mature citrus fruit.

Pthlh, a promising cancer modifier gene in rat tongue carcinogenesis.

Susceptibly to the induction of rat tongue cancer (TC) by oral 4-nitroquinoline 1-oxide (4NQO) exposure is a polygenic trait. Among several quantitative trait loci identified by crosses between TC-susceptible Dark Agouti (DA) rats and TC-resistant Wistar-Furth (WF) rats, we focused on tongue cancer susceptibility locus (Tcas3) of chromosome 4. We examined tongue carcinogenesis in the reciprocal congenic strains DA.WF-Tcas3 and WF.DA-Tcas3 and in their parental strains. The Tcas3DA allele, and not the Tcas3WF allele, significantly favored tumor latency, incidence and TC number/size. In genomic DNA of TCs induced in (DA x WF) F1 rats, the resistant Tcas3WF allele was frequently and selectively lost, particularly in larger tumors. Thus, we searched the possible candidate genes in the Tcas3 region using microarray analysis of TCs in F1 rats and revealed significant upregulation of 2 cancer-related genes, parathyroid hormone-like hormone (Pthlh) and Kras2. The relevance of the WF allele of Pthlh as a cancer modifier was indicated by 3 single nucleotide polymorphisms specific to this strain. In contrast, no consistent strain-specific variations were found in Kras2. Moreover, the plasma Ca2+ level was consistently higher in DA rats when compared to the level in WF rats bearing TCs; moreover, the Pthlh-mRNA expression level was >30-fold higher in TCs when compared to this level in the normal tongue mucosa. Immunostaining experiments showed strong PTHrP protein expression in TCs of DA rats, and the signal was intensified in larger TCs. Kras2 was also upregulated in TCs, but to a lesser degree than PTHrP. Thus, Pthlh is a promising candidate modifier gene in the development and progression of rat TCs.

Quinoline-3-carbothioamides and related compounds as novel immunomodulating agents.

A series of quinoline-3-carbothioamides and their analogues was prepared via four synthetic routes and evaluated for their antinephritic and immunomodulating activities. The optimal compound 9g strongly inhibited the T-cell independent antibody production in mice immunized with TNP-LPS and was highly effective in two nephritis models, namely chronic graft-versus-host disease and autoimmune MRL/l mice.