Female sample screening using colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting non-coding RNA XIST.

Forensic sample screening is important for establishing an effective DNA typing workflow. The detection of sex-specific markers in forensic samples highlights the necessity for further analysis. Y-chromosome DNA can confirm male contributions, but female contributions are difficult to confirm using DNA-based methods. To address this, we developed a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the long non-coding RNA X-inactive specific transcript (XIST) to screen female samples. Operating at 65 °C for 30 min, the assay yielded results discernible from the color change of the pH indicator dye. The assay showed a detection limit of approximately 0.5 µL of blood. The assay also detected XIST RNA in mixed body fluids and mock samples, indicating its potential applicability to casework samples. Taken together, our assay provides a rapid and simple strategy for screening female samples.

RNA-based short tandem repeat analysis on the D18S51 locus.

DNA typing based on short tandem repeat (STR) analysis is an effective forensic method for human identification. Some STRs are contained within the introns of protein-coding genes and are transcribed as pre-mRNAs. However, the possibility of using RNA for STR analysis is yet to be fully explored. Considering that RNA in forensic samples is relatively stable, especially under dry- and low-temperature conditions, we hypothesized that STR information could be obtained from RNA. Here, we investigated the possibility of conducting RNA-based STR analysis using the D18S51 locus as a model.

One-tube, two-step isothermal amplification of histatin 3 mRNA for saliva screening.

Saliva samples are frequently collected at crime scenes. Salivary mRNA profiling, such as that of histatin 3 (HTN3), is a highly specific approach that overcomes the limitation of traditional amylase tests. However, typical mRNA detection methods based on reverse transcription PCR (RT-PCR) are time-consuming and labor-intensive. Here, we report a one-tube, two-step isothermal amplification assay for HTN3 mRNA, which enables rapid, simple, and sensitive screening of saliva. The first step is an RT-recombinase polymerase amplification (RT-RPA) assay at 42 °C for 20 min; the second step is a loop-mediated isothermal amplification (LAMP) assay at 65 °C for 30 min. The reactions can be performed in a closed tube, and the products are detected using real-time fluorescence analysis. The assay sensitivity was 0.5 µL of saliva samples. It also detected HTN3 mRNA in mixed and mock samples, demonstrating its applicability to actual forensic samples. These findings suggest that our strategy is promising for screening of saliva from forensic samples.

[Intraoperative Subsegmental Pulmonary Artery Embolization for Catheter Induced Pulmonary Pseudoaneurysm].

An 82-year-old female patient with severe aortic stenosis underwent aortic valve replacement. After weaning from cardiopulmonary bypass, it was noticed that Swan-Ganz( SG) catheter tip was located 50 cm distally to the right neck. After the catheter was pulled back, massive hemoptysis occurred. Fiberoptic bronchoscopy revealed bleeding from the left main bronchus. The tracheal tube was exchanged to a left selective bronchial tube and protamine sulfate was administered. However, massive hemorrhage continued. Intraoperative selective pulmonary angiography identified a pseudoaneurysm in A10. Efferent arteries, A10a, A10b, A10c, and an afferent artery, A10, were successfully embolized to obtain hemostasis. The patient remained hypoxemic despite inhalation with 100% oxygen and high positive end-expiratory pressure, so veno-arterial extracorporeal membrance oxygenation (VA-ECMO) was initiated. The patient was then transferred to intensive care unit (ICU) with VA-ECMO and the sternum was left open. Strenuous bronchial lavage was performed and VA-ECMO was discontinued at 2 days later. The patient was weaned from ventilator 14 days and discharged 63 days postoperatively.

Dearomative triple elementalization of quinolines driven by visible light.

Organoboron and organosilicon compounds are used not only as synthetic building blocks but also as functional materials and pharmaceuticals, and compounds with multiple boryl and silyl groups are beginning to be used for these purposes. Especially in drug discovery, methodology providing easy stereoselective access to aliphatic nitrogen heterocycles bearing multiple boryl or silyl groups from readily available aromatic nitrogen heterocycles would be attractive. However, such transformations remain challenging, and available reactions have been mostly limited to dearomative hydroboration or hydrosilylation reactions. Here, we report the dearomative triple elementalization (carbo-sila-boration) of quinolines via the addition of organolithium followed by photo-boosted silaboration, affording the desired products with complete chemo-, regio-, and stereoselectivity. The reaction proceeds via the formation of silyl radicals instead of silyl anions. We also present preliminary studies to illustrate the potential of silaboration products as synthetic platforms.

CRTC1 deficiency, specifically in melanocortin-4 receptor-expressing cells, induces hyperphagia, obesity, and insulin resistance.

Melanocortin-4 receptor (MC4R) is a critical regulator of appetite and energy expenditure in rodents and humans. MC4R deficiency causes hyperphagia, reduced energy expenditure, and impaired glucose metabolism. Ligand binding to MC4R activates adenylyl cyclase, resulting in increased levels of intracellular cyclic adenosine monophosphate (cAMP), a secondary messenger that regulates several cellular processes. Cyclic adenosine monophosphate responsive element-binding protein-1-regulated transcription coactivator-1 (CRTC1) is a cytoplasmic coactivator that translocates to the nucleus in response to cAMP and is reportedly involved in obesity. However, the precise mechanism through which CRTC1 regulates energy metabolism remains unknown. Additionally, there are no reports linking CRTC1 and MC4R, although both CRTC1 and MC4R are known to be involved in obesity. Here, we demonstrate that mice lacking CRTC1, specifically in MC4R cells, are sensitive to high-fat diet (HFD)-induced obesity and exhibit hyperphagia and increased body weight gain. Moreover, the loss of CRTC1 in MC4R cells impairs glucose metabolism. MC4R-expressing cell-specific CRTC1 knockout mice did not show changes in body weight gain, food intake, or glucose metabolism when fed a normal-chow diet. Thus, CRTC1 expression in MC4R cells is required for metabolic adaptation to HFD with respect to appetite regulation. Our results revealed an important protective role of CRTC1 in MC4R cells against dietary adaptation.

Stimulation of Gs signaling in MC4R cells by DREADD increases energy expenditure, suppresses food intake, and increases locomotor activity in mice.

The melanocortin 4 receptor (MC4R) plays an important role in the regulation of appetite and energy expenditure in humans and rodents. Impairment of MC4R signaling causes severe obesity. MC4R mainly couples to the G-protein Gs. Ligand binding to MC4R activates adenylyl cyclase resulting in increased intracellular cAMP levels. cAMP acts as a secondary messenger, regulating various cellular processes. MC4R can also couple with Gq and other signaling pathways. Therefore, the contribution of MC4R/Gs signaling to energy metabolism and appetite remains unclear. To study the effect of Gs signaling activation in MC4R cells on whole body energy metabolism and appetite, we generated a novel mouse strain that expresses a Gs-coupled designer receptors exclusively activated by designer drugs [Gs-DREADD (GsD)] selectively in MC4R-expressing cells (GsD-MC4R mice). Chemogenetic activation of the GsD by a designer drug [deschloroclozapine (DCZ); 0.01∼0.1 mg/kg body wt] in MC4R-expressing cells significantly increased oxygen consumption and locomotor activity. In addition, GsD activation significantly reduced the respiratory exchange ratio, promoting fatty acid oxidation, but did not affect core (rectal) temperature. A low dose of DCZ (0.01 mg/kg body wt) did not suppress food intake, but a high dose of DCZ (0.1 mg/kg body wt) suppressed food intake in MC4R-GsD mice, although either DCZ dose (0.01 or 0.1 mg/kg body wt) did not affect food intake in the control mice. In conclusion, the current study demonstrated that the stimulation of Gs signaling in MC4R-expressing cells increases energy expenditure and locomotor activity and suppresses appetite.NEW & NOTEWORTHY We report that Gs signaling in melanocortin 4 receptor (MC4R)-expressing cells regulates energy expenditure, appetite, and locomotor activity. These findings shed light on the mechanism underlying the regulation of energy metabolism and locomotor activity by MC4R/cAMP signaling.

Disruption of CRTC1 and CRTC2 in Sim1 cells strongly increases high-fat diet intake in female mice but has a modest impact on male mice.

cAMP responsive element binding protein (CREB)-regulated transcription coactivators (CRTCs) regulate gene transcription in response to an increase in intracellular cAMP or Ca2+ levels. To date, three isoforms of CRTC have been identified in mammals. All CRTCs are widely expressed in various regions of the brain. Numerous studies have shown the importance of CREB and CRTC in energy homeostasis. In the brain, the paraventricular nucleus of the hypothalamus (PVH) plays a critical role in energy metabolism, and CRTC1 and CRTC2 are highly expressed in PVH neuronal cells. The single-minded homolog 1 gene (Sim1) is densely expressed in PVH neurons and in some areas of the amygdala neurons. To determine the role of CRTCs in PVH on energy metabolism, we generated mice that lacked CRTC1 and CRTC2 in Sim1 cells using Sim-1 cre mice. We found that Sim1 cell-specific CRTC1 and CRTC2 double-knockout mice were sensitive to high-fat diet (HFD)-induced obesity. Sim1 cell-specific CRTC1 and CRTC2 double knockout mice showed hyperphagia specifically for the HFD, but not for the normal chow diet, increased fat mass, and no change in energy expenditure. Interestingly, these phenotypes were stronger in female mice than in male mice, and a weak phenotype was observed in the normal chow diet. The lack of CRTC1 and CRTC2 in Sim1 cells changed the mRNA levels of some neuropeptides that regulate energy metabolism in female mice fed an HFD. Taken together, our findings suggest that CRTCs in Sim1 cells regulate gene expression and suppress excessive fat intake, especially in female mice.

Photo-Induced ortho-C-H Borylation of Arenes through In Situ Generation of Rhodium(II) Ate Complexes.

Photoinduced in situ "oxidation" of half-sandwich metal complexes to "high-valent" cationic metal complexes has been used to accelerate catalytic reactions. Here, we report the unprecedented photoinduced in situ "reduction" of half-sandwich metal [Rh(III)] complexes to "low-valent" anionic metal [Rh(II)] ate complexes, which facilitate ligand exchange with electron-deficient elements (diboron). This strategy was realized by using a functionalized cyclopentadienyl (CpA3) Rh(III) catalyst we developed, which enabled the basic group-directed room temperature ortho-C-H borylation of arenes.

Loss of CREB Coactivator CRTC1 in SF1 Cells Leads to Hyperphagia and Obesity by High-fat Diet But Not Normal Chow Diet.

Cyclic adenosine monophosphate responsive element-binding protein-1-regulated transcription coactivator-1 (CRTC1) is a cytoplasmic coactivator that translocates to the nucleus in response to cyclic adenosine monophosphate. Whole-body knockdown of Crtc1 causes obesity, resulting in increased food intake and reduced energy expenditure. CRTC1 is highly expressed in the brain; therefore, it might play an important role in energy metabolism via the neuronal pathway. However, the precise mechanism by which CRTC1 regulates energy metabolism remains unknown. Here, we showed that mice lacking CRTC1, specifically in steroidogenic factor-1 expressing cells (SF1 cells), were sensitive to high-fat diet (HFD)-induced obesity, exhibiting hyperphagia and increased body weight gain. The loss of CRTC1 in SF1 cells impaired glucose metabolism. Unlike whole-body CRTC1 knockout mice, SF1 cell-specific CRTC1 deletion did not affect body weight gain or food intake in normal chow feeding. Thus, CRTC1 in SF1 cells is required for normal appetite regulation in HFD-fed mice. CRTC1 is primarily expressed in the brain. Within the hypothalamus, which plays an important role for appetite regulation, SF1 cells are only found in ventromedial hypothalamus. RNA sequencing analysis of microdissected ventromedial hypothalamus samples revealed that the loss of CRTC1 significantly changed the expression levels of certain genes. Our results revealed the important protective role of CRTC1 in SF1 cells against dietary metabolic imbalance.

microRNA-33 maintains adaptive thermogenesis via enhanced sympathetic nerve activity.

Adaptive thermogenesis is essential for survival, and therefore is tightly regulated by a central neural circuit. Here, we show that microRNA (miR)-33 in the brain is indispensable for adaptive thermogenesis. Cold stress increases miR-33 levels in the hypothalamus and miR-33-/- mice are unable to maintain body temperature in cold environments due to reduced sympathetic nerve activity and impaired brown adipose tissue (BAT) thermogenesis. Analysis of miR-33f/f dopamine-ß-hydroxylase (DBH)-Cre mice indicates the importance of miR-33 in Dbh-positive cells. Mechanistically, miR-33 deficiency upregulates gamma-aminobutyric acid (GABA)A receptor subunit genes such as Gabrb2 and Gabra4. Knock-down of these genes in Dbh-positive neurons rescues the impaired cold-induced thermogenesis in miR-33f/f DBH-Cre mice. Conversely, increased gene dosage of miR-33 in mice enhances thermogenesis. Thus, miR-33 in the brain contributes to maintenance of BAT thermogenesis and whole-body metabolism via enhanced sympathetic nerve tone through suppressing GABAergic inhibitory neurotransmission. This miR-33-mediated neural mechanism may serve as a physiological adaptive defense mechanism for several stresses including cold stress.

Rhodium(III)-Catalyzed Oxidative ortho-Olefination of Phenyl Carbamates with Alkenes: Elucidation of Acceleration Mechanisms by Using an Unsubstituted Cyclopentadienyl Ligand.

It has been established that an unsubstituted cyclopentadienyl (Cp) Rh(III) complex is an effective catalyst for the oxidative ortho-olefination of phenyl carbamates with both acrylates and styrenes under mild conditions. In addition, diolefination of a protected BINOL (1,1'-binaphthalene-2,2'-diol) proceeded in high yields and disubstituted acrylates could participate in this catalysis. Experimental and theoretical mechanistic studies elucidated that an electron-deficient nature of the unsubstituted CpRh(III) complex accelerates both the electrophilic aryl C-H rhodation and the rate-limiting alkene insertion steps.

Rhodium-Catalyzed ortho-Bromination of O-Phenyl Carbamates Accelerated by a Secondary Amide-Pendant Cyclopentadienyl Ligand.

It has been established that a newly developed cyclopentadienyl rhodium(III) [CpA RhIII ] complex, bearing an acidic secondary amide moiety on the Cp ring, is able to catalyze the ortho-bromination of O-phenyl carbamates with N-bromosuccinimide (NBS) at room temperature. The presence of the acidic secondary amide moiety on the CpA ligand accelerates the bromination by the hydrogen bond between the acidic NH group of the CpA ligand and the carbonyl group of NBS.

Analysis of negative historical control group data from the in vitro micronucleus assay using TK6 cells.

The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity.

Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay.

Screening for male-derived biological material from collected samples plays an important role in criminal investigations, especially those involving sexual assaults. We have developed a loop-mediated isothermal amplification (LAMP) assay targeting multi-repeat sequences of the Y chromosome for detecting male DNA. Successful amplification occurred with 0.5 ng of male DNA under isothermal conditions of 61 to 67 °C, but no amplification occurred with up to 10 ng of female DNA. Under the optimized conditions, the LAMP reaction initiated amplification within 10 min and amplified for 20 min. The LAMP reaction was sensitive at levels as low as 1-pg male DNA, and a quantitative LAMP assay could be developed because of the strong correlation between the reaction time and the amount of template DNA in the range of 10 pg to 10 ng. Furthermore, to apply the LAMP assay to on-site screening for male-derived samples, we evaluated a protocol using a simple DNA extraction method and a colorimetric intercalating dye that allows detection of the LAMP reaction by evaluating the change in color of the solution. Using this protocol, samples of male-derived blood and saliva stains were processed in approximately 30 min from DNA extraction to detection. Because our protocol does not require much hands-on time or special equipment, this LAMP assay promises to become a rapid and simple screening method for male-derived samples in forensic investigations.

Reference control data obtained from an in vivo comet-micronucleus combination assay using Sprague Dawley rats.

According to the International Conference on Harmonization Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (ICH S2(R1)), a positive response in any in vitro assay necessitates additional in vivo test(s) (other tissue/endpoint) in addition to the erythrocyte micronucleus test when Option 1 of the test battery is selected. When Option 2 of the test battery is selected, a bacterial gene mutation test and two in vivo tests with different tissues/endpoint are required. The in vivo alkaline comet assay is recommended as the second in vivo test because it can detect a broad spectrum of DNA damage in any tissue and can be combined with the erythrocyte micronucleus test. Considering animal welfare, a combination assay is preferable to an individual assay. Thus, we validated the protocol for the in vivo comet-micronucleus combination assay in rats with three daily administrations and determined the dose of the positive control (ethyl methanesulfonate; EMS, 200mg/kg/day). We also collected the negative control (vehicle) and positive control (EMS) data from the comet (liver, stomach, and kidney) and micronucleus (bone marrow) combination assay using male Sprague Dawley (SD) rats. The negative control data were comparable to our historical control data obtained from stand-alone assays. The positive control data showed clear and consistent positive responses in both endpoints.

Comet assay evaluation of six chemicals of known genotoxic potential in rats.

As a part of an international validation of the in vivo rat alkaline comet assay (comet assay) initiated by the Japanese Center for the Validation of Alternative Methods (JaCVAM) we examined six chemicals for potential to induce DNA damage: 2-acetylaminofluorene (2-AAF), N-nitrosodimethylamine (DMN), o-anisidine, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), sodium chloride, and sodium arsenite. DNA damage was evaluated in the liver and stomach of 7- to 9-week-old male Sprague Dawley rats. Of the five genotoxic carcinogens tested in our laboratory, DMN and 1,2-DMH were positive in the liver and negative in the stomach, 2-AAF and o-anisidine produced an equivocal result in liver and negative results in stomach, and sodium arsenite was negative in both liver and stomach. 1,2-DMH and DMN induced dose-related increases in hedgehogs in the same tissue (liver) that exhibited increased DNA migration. However, no cytotoxicity was indicated by the neutral diffusion assay (assessment of highly fragmented DNA) or histopathology in response to treatment with any of the tested chemicals. Therefore, the increased DNA damage resulting from exposure to DMN and 1,2-DMH was considered to represent a genotoxic response. Sodium chloride, a non-genotoxic non-carcinogen, was negative in both tissues as would be predicted. Although only two (1,2-DMH and DMN) out of five genotoxic carcinogens produced clearly positive results in the comet assay, the results obtained for o-anisidine and sodium arsenite in liver and stomach cells are consistent with the known mode of genotoxicity and tissue specificity exhibited by these carcinogens. In contrast, given the known genotoxic mode-of-action and target organ carcinogenicity of 2-AAF, it is unclear why this chemical failed to convincingly increase DNA migration in the liver. Thus, the results of the comet assay validation studies conducted in our laboratory were considered appropriate for five out of the six test chemicals.

Critical issues with the in vivo comet assay: A report of the comet assay working group in the 6th International Workshop on Genotoxicity Testing (IWGT).

As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use.

Repeated-dose liver and gastrointestinal tract micronucleus assays for quinoline in rats.

Repeated-dose liver, bone marrow, and gastrointestinal tract micronucleus assays that use young adult rats were evaluated in a collaborative study that was organized by the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group. A genotoxic hepatocarcinogen quinoline was orally administered to independent groups of five Crl:CD (SD) male rats at doses of 30, 60 and 120mg/kg for 14 days and at doses of 15, 30 and 60mg/kg for 28 days. After treatment, the livers were harvested and hepatocytes were isolated by collagenase treatment. The frequency of micronucleated hepatocytes (MNHEPs) increased significantly in both the 14- and 28-day repeated dose studies. However, the frequency of micronucleated cells did not increase in the bone marrow, stomach or colon cells, which were not quinoline-induced carcinogenic target organs in the rats. These results indicate that a repeated-dose liver micronucleus (RDLMN) assay using young adult rats is capable of detecting the genotoxicity of quinoline at the target organ of carcinogenicity. The protocol may also permit the integration of the genotoxic endpoint into general repeated-dose toxicity studies. Furthermore, we elucidated that conducting the micronucleus assay in multiple organs could potentially assess organ specificity.

Pulmonary sclerosing hemangioma with lymph node metastasis: A case report and literature review.

Pulmonary sclerosing hemangioma (SH) is an uncommon benign or low-grade malignant tumor. Multicentric SH and SH with lymph node metastasis have rarely been reported. The present report describes a case of pulmonary SH with lymph node metastasis in a middle-aged female. A nodule was found incidentally in the lower left lung. The patient underwent left lower pulmonary lobectomy and lymph node dissection. Histologically, the nodule demonstrated the characteristic features of SH and one of the resected lymph nodes contained a metastasis of this tumor. Thus, pulmonary SH has the potential to metastasize, a potential not suggested by histological features.