Exome-wide benchmark of difficult-to-sequence regions using short-read next-generation DNA sequencing.

Next-generation DNA sequencing (NGS) in short-read mode has recently been used for genetic testing in various clinical settings. NGS data accuracy is crucial in clinical settings, and several reports regarding quality control of NGS data, primarily focusing on establishing NGS sequence read accuracy, have been published thus far. Variant calling is another critical source of NGS errors that remains unexplored at the single-nucleotide level despite its established significance. In this study, we used a machine-learning-based method to establish an exome-wide benchmark of difficult-to-sequence regions at the nucleotide-residue resolution using 10 genome sequence features based on real-world NGS data accumulated in The Genome Aggregation Database (gnomAD) of the human reference genome sequence (GRCh38/hg38). The newly acquired metric, designated the 'UNMET score,' along with additional lines of structural information from the human genome, allowed us to assess the sequencing challenges within the exonic region of interest using conventional short-read NGS. Thus, the UNMET score could provide a basis for addressing potential sequential errors in protein-coding exons of the human reference genome sequence GRCh38/hg38 in clinical sequencing.

RNA Oncological Therapeutics: Intracellular Hairpin RNA Assembly Enables MicroRNA-Triggered Anticancer Functionality.

RNA therapeutics are of global interest because of their versatility in targeting a variety of intracellular and extracellular biomolecules. In that context, long double-stranded RNA (dsRNA) has been studied as an antitumor agent that activates the immune response. However, its performance is constrained by poor cancer selectivity and cell-penetration ability. Here, we designed and synthesized an oncolytic RNA hairpin pair (oHP) that was selectively cytotoxic toward cancer cells expressing abundant oncogenic microRNA-21 (miR-21). Although the structure of each hairpin RNA was thermodynamically metastable, catalytic miR-21 input triggered it to open to generate a long nicked dsRNA. We demonstrated that oHP functioned as a cytotoxic amplifier of information in the presence of miR-21 in various cancer cells and tumor-bearing mice. This work represents the first example of the use of short RNA molecules as build-up-type anticancer agents that are triggered by an oncogenic miRNA.

Androgen receptor suppresses ß-adrenoceptor-mediated CREB activation and thermogenesis in brown adipose tissue of male mice.

Thermoregulation is a process by which core body temperature is maintained in mammals. Males typically have a lower body temperature than females. However, the effects of androgens, which show higher levels in males, on adrenergic receptor-mediated thermogenesis remain unclear. Here, we demonstrate that androgen-androgen receptor (AR) signaling suppresses the ß-adrenergic agonist-induced rise of core body temperature using castrated and AR knockout (ARKO) male mice. Furthermore, in vitro mechanistic studies show that activated AR inhibits cAMP response element (CRE)-mediated transcription by suppressing cAMP response element-binding protein (CREB) phosphorylation. The elevation of body temperature induced by the ß-adrenergic agonist CL316243 was higher in ARKO and castrated mice than in the control mice. Similarly, CL316243 induced a greater increase in Uncoupling protein 1 (Ucp1) expression and CREB phosphorylation in the brown adipose tissue of ARKO mice than in that of controls. We determined that activation of AR by dihydrotestosterone suppressed ß3-agonist- or forskolin-induced CRE-mediated transcription, which was prevented by AR antagonist. AR activation also suppressed CREB phosphorylation induced by forskolin. Moreover, we found AR nuclear localization, but not transcriptional activity, was necessary for the suppression of CRE-mediated transcription. Finally, modified mammalian two-hybrid and immunoprecipitation analyses suggest nuclear AR and CREB form a protein complex both in the presence and absence of dihydrotestosterone and forskolin. These results suggest androgen-AR signaling suppresses ß-adrenoceptor-induced UCP1-mediated brown adipose tissue thermogenesis by suppressing CREB phosphorylation, presumably owing to a protein complex with AR and CREB. This mechanism explains sexual differences in body temperature, at least partially.

Oncolytic Hairpin DNA Pair: Selective Cytotoxic Inducer through MicroRNA-Triggered DNA Self-Assembly.

Artificial nucleic acids have attracted much attention as potential cancer immunotherapeutic materials because they are recognized by a variety of extracellular and intracellular nucleic acid sensors and can stimulate innate immune responses. However, their low selectivity for cancer cells causes severe systemic immunotoxicity, making it difficult to use artificial nucleic acid molecules for immune cancer therapy. To address this challenge, we herein introduce a hairpin DNA assembly technology that enables cancer-selective immune activation to induce cytotoxicity. The designed artificial DNA hairpins assemble into long nicked double-stranded DNA triggered by intracellular microRNA-21 (miR-21), which is overexpressed in various types of cancer cells. We found that the products from the hairpin DNA assembly selectively kill miR-21-abundant cancer cells in vitro and in vivo based on innate immune activation. Our approach is the first to allow selective oncolysis derived from intracellular DNA self-assembly, providing a powerful therapeutic modality to treat cancer.

Bacterial Community Composition Under Paddy Conditions Is More Strongly Affected by the Difference in Soil Type than by Field Management.

In this study, we aimed to investigate the effects of soil type and field management on bacterial communities in paddy soils, taking into account the differences in soil physicochemical properties. We collected soil samples from 51 paddy fields in six prefectures in Japan. The paddy fields were managed under organic regimes (26 fields), natural-farming regimes (12 fields), or conventional regimes (13 fields). The paddy fields were classified into four soil types: andosol, gray lowland soil, gley soil, and gray upland soil. Soil DNA was extracted from the soil samples collected 2 to 10 weeks after the flooding, and the 16S rRNA gene amplicon sequencing analysis was performed. The bacterial community compositions were dominated by the phylum Proteobacteria, Chloroflexi, Actinobacteria, Acidobacteria, and Firmicutes in all fields. The difference in soil type had significant effects on α-diversities of the bacterial communities, although the field management had no effect. The soil bacterial communities in the gley soils and gray upland soils individually formed different groups from those in the other soils, while the andosol and gray lowland soils tended to form relatively similar bacterial communities. On the other hand, the effects of the field management were estimated to be smaller than those of soil type. The ß-diversity of the bacterial community compositions were significantly correlated with soil pH, total nitrogen content, total carbon content, and divalent iron content. Our results suggest that the soil microbial community in paddy fields may be strongly influenced by soil physiochemical properties derived from differences in soil type.

Methods for preparing tissue microarray slides using xenografts with different levels of HER2 expression to standardize HER2 detection.

Gene amplification and protein overexpression of human epidermal growth factor receptor type 2 (HER2) are specific targets for HER2-targeting drugs in breast, gastric, salivary gland, and colorectal cancers. The histopathological determination of HER2 status is crucial for treatment, highlighting the importance of improving HER2 detection accuracy in clinical practice. We prepared tissue microarray (TMA) slides for use as control slides for the standardization of gastric HER2 testing. Four human gastric cancer cell lines with HER2 scores of 3+, 2+, 1+, and 0 were xenografted in NOG mice. The TMA slides were constructed using samples from three different areas in these tumors. Staining properties were determined using six clinical kits for HER2. In TMA, HER2-positive tumors with HER2 scores of 3+ and 2+ showed good staining with all diagnostic kits, and the tissue images were similar to those of clinical samples. Xenograft tumor slides could potentially be used as external controls to standardize staining conditions for a variety of kits and may improve the accuracy of HER2 detection in clinical practice.

All-Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion.

All-trans retinoic acid (ATRA) promotes myoblast differentiation into myotubes. Leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) is a candidate ATRA-responsive gene; however, its role in skeletal muscles remains unclear. Here, we demonstrated that during the differentiation of murine C2C12 myoblasts into myotubes, Lgr6 mRNA expression transiently increased before the increase in the expression of the mRNAs encoding myogenic regulatory factors, such as myogenin, myomaker, and myomerger. The loss of LGR6 decreased the differentiation and fusion indices. The exogenous expression of LGR6 up to 3 and 24 h after the induction of differentiation increased and decreased the mRNA levels of myogenin, myomaker, and myomerger, respectively. Lgr6 mRNA was transiently expressed after myogenic differentiation in the presence of a retinoic acid receptor α (RARα) agonist and an RARγ agonist in addition to ATRA, but not in the absence of ATRA. Furthermore, a proteasome inhibitor or Znfr3 knockdown increased exogenous LGR6 expression. The loss of LGR6 attenuated the Wnt/ß-catenin signaling activity induced by Wnt3a alone or in combination with Wnt3a and R-spondin 2. These results indicate that LGR6 promotes myogenic differentiation and that ATRA is required for the transient expression of LGR6 during differentiation. Furthermore, LGR6 expression appeared to be downregulated by the ubiquitin-proteasome system involving ZNRF3.

[Two Cases of the Significant Liver Damage by Regorafenib].

There is a liver damage in a serious side effect of regorafenib. Case 1 was a 54-year-old woman, and she had an operation of rectal cancer and metastasized to multiple organs afterwards and started regorafenib as third-line. Erythema exudativum multiform developed on the 8th day after a start and regorafenib was canceled once and reduced on the 21st day when a skin symptom was relieved and restarted. However, because a significant rise of AST, ALT, T -Bil was recognized afterwards, regorafenib was canceled on the 27th day and enforced steroid pulse therapy and was relieved afterwards. Case 2 was a 61-year-old woman, and she had an operation of ascending colon cancer, ovarian metastasis and peritoneum dissemination. Regorafenib was started by frequent occurrence lung metastasis, cancerous pleurisy afterwards as fifth-line. Dissemination erythema developed on the 16th day and a liver damage developed on the 22nd day. Because a rise of AST, ALT went and was prolonged, liver biopsy was enforced in a cause close inspection purpose on the 45th day. A medicamentosus liver damage was diagnosed. The liver enzyme decreased afterwards. It may be easy to make the liver damage by regorafenib serious, and attention is necessary.

Curcumin activates G protein-coupled receptor 97 (GPR97) in a manner different from glucocorticoid.

Curcumin is a yellow pigment in turmeric (Curcuma longa) with various physiological effects in the body. To elucidate the molecular mechanisms by which bioactive compounds exert their function, identification of their molecular targets is crucial. In this study, we show that curcumin activates G protein-coupled receptor 97 (GPR97). Curcumin dose-dependently activated serum-response element-, but not serum-response factor-response element-, nuclear factor of activated T-cell-response element-, or cAMP-response element-, mediated transcription in cells overexpressed with GPR97. The structure-activity relationship indicated that (i) the double-bonds of the central 7-carbon chain were essential for activation; (ii) a methoxy group on the aromatic ring was required for maximal activity; (iii) the addition of glucuronic acid moiety or a methoxy group to the aromatic ring, but not the methylation of the aromatic p-hydroxy group, eliminated the activity; (iv) the stability of curcumin would be related to receptor activation. Both mutant GPR97(T250A) lacking the cleavage at GPCR proteolysis site and mutant GPR97(ΔN) lacking the N-terminal extracellular region were activated by curcumin and its related compounds similar to wild-type GPR97. In contrast, the synthetic glucocorticoid beclomethasone dipropionate and l-Phe activated wild-type GPR97 and GPR97(T250A), but not GPR97(ΔN). Moreover, curcumin exerted an additive effect on the activation of wild-type GPR97 with beclomethasone dipropionate, but not with l-Phe. Taken together, these results indicate that curcumin activates GPR97 coupled to Gi/Go subunit, and suggest that curcumin and glucocorticoid activate GPR97 in a different manner.

Sensitivity of eight types of ALK fusion variant to alectinib in ALK-transformed cells.

Tyrosine kinase inhibitors of anaplastic lymphoma kinase (ALK-TKIs) including alectinib have been the standard therapy against ALK fusion gene-positive non-small cell lung cancers (NSCLCs). Many ALK fusion variants have been identified in NSCLCs, and the predominant variants are echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) variant 1 (V1), V2 and V3a/b. However, there have been conflicting reports on the clinical responses of these variants to ALK-TKIs, and there are few reports on other less common ALK variants. To examine the influence of ALK variants on the efficacy of ALK-TKIs, we analyzed the sensitivity to alectinib of eight types of ALK variant: three major variants (V1, V2 and V3a) and five less common variants (V4; kinesin family member 5-ALK; kinesin light chain 1-ALK; striatin, calmodulin-binding protein-ALK; and tropomyosin-receptor kinase fused gene-ALK). Analysis was done by cell-free kinase assays using the recombinant proteins and by cell, growth assays using murine Ba/F3 cells expressing ALK variants. The kinase activity of each recombinant protein was significantly inhibited by alectinib. Intracellular ALK phosphorylation levels and its downstream signaling mediators, STAT3 and ERK, were suppressed by alectinib in each ALK variant-expressing Ba/F3 cell. Each cellular proliferation was markedly inhibited by alectinib treatment. There was no significant difference in the IC50 values between cells, with a <3.6-fold difference in responsiveness. In conclusion, these eight ALK variants had similar sensitivity to alectinib in vitro, indicating that it may not be possible to predict the response to alectinib just by determination of the ALK variant type in ALK fusion-positive NSCLCs.

Resistance to obinutuzumab-induced antibody-dependent cellular cytotoxicity caused by abnormal Fas signaling is overcome by combination therapies.

BACKGROUND: Obinutuzumab, a Type II anti-CD20 antibody, is used to treat follicular lymphoma. A major mode of action of obinutuzumab is antibody-dependent cellular cytotoxicity (ADCC). Knowledge of the mechanisms of resistance to obinutuzumab is important for the development of next-line strategies to follow obinutuzumab-containing therapy, including obinutuzumab retreatment. Unfortunately, the mechanisms by which tumor cells acquire resistance to ADCC are still poorly understood. To address this, we examined the mechanisms of resistance to obinutuzumab-induced ADCC and the combination efficacy of obinutuzumab and clinically available agents in the established resistant cells. METHODS AND RESULTS: We established cells resistant to obinutuzumab-induced ADCC using the non-Hodgkin lymphoma cell line RL and examined their mechanisms of resistance and the combination efficacy of obinutuzumab and clinically available agents. Comprehensive analysis by RNA sequencing of resistance mechanisms revealed that abnormal Fas signaling decreased sensitivity to ADCC in resistant clones. Combination treatment with prednisolone, a component of CHOP and CVP, was found to enhance ADCC sensitivity of RL cells and resistant clones and to significantly suppress tumor growth in xenograft models. Treatment with prednisolone upregulated expression of CD20 and an apoptosis-inducing protein BIM, which might augment perforin/granzyme B-mediated cell death. Furthermore, pretreatment of the effector cells with bendamustine enhanced ADCC activity, and treatment with obinutuzumab plus bendamustine showed significant antitumor efficacy in xenograft models. It was speculated that bendamustine upregulates ADCC activity by potentiating granules-mediated cell killing. CONCLUSIONS: Our study revealed a novel mechanism underlying obinutuzumab-induced ADCC resistance and indicated that ADCC resistance could be overcome by combining obinutuzumab with prednisolone or bendamustine. This study provides a scientific rationale for obinutuzumab-retreatment in combination with clinically available chemotherapeutic agents for obinutuzumab resistant follicular lymphoma.

Dietary oleamide attenuates obesity induced by housing mice in small cages.

Physical inactivity due to prolonged sedentary behavior induces obesity. Therefore, we investigated whether housing mice in small cages to mimic sedentary behavior induced obesity and whether dietary oleamide (cis-9,10-octadeceneamide) suppressed the induced obesity. A single oral administration of oleamide (50 mg/kg) to mice resulted in the accumulation of the exogenous oleamide in abdominal visceral fat. Next, mice were housed in small cages and oleamide (50 mg/kg/d) was orally administered for 12 weeks. Housing mice in small cages impaired glucose tolerance and increased food efficiency. It also increased body weight and abdominal fat mass. Dietary oleamide improved the impairment and inhibited their increase in mice housed in small cages. Furthermore, dietary oleamide suppressed the mRNA expression of inflammation-related factors in the abdominal fat of mice housed in small cages. Hence, these results indicate that although housing mice in small cages induces obesity and increases abdominal fat mass, dietary oleamide suppresses the obesity.

Carotenoid transporter CD36 expression depends on hypoxia-inducible factor-1α in mouse soleus muscles.

Dietary ß-carotene induces muscle hypertrophy and prevents muscle atrophy in red slow-twitch soleus muscles, but not in white fast-twitch extensor digitorum longus (EDL) muscles and gastrocnemius muscles. However, it remains unclear why these beneficial effects of ß-carotene are elicited in soleus muscles. To address this issue, we focused on carotenoid transporters in skeletal muscles. In mice, Cd36 mRNA levels were higher in red muscle than in white muscle. The siRNA-mediated knockdown of CD36 decreased ß-carotene uptake in C2C12 myotubes. In soleus muscles, CD36 knockdown inhibited ß-carotene-induced increase in muscle mass. Intravenous injection of the hypoxia marker pimonidazole produced more pimonidazole-bound proteins in soleus muscles than in EDL muscles, and the hypoxia-inducible factor-1 (HIF-1) α protein level was higher in soleus muscles than in EDL muscles. In C2C12 myotubes, hypoxia increased the expression of CD36 and HIF-1α at the protein and mRNA levels, and HIF-1α knockdown reduced hypoxia-induced increase in Cd36 mRNA level. In soleus muscles, HIF-1α knockdown reduced Cd36 mRNA level. These results indicate that CD36 is predominantly involved in ß-carotene-induced increase in soleus muscle mass of mice. Furthermore, we demonstrate that CD36 expression depends on HIF-1α in the soleus muscles of mice, even under normal physiological conditions.

Oleamide rescues tibialis anterior muscle atrophy of mice housed in small cages.

Skeletal muscle atrophy causes decreased physical activity and increased risk of metabolic diseases. We investigated the effects of oleamide (cis-9,10-octadecanamide) treatment on skeletal muscle health. The plasma concentration of endogenous oleamide was approximately 30 nm in male ddY mice under normal physiological conditions. When the stable isotope-labelled oleamide was orally administered to male ddY mice (50 mg/kg), the plasma concentration of exogenous oleamide reached approximately 170 nm after 1 h. Male ddY mice were housed in small cages (one-sixth of normal size) to enforce sedentary behaviour and orally administered oleamide (50 mg/kg per d) for 4 weeks. Housing in small cages decreased tibialis anterior (TA) muscle mass and the cross-sectional area of the myofibres in TA muscle. Dietary oleamide alleviated the decreases in TA muscle and resulted in plasma oleamide concentration of approximately 120 nm in mice housed in small cages. Housing in small cages had no influence on the phosphorylation levels of Akt serine/threonine kinase (Akt), mechanistic target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K) in TA muscle; nevertheless, oleamide increased the phosphorylation levels of the proteins. Housing in small cages increased the expression of microtubule-associated protein 1 light chain 3 (LC3)-II and sequestosome 1 (p62), but not LC3-I, in TA muscle, and oleamide reduced LC3-I, LC3-II and p62 expression levels. In C2C12 myotubes, oleamide increased myotube diameter at ≥100 nm. Furthermore, the mTOR inhibitor, Torin 1, suppressed oleamide-induced increases in myotube diameter and protein synthesis. These results indicate that dietary oleamide rescued TA muscle atrophy in mice housed in small cages, possibly by activating the phosphoinositide 3-kinase/Akt/mTOR signalling pathway and restoring autophagy flux.

Role of gut microbiota in sex- and diet-dependent metabolic disorders that lead to early mortality of androgen receptor-deficient male mice.

The gut microbiota is involved in metabolic disorders induced by androgen deficiency after sexual maturation in males (late-onset hypogonadism). However, its role in the energy metabolism of congenital androgen deficiency (e.g., androgen-insensitive syndrome) remains elusive. Here, we examined the link between the gut microbiota and metabolic disease symptoms in androgen receptor knockout (ARKO) mouse by administering high-fat diet (HFD) and/or antibiotics. HFD-fed male, but not standard diet-fed male or HFD-fed female, ARKO mice exhibited increased feed efficiency, obesity with increased visceral adipocyte mass and hypertrophy, hepatic steatosis, glucose intolerance, insulin resistance, and loss of thigh muscle. In contrast, subcutaneous fat mass accumulated in ARKO mice irrespective of the diet and sex. Notably, all HFD-dependent metabolic disorders observed in ARKO males were abolished after antibiotics administration. The ratios of fecal weight-to-food weight and cecum weight-to-body weight were specifically reduced by ARKO in HFD-fed males. 16S rRNA sequencing of fecal microbiota from HFD-fed male mice revealed differences in microbiota composition between control and ARKO mice. Several genera or species (e.g., Turicibacter and Lactobacillus reuteri, respectively) were enriched in ARKO mice, and antibiotics treatment spoiled the changes. Furthermore, the life span of HFD-fed ARKO males was shorter than that of control mice, indicating that androgen deficiency causes metabolic dysfunctions leading to early death. These findings also suggest that AR signaling plays a role in the prevention of metabolic dysfunctions, presumably by influencing the gut microbiome, and improve our understanding of health consequences in subjects with hypogonadism and androgen insensitivity.

Trastuzumab in combination with paclitaxel enhances antitumor activity by promoting apoptosis in human epidermal growth factor receptor 2-positive trastuzumab-resistant gastric cancer xenograft models.

Trastuzumab, a humanized anti-human epidermal growth factor receptor 2 antibody drug, is the first-line therapy for human epidermal growth factor receptor 2-positive breast and gastric cancer. For breast cancer, the benefit of continuous treatment with trastuzumab after it becomes refractory to first-line therapy has been demonstrated. However, it is unclear whether trastuzumab can show similar efficacy as a second-line treatment for gastric cancer. Here, we report that trastuzumab in combination with paclitaxel exhibits increased antitumor efficacy even for trastuzumab-resistant xenografted tumors. We derived the trastuzumab-resistant models from previously established human epidermal growth factor receptor 2-positive gastric cancer patient-derived cells. Human epidermal growth factor receptor 2 expression, PIK3CA mutation, and phosphatase and tensin homolog expression in these resistant models was equivalent to those in the trastuzumab-sensitive parental model, whereas cyclin-dependent kinase inhibitors, such as p16, p15, and p21, were downregulated. Trastuzumab in combination with paclitaxel enhanced antitumor activity in both the sensitive and resistant models. In the trastuzumab-sensitive model, the combination of trastuzumab and paclitaxel resulted in suppression of the AKT-p27-retinoblastoma protein pathway and induction of apoptosis. Although this combination did not suppress retinoblastoma protein phosphorylation in the trastuzumab-resistant model, it did markedly decrease epidermal growth factor receptor and human epidermal growth factor receptor 2 phosphorylation and further enhance paclitaxel-mediated apoptosis. These results suggested that trastuzumab in combination with paclitaxel can still exert more potent antitumor efficacy than each agent alone in trastuzumab-resistant models, providing evidence that trastuzumab remains beneficial in the treatment of trastuzumab-resistant tumors.

Biological Activity of Pseudovitamin B12 on Cobalamin-Dependent Methylmalonyl-CoA Mutase and Methionine Synthase in Mammalian Cultured COS-7 Cells.

Adenyl cobamide (commonly known as pseudovitamin B12) is synthesized by intestinal bacteria or ingested from edible cyanobacteria. The effect of pseudovitamin B12 on the activities of cobalamin-dependent enzymes in mammalian cells has not been studied well. This study was conducted to investigate the effects of pseudovitamin B12 on the activities of the mammalian vitamin B12-dependent enzymes methionine synthase and methylmalonyl-CoA mutase in cultured mammalian COS-7 cells to determine whether pseudovitamin B12 functions as an inhibitor or a cofactor of these enzymes. Although the hydoroxo form of pseudovitamin B12 functions as a coenzyme for methionine synthase in cultured cells, pseudovitamin B12 does not activate the translation of methionine synthase, unlike the hydroxo form of vitamin B12 does. In the second enzymatic reaction, the adenosyl form of pseudovitamin B12 did not function as a coenzyme or an inhibitor of methylmalonyl-CoA mutase. Experiments on the cellular uptake were conducted with human transcobalamin II and suggested that treatment with a substantial amount of pseudovitamin B12 might inhibit transcobalamin II-mediated absorption of a physiological trace concentration of vitamin B12 present in the medium.

Fetal androgen signaling defects affect pancreatic ß-cell mass and function, leading to glucose intolerance in high-fat diet-fed male rats.

We previously demonstrated that androgen signaling expands pancreatic ß-cell mass in the sexual maturation period (Am J Physiol Endocrinol Metab 314: E274-E286, 2018). The aim of this study was to elucidate whether fetal androgen signaling plays important roles in ß-cell mass development and ß-cell function in adulthood, defects of which are associated with type 2 diabetes mellitus. In the pancreas of male fetuses, androgen receptor (AR) was strongly expressed in the cytoplasm and at the cell membrane of Nkx6.1-positive ß-cell precursor cells but was markedly reduced in insulin-positive ß-cells. Administration of the anti-androgen flutamide to pregnant dams during late gestation reduced ß-cell mass and Ki67-positive proliferating ß-cells at birth in a male-specific manner without affecting body weight. The decrease of ß-cell mass in flutamide-exposed male rats was not recovered when rats were fed a standard diet, whereas it was fully recovered when rats were fed a high-fat diet (HFD), at 6 and 12 wk of age. Flutamide exposure in utero led to the development of glucose intolerance in male rats due to a decrease in insulin secretion when fed HFD but not standard diet. Insulin sensitivity did not differ between the two groups irrespective of diet. These results indicated that the action of fetal androgen contributed to ß-cell mass expansion in a sex-specific manner at birth and to the development of glucose intolerance by decreasing the secretion of insulin in HFD-fed male rats. Our data demonstrated the involvement of fetal androgen signaling in hypothesized sex differences in the developmental origins of health and disease by affecting pancreatic ß-cell function.

Expression of C-terminal ALK, RET, or ROS1 in lung cancer cells with or without fusion.

BACKGROUND: Genetic alterations, including mutation of epidermal growth factor receptor or v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog and fusion of anaplastic lymphoma kinase (ALK), RET proto-oncogene (RET), or v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1), occur in non-small cell lung cancers, and these oncogenic drivers are important biomarkers for targeted therapies. A useful technique to screen for these fusions is the detection of native carboxy-terminal (C-terminal) protein by immunohistochemistry; however, the effects of other genetic alterations on C-terminal expression is not fully understood. In this study, we evaluated whether C-terminal expression is specifically elevated by fusion with or without typical genetic alterations of lung cancer. METHODS: In 37 human lung cancer cell lines and four tissue specimens, protein and mRNA levels were measured by capillary western blotting and reverse transcription-PCR, respectively. RESULTS: Compared with the median of all 37 cell lines, mRNA levels at the C-terminus of all five of the fusion-positive cell lines tested (three ALK, one RET, and one ROS1) were elevated at least 2000-, 300-, or 2000-fold, respectively, and high C-terminal protein expression was detected. In an ALK fusion-positive tissue specimen, the mRNA and protein levels of C-terminal ALK were also markedly elevated. Meanwhile, in one of 36 RET fusion-negative cell lines, RET mRNA levels at the C-terminus were elevated at least 500-fold compared with the median of all 37 cell lines, and high C-terminal protein expression was detected despite the absence of RET fusion. CONCLUSIONS: This study of 37 cell lines and four tissue specimens shows the detection of C-terminal ALK or ROS1 proteins could be a comprehensive method to determine ALK or ROS1 fusion, whereas not only the detection of C-terminal RET protein but also other methods would be needed to determine RET fusion.

Salt Tolerance Improvement in Rice through Efficient SNP Marker-Assisted Selection Coupled with Speed-Breeding.

Salinity critically limits rice metabolism, growth, and productivity worldwide. Improvement of the salt resistance of locally grown high-yielding cultivars is a slow process. The objective of this study was to develop a new salt-tolerant rice germplasm using speed-breeding. Here, we precisely introgressed the hst1 gene, transferring salinity tolerance from "Kaijin" into high-yielding "Yukinko-mai" (WT) rice through single nucleotide polymorphism (SNP) marker-assisted selection. Using a biotron speed-breeding technique, we developed a BC3F3 population, named "YNU31-2-4", in six generations and 17 months. High-resolution genotyping by whole-genome sequencing revealed that the BC3F2 genome had 93.5% similarity to the WT and fixed only 2.7% of donor parent alleles. Functional annotation of BC3F2 variants along with field assessment data indicated that "YNU31-2-4" plants carrying the hst1 gene had similar agronomic traits to the WT under normal growth condition. "YNU31-2-4" seedlings subjected to salt stress (125 mM NaCl) had a significantly higher survival rate and increased shoot and root biomasses than the WT. At the tissue level, quantitative and electron probe microanalyzer studies indicated that "YNU31-2-4" seedlings avoided Na+ accumulation in shoots under salt stress. The "YNU31-2-4" plants showed an improved phenotype with significantly higher net CO2 assimilation and lower yield decline than WT under salt stress at the reproductive stage. "YNU31-2-4" is a potential candidate for a new rice cultivar that is highly tolerant to salt stress at the seedling and reproductive stages, and which might maintain yields under a changing global climate.