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Unlike protein-coding genes, the majority of human long non-coding RNAs (lncRNAs) are considered non-conserved. Although lncRNAs have been shown to function in diverse pathophysiological processes in mice, it remains largely unknown whether human lncRNAs have such in vivo functions. Here, we describe an integrated pipeline to define the in vivo function of non-conserved human lncRNAs. We first identify lncRNAs with high function potential using multiple indicators derived from human genetic data related to cardiometabolic traits, then define lncRNA’s function and specific target genes by integrating its correlated biological pathways in humans and co-regulated genes in a humanized mouse model. Finally, we demonstrate that the in vivo function of human-specific lncRNAs can be successfully examined in the humanized mouse model, and experimentally validate the predicted function of an obesity-associated lncRNA, LINC01018, in regulating the expression of genes in fatty acid oxidation in humanized livers through its interaction with RNA-binding protein HuR. Majority of human long non-coding RNAs (lncRNAs) are not conserved in mouse. Here the authors identify metabolic trait-associated lncRNA genes and show a functional role of a non-conserved human lncRNA, LINC01018, in lipid metabolism using a humanized mouse model.

A growing number of long noncoding RNAs (lncRNAs) have emerged as vital metabolic regulators. However, most human lncRNAs are nonconserved and highly tissue specific, vastly limiting our ability to identify human lncRNA metabolic regulators (hLMRs). In this study, we established a pipeline to identify putative hLMRs that are metabolically sensitive, disease relevant, and population applicable. We first progressively processed multilevel human transcriptome data to select liver lncRNAs that exhibit highly dynamic expression in the general population, show differential expression in a nonalcoholic fatty liver disease (NAFLD) population, and respond to dietary intervention in a small NAFLD cohort. We then experimentally demonstrated the responsiveness of selected hepatic lncRNAs to defined metabolic milieus in a liver-specific humanized mouse model. Furthermore, by extracting a concise list of protein-coding genes that are persistently correlated with lncRNAs in general and NAFLD populations, we predicted the specific function for each hLMR. Using gain- and loss-of-function approaches in humanized mice as well as ectopic expression in conventional mice, we validated the regulatory role of one nonconserved hLMR in cholesterol metabolism by coordinating with an RNA-binding protein, PTBP1, to modulate the transcription of cholesterol synthesis genes. Our work overcame the heterogeneity intrinsic to human data to enable the efficient identification and functional definition of disease-relevant human lncRNAs in metabolic homeostasis.

A growing number of long noncoding RNAs (lncRNAs) have emerged as vital metabolic regulators. However, most human lncRNAs are nonconserved and highly tissue specific, vastly limiting our ability to identify human lncRNA metabolic regulators (hLMRs). In this study, we established a pipeline to identify putative hLMRs that are metabolically sensitive, disease relevant, and population applicable. We first progressively processed multilevel human transcriptome data to select liver lncRNAs that exhibit highly dynamic expression in the general population, show differential expression in a nonalcoholic fatty liver disease (NAFLD) population, and respond to dietary intervention in a small NAFLD cohort. We then experimentally demonstrated the responsiveness of selected hepatic lncRNAs to defined metabolic milieus in a liver-specific humanized mouse model. Furthermore, by extracting a concise list of protein-coding genes that are persistently correlated with lncRNAs in general and NAFLD populations, we predicted the specific function for each hLMR. Using gain- and loss-offunction approaches in humanized mice as well as ectopic expression in conventional mice, we validated the regulatory role of one nonconserved hLMR in cholesterol metabolism by coordinating with an RNA-binding protein, PTBP1, to modulate the transcription of cholesterol synthesis genes. Our work overcame the heterogeneity intrinsic to human data to enable the efficient identification and functional definition of disease-relevant human lncRNAs in metabolic homeostasis.

Direct knowledge of gene regulation in human liver by metabolic stimuli could fundamentally advance our understanding of metabolic physiology. This information, however, is largely unknown, and this void is deeply rooted in a paradox that is caused by the inaccessibility of human liver to treatments and the insufficient annotation of liver transcriptome. Recent advances have uncovered immense complexity of transcriptome, i.e., multiple transcripts produced from one gene and extensive RNA modifications, which are all highly regulated and often condition-dependent. Establishing an inclusive annotation to study liver transcriptome dynamics thus requires human liver samples of diverse conditions, which are paradoxically unavailable due to the inaccessibility of human liver to treatments. In this work, we addressed these challenges by coupling an isogenic humanized mouse model with Nanopore single-molecule direct RNA sequencing (DRS). We first generated mice that carried humanized livers of identical genetic background, which were equivalent to clones of a single human liver, and then subjected the mice to representative metabolic treatments. We then analyzed the humanized livers with Nanopore DRS, which directly reads full-length native RNAs to determine the expression level, m6A modification and poly(A) tail length of all RNA transcript isoforms. Thus, our system allows for constructing a de novo annotation of human liver transcriptomes reflecting metabolic responses and studying transcriptome dynamics in conjunction. Our analysis uncovered a vast number of novel genes and transcripts that have not been previously reported. Our transcript-level analysis of human liver transcriptomes also identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional short read RNA-seq. We also revealed for the first time the dynamic changes in m6A and poly(A) tail length of human liver transcripts many of which are transcribed from key metabolic genes. Furthermore, we performed comparative analyses of gene regulation between human and mouse and between two individuals using the liver-specific humanized mice. This revealed that transcriptome dynamics are highly species- and genetic background-dependent, which may only be faithfully studied in a humanized system that entails clones of the same human liver. Hence our work revealed a complex metabolic responsive landscape of human liver transcriptome and also provided a framework to understand transcriptome dynamics of human liver in response to physiologically relevant metabolic stimuli.Competing Interest StatementThe authors have declared no competing interest.

A growing number of long non-coding RNAs (lncRNAs) have emerged as vital metabolic regulators in research animals suggesting that lncRNAs could also play an important role in human metabolism. However, most human lncRNAs are non-conserved, vastly limiting our ability to identify human lncRNA metabolic regulators (hLMRs). As the sequence-function relation of lncRNAs has yet to be established, the identification of lncRNA metabolic regulators in animals often relies on their regulations by experimental metabolic conditions. But it is very challenging to apply this strategy to human lncRNAs because well-controlled human data are much limited in scope and often confounded by genetic heterogeneity. In this study, we establish an efficient pipeline to identify putative hLMRs that are metabolically sensitive, disease-relevant, and population applicable. We first progressively processed human transcriptome data to select human liver lncRNAs that exhibit highly dynamic expression in the general population, show differential expression in a metabolic disease population, and response to dietary intervention in a small disease cohort. We then experimentally demonstrated the responsiveness of selected hepatic lncRNAs to defined metabolic milieus in a liver-specific humanized mouse model. Furthermore, by extracting a concise list of protein-coding genes that are persistently correlated with lncRNAs in general and metabolic disease populations, we predicted the specific function for each hLMR. Using gain- and loss-of-function approaches in humanized mice as well as ectopic expression in conventional mice, we were able to validate the regulatory role of one non-conserved hLMR in cholesterol metabolism. Mechanistically, this hLMR binds to an RNA-binding protein, PTBP1, to modulate the transcription of cholesterol synthesis genes. In summary, our study provides a pipeline to overcome the variabilities intrinsic to human data to enable the efficient identification and functional definition of hLMRs. The combination of this bioinformatic framework and humanized murine model will enable broader systematic investigation of the physiological role of disease-relevant human lncRNAs in metabolic homeostasis.

Ficifolidione, a natural insecticidal compound isolated from the essential oils of Myetaceae species, is a spiro phloroglucinol with an isobutyl group at the C-4 position. We found that ficifolidione showed cytotoxicity against cancer cells via apoptosis. Replacement of the isobutyl group by n-propyl group did not influence the potency, but the effect of the replacement of this group by a shorter or longer alkyl group on the biological activity remains unknown. In this study, ficifolidione derivatives with alkyl groups such as methyl, n-pentyl, and n-heptyl group—instead of the isobutyl group at the C-4 position—were synthesized to evaluate their cytotoxicity against the human promyelocytic leukaemia cell line HL60 and their insecticidal activity against mosquito larvae. The biological activities of their corresponding 4-epimers were also evaluated. As a result, the conversion of the isobutyl group to another alkyl group did not significantly influence the cytotoxicity or insecticidal activity. In HL60 cells treated with the n-heptyl-ficifolidione derivative, the activation of caspase 3/7 and the early stages of apoptosis were detected by using immunofluorescence and flow cytometric techniques, respectively, suggesting that the cytotoxicity should be induced by apoptosis even though the alkyl group was changed.

BackgroundThe practice patterns of exercise restrictions for patients with chronic kidney disease have not been adequately evaluated yet; thus, we examined them using a cross-sectional design and explored the factors related with those restrictions.MethodsThe Chronic Kidney Disease Japan Cohort study was a multicentre cohort study of Japanese patients (age 20–75 years) living in Japan. We used the information in the questionnaire on the restriction of physical activities offered by physicians to the patients during enrolment. We initially considered and used the following data as the clinical factors that the physician used for decision making on the directions of restriction of physical activities: age, sex, cause of chronic kidney disease (CKD), comorbid diseases, body mass index (BMI), systolic blood pressure, estimated glomerular filtration rate (eGFR) and urine albumin. The logistic regression model was used to explore the factors and estimate their adjusted odds ratio with regard to physician’s direction of restriction of physical activities.ResultsPhysician’s direction of exercise restrictions was implemented in 9.9% of the participants. In 17 facilities, the proportion of physician’s direction of exercise restriction ranged from 2.9 to 17.8%. The logistic regression analysis showed that the proportion of the factors such as younger age, cardiovascular disease, congestive heart failure and lower eGFR was higher in patients with physician’s direction of exercise restrictions.ConclusionsThe findings from this study suggested the factors related with prescribing exercise restrictions. Further studies examining which patients with CKD need direction of exercise restrictions are needed.

Chloramphenicol (Cm) is a broad-spectrum classic antibiotic active against prokaryotic organisms. However, Cm has severe side effects in eukaryotes of which the cause remains unknown. The plant pathogenic fungus Magnaporthe oryzae , which causes rice blast, forms an appressorium to infect the host cell via single-cell differentiation. Chloramphenicol specifically inhibits appressorium formation, which indicates that Cm has a novel molecular target (or targets) in the rice blast fungus. Application of the T7 phage display method inferred that MoDullard, a Ser/Thr-protein phosphatase, may be a target of Cm. In animals Dullard functions in cell differentiation and protein synthesis, but in fungi its role is poorly understood. In vivo and in vitro analyses showed that MoDullard is required for appressorium formation, and that Cm can bind to and inhibit MoDullard function. Given that human phosphatase CTDSP1 complemented the MoDullard function during appressorium formation by M. oryzae , CTDSP1 may be a novel molecular target of Cm in eukaryotes.

Background Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal storage disorder (LSD) caused by recessive mutations in the α-L-iduronidase ( IDUA ) gene. Enzyme replacement therapy (ERT) utilizing terminal mannose-6-phosphate (M6P)-carrying N -glycans attached to therapeutic enzymes produced by mammalian cell lines has been clinically applied to several LSDs. Recent studies suggested an unidentified delivery pathway mediated by sialic acid-containing N -glycans. However, more economical platform development is required to produce large quantities of recombinant enzymes. Transgenic silkworms have been established as low-cost systems for expressing recombinant glycoproteins. Microbial endo-β- N -acetylglucosaminidases (ENGases) enable the transglycosylation of N -glycans to other types. Methods We purified recombinant human IDUA from IDUA transgenic silkworm cocoons and performed ENGase-mediated transglycosylation. Furthermore, we performed intravenous enzyme replacement therapy in a Japanese macaque MPS I non-human primate model carrying a homozygous IDUA missense mutation. Results Here we show the establishment of IDUA transgenic silkworms and purification of recombinant human IDUA from cocoons. As M6P- and sialic acid-containing N -glycans are not attached to purified hIDUA, we perform ENGase-mediated transglycosylation to obtain hIDUAs with M6P- and sialic acid-containing N -glycans (neoglyco-hIDUAs). Furthermore, we perform intravenous neoglyco-hIDUA replacement therapy in MPS I non-human primate model and succeed in improving the clinical signs and reducing the urinary glycosaminoglycan (GAG) levels. Conclusions These glycotechnologies using transgenic silkworms and ENGases are expected to serve as platforms for developing therapeutic glycoproteins. Shinoda and Kitakaze et al. prepared recombinant hIDUA from transgenic silkworm cocoons and transglycosylated to mannose-6-phosphate- or α2,6-sialyl glycan-type using ENGases. These enzymes were administrated to a mucopolysaccharidosis type I macaque and succeeded in improving the clinical signs and reducing the urinary glycosaminoglycan levels. Plain language summary Lysosomal storage disorders (LSDs) are a group of inherited diseases related to metabolism. These can result in buildup of toxic material in the body due to defects in enzymes (a type of protein). One of the current treatments for LSDs is enzyme replacement therapy (ERT), in which functional human enzymes are given to patients. However, producing large quantities of these therapeutic enzymes can be challenging. Here, we describe a method to produce certain types of proteins using silkworms. We used this method to evaluate its therapeutic effects on a non-human primate model of one type of LSD and succeeded in improving the animal’s symptoms. This method could be a promising approach for producing treatments for humans.

ObjectivesWe aimed to examine the validity of the quick Sequential Organ Failure Assessment (qSOFA) score for mortality and bacteraemia risk assessment in Japanese haemodialysis patients.DesignThis is a retrospective multicentre cohort study.SettingThe six participating hospitals are tertiary-care institutions that receive patients on an emergency basis and provide primary, secondary and tertiary care. The other participating hospital is a secondary-care institution that receives patients on an emergency basis and provides both primary and secondary care.ParticipantsThis study included haemodialysis outpatients admitted for bacteraemia suspicion, who had blood drawn for cultures within 48 hours of their initial admission.Primary and secondary outcome measuresThe primary outcome measure was overall in-hospital mortality. Secondary outcomes included 28-day in-hospital mortality and the incidence of bacteraemia diagnosed based on blood culture findings. The discrimination, calibration and test performance of the qSOFA score were assessed. Missing data were handled using multiple imputation.ResultsAmong the 507 haemodialysis patients admitted with bacteraemia suspicion between August 2011 and July 2013, the overall in-hospital mortality was 14.6% (74/507), the 28-day in-hospital mortality was 11.1% (56/507) and the incidence of bacteraemia, defined as a positive blood culture, was 13.4% (68/507). For predicting in-hospital mortality among haemodialysis patients, the area under the receiver operating characteristic curve was 0.61 (95% CI 0.56–0.67) for a qSOFA score ≥2. The Hosmer-Lemeshow χ2 statistics for the qSOFA score as a predictor of overall and 28-day in-hospital mortality were 5.72 (p=0.02) and 7.40 (p<0.01), respectively.ConclusionOn external validation, the qSOFA score exhibited low diagnostic accuracy and miscalibration for in-hospital mortality and bacteraemia among haemodialysis patients.