Versatile strategy using vaccinia virus-capping enzyme to synthesize functional 5' cap-modified mRNAs.

The potential of synthetic mRNA as a genetic carrier has increased its application in scientific fields. Because the 5' cap regulates the stability and translational activity of mRNAs, there are concerted efforts to search for and synthesize chemically-modified 5' caps that improve the functionality of mRNA. Here, we report an easy and efficient method to synthesize functional mRNAs by modifying multiple 5' cap analogs using a vaccinia virus-capping enzyme. We show that this enzyme can introduce a variety of GTP analogs to the 5' end of RNA to generate 5' cap-modified mRNAs that exhibit different translation levels. Notably, some of these modified mRNAs improve translation efficiency and can be conjugated to chemical structures, further increasing their functionality. Our versatile method to generate 5' cap-modified mRNAs will provide useful tools for RNA therapeutics and biological research.

Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information.

Treatment with granulocyte colony-stimulating factor in the refeeding phase of anorexia nervosa complicated with severe neutropenia and sepsis: a case report.

Leukopenia is frequently observed in patients with anorexia nervosa (AN). However, serious infectious diseases are infrequent in patients with AN. We present the case of a 13-year-old girl with AN complicated with severe neutropenia and sepsis upon refeeding. Her body mass index was 11 kg/m2 and her absolute neutrophil count (ANC) was 1555/µL on admission. After the initiation of refeeding, her ANC gradually declined to 346/µL. High fever occurred and a blood culture tested positive for Enterobacter cloacae. Although fever subsided with administration of antibiotics, human recombinant granulocyte colony-stimulating factor (G-CSF) was administered to improve her severe neutropenia. After administration of G-CSF, initially for 5 days and again for 3 days, her ANC became normal and stable. The combination of neutropenia and low body mass index may contribute to severe bacterial infection. Usually, hematological abnormalities such as neutropenia improve spontaneously with the recovery of nutritional status in AN patients. Therefore, it is difficult to determine the indication for treatment with G-SCF. Although a careful consideration is required, treatment with G-CSF is effective in AN patients with sepsis caused by severe neutropenia.

Quantitative and simultaneous translational control of distinct mammalian mRNAs.

The introduction of multiple genes into cells is increasingly required for understanding and engineering biological systems. Small-molecule-responsive transcriptional regulation has been widely used to control transgene expression. In contrast, methods for specific and simultaneous regulation of multiple genes with a single regulatory protein remain undeveloped. In this report, we describe a method for quantitatively tuning the expression of multiple transgenes with a translational regulatory protein. A protein that binds a specific RNA motif inserted in the 5'-untranslated region (UTR) of an mRNA modulates the translation of that message in mammalian cells. We provide two independent mechanisms by which to rationally fine-tune the output: the efficiency of translation correlates well with the distance between the inserted motif and the 5' terminus of the mRNA and is further modulated by the tandem insertion of multiple RNA motifs. The combination of these two approaches allowed us to fine-tune the translational efficiency of target mRNAs over a wide dynamic range. Moreover, we controlled the expression of two transgenes simultaneously and specifically by engineering each cis-regulatory 5'-UTR. The approach provides a useful alternative regulatory layer for controlling gene expression in biological research and engineering.

Neuronal activity topography parameters as a marker for differentiating vascular cognitive impairment in carotid stenosis.

Previously, we reported on the differentiation between patients with Alzheimer disease and normal controls using a quantitative electroencephalographic technique called neuronal activity topography (NAT). In this technique, cerebral neuronal activities are characterized by the signal intensity and coherence (sNAT and vNAT, respectively). In the present study, we examined 47 patients with vascular cognitive impairment in carotid stenosis and 52 normal controls. All subjects underwent electroencephalography in a resting state with closed eyes for 5 minutes. Electroencephalographic markers of the differential likelihood, that is, the sensitivity-versus-specificity characteristics, sL(x:VCI-NLc) and vL(x:VCI-NLc), were assessed with neuronal activity topography and were compared between the 2 groups. sL(x:VCI-NLc) and vL(x:VCI-NLc) crossed each other at a cutoff value of the differential likelihood. Separation of the patients and controls was made with a sensitivity of 92% and 88%, as well as a false-positive rate of 8% and 12% for sL(x:VCI-NLc) and vL(x:VCI-NLc), respectively. Using sNAT, we accurately differentiated 92% patients with vascular cognitive impairment. We recommend that sNAT, rather than vNAT, should be used in detecting vascular cognitive impaired patients.

A let-7 microRNA-RALB axis links the immune properties of iPSC-derived megakaryocytes with platelet producibility.

We recently achieved the first-in-human transfusion of induced pluripotent stem cell-derived platelets (iPSC-PLTs) as an alternative to standard transfusions, which are dependent on donors and therefore variable in supply. However, heterogeneity characterized by thrombopoiesis-biased or immune-biased megakaryocytes (MKs) continues to pose a bottleneck against the standardization of iPSC-PLT manufacturing. To address this problem, here we employ microRNA (miRNA) switch biotechnology to distinguish subpopulations of imMKCLs, the MK cell lines producing iPSC-PLTs. Upon miRNA switch-based screening, we find imMKCLs with lower let-7 activity exhibit an immune-skewed transcriptional signature. Notably, the low activity of let-7a-5p results in the upregulation of RAS like proto-oncogene B (RALB) expression, which is crucial for the lineage determination of immune-biased imMKCL subpopulations and leads to the activation of interferon-dependent signaling. The dysregulation of immune properties/subpopulations, along with the secretion of inflammatory cytokines, contributes to a decline in the quality of the whole imMKCL population.

Feasibility of Laparoscopic Sleeve Gastrectomy for Patients with Obesity and Disorders of Intellectual Development: a Single Institutional Experience.

BACKGROUND: Owing to their difficulty following clinical advice for procedural safety and ideal surgical outcomes, bariatric and metabolic surgery (BMS) for patients with disorders of intellectual development (DID) is concerning. Studies reporting the feasibility of BMS for this population remain scarce. This study aims to clarify the feasibility of laparoscopic sleeve gastrectomy (LSG) for patients with clinically severe obesity and DID. METHODS: A retrospective analysis of a single institutional prospective database collected from 2010 to 2022 was performed. The Wechsler Adult Intelligence Scale (WAIS) was used to measure intellectual ability before LSG. A multidisciplinary team approach was implemented to give special support and care to patients with DID. Patients were categorized into groups according to their WAIS scores. LSG outcomes were statistically compared between the DID and average intellectual ability groups. RESULTS: Using the WAIS to measure intellectual ability among patients who underwent LSG, we identified 14 patients with DID (IQ score: < 69, mean IQ: 63.4) and 71 with average intellectual ability (IQ score: 90-109, mean IQ: 98.9). Operative outcomes were comparable between the groups as follows: operation time (DID: 163 ± 41 min, average intelligence: 162 ± 30 min), hospital stay (DID: 4 [4-5] days, average intelligence: 5 [4-6] days), and total comorbidities (DID: 7.1%, average intelligence: 8.4%). No reoperations were performed, and no mortalities were observed. CONCLUSIONS: With medical and social support and care, performing LSG on patients with clinically severe obesity and DID is safe, with good short-term results.

Synthetic translational regulation by an L7Ae-kink-turn RNP switch.

The regulation of cell signaling pathways and the reconstruction of genetic circuits are important aspects of bioengineering research. Both of these goals require molecular devices to transmit information from an input biomacromolecule to the desired outputs. Here, we show that an RNA-protein (RNP)-containing L7Ae-kink-turn interaction can be used to construct translational regulators under control of an input protein that regulates the expression of desired output proteins. We built a system in which L7Ae, an archaeal ribosomal protein, regulates the translation of a designed mRNA in vitro and in human cells. The translational regulator composed of the RNP might provide new therapeutic strategies based on the detection, repair or rewiring of intrinsic cellular defects, and it may also serve as an invaluable tool for the dissection of the behavior of complex, higher-order circuits in the cell.

A versatile and robust cell purification system with an RNA-only circuit composed of microRNA-responsive ON and OFF switches.

Human induced pluripotent stem cells (iPSCs) are promising cell resources for cell therapy and drug discovery. However, iPSC-derived differentiated cells are often heterogenous and need purification using a flow cytometer, which has high cost and time consumption for large-scale purification. MicroRNAs (miRNAs) can be used as cell selection markers, because their activity differs between cell types. Here, we show miRNA-responsive ON and OFF switch mRNAs for robust cell purification. The ON switch contains a miRNA-target sequence after the polyadenylate tail, triggering translational activation by sensing the target miRNA. By designing RNA-only circuits with miRNA-ON and -OFF switch mRNAs that encode a lethal ribonuclease, Barnase, and its inhibitor, Barstar, we efficiently purified specific cell types, including human iPSCs and differentiated cardiomyocytes, without flow cytometry. Synthetic mRNA circuits composed of ON and OFF switches provide a safe, versatile, and time-saving method to purify various cell types for biological and clinical applications.

Metabolic surgery in treatment of obese Japanese patients with type 2 diabetes: a joint consensus statement from the Japanese Society for Treatment of Obesity, the Japan Diabetes Society, and the Japan Society for the Study of Obesity.

Bariatric surgery has been shown to have a variety of metabolically beneficial effects for patients with type 2 diabetes (T2D), and is now also called metabolic surgery. At the 2nd Diabetes Surgery Summit held in 2015 in London, the indication for bariatric and metabolic surgery was included in the "algorithm for patients with type T2D". With this background, the Japanese Society for Treatment of Obesity (JSTO), the Japan Diabetes Society (JDS) and the Japan Society for the Study of Obesity (JASSO) have formed a joint committee to develop a consensus statement regarding bariatric and metabolic surgery for the treatment of Japanese patients with T2D. Eventually, the consensus statement was announced at the joint meeting of the 38th Annual Meeting of JSTO and the 41st Annual Meeting of JASSO convened in Toyama on March 21, 2021. In preparing the consensus statement, we used Japanese data as much as possible as scientific evidence to consider the indication criteria, and set two types of recommendation grades, "recommendation" and "consideration", for items for which recommendations are possible. We hope that this statement will be helpful in providing evidence-based high-quality care through bariatric and metabolic surgery for the treatment of obese Japanese patients with T2D. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13340-021-00551-0.

Correction to: Metabolic surgery in treatment of obese Japanese patients with type 2 diabetes: a joint consensus statement from the Japanese Society for Treatment of Obesity, the Japan Diabetes Society, and the Japan Society for the Study of Obesity.

[This corrects the article DOI: 10.1007/s13340-021-00551-0.].

Correction to: Metabolic surgery in treatment of obese Japanese patients with type 2 diabetes: a joint consensus statement from the Japanese Society for Treatment of Obesity, the Japan Diabetes Society, and the Japan Society for the Study of Obesity.

[This corrects the article DOI: 10.1007/s13340-021-00551-0.].

Light-controllable RNA-protein devices for translational regulation of synthetic mRNAs in mammalian cells.

The photo-regulation of transgene expression is one effective approach in mammalian synthetic biology due to its high spatial and temporal resolution. While DNAs are mainly used as vectors, modified RNAs (modRNAs) are also useful for medical applications of synthetic biology, because they can avoid insertional mutagenesis and immunogenicity. However, the optogenetic control of modRNA-delivered transgenes is much more difficult than that of DNA-delivered transgenes. Here, we develop two types of photo-controllable translational activation systems that are compatible with modRNAs. One is composed of a heterodimerization domain-fused split translational activator protein and a photocaged heterodimerizer. The other is composed of a destabilizing domain-fused translational activator protein and a photocaged stabilizer. The destabilized type can be used for not only translational activation but also translational repression of the modRNAs. These photo-controllable translation systems will expand the application of mammalian synthetic biology research.

Impact of Mental Health Background and Nutrition Intake on Medium-Term Weight Loss in Japanese Patients Undergoing Laparoscopic Sleeve Gastrectomy.

OBJECTIVES: Bariatric surgery is the most effective weight loss therapy, and recently laparoscopic sleeve gastrectomy (LSG) is gaining popularity worldwide. On the other hand, patients undergoing bariatric surgery have a high prevalence of mental disorders. A Japanese nationwide survey reported high prevalence of mental disorders in patients with low percent total weight loss (%TWL) and also in those with high %TWL. The aim of this study was to investigate the relationship of 1-year %TWL with background mental health status, 3-year outcomes, and nutrition intake in Japanese patients after LSG. METHODS: This study was a single-center retrospective database analysis. A total of 89 Japanese patients who underwent LSG and were followed for 3 years were enrolled (mean age 41.9 years, baseline body mass index 44.9, baseline glycosylated hemoglobin, HbA1c, 7.0%). The patients were divided into 3 groups according to 1-year %TWL as follows: ≤19.9% (insufficient group), 20.0-34.9% (average group) and ≥35.0% (excessive group). Psychosocial and nutritional status as well as physical data were collected from all patients. RESULTS: The prevalence of mental disorders was 51.7%, and 1-year %TWL was 28.1% in all patients. No significant differences were observed in the changes in body weight and HbA1c between patients with and those without mental disorders. The prevalence of mental disorders was particularly high in the insufficient and excessive groups. In the insufficient group, mood disorders and mental retardation/developmental disorders were frequent, and snacking and eating out habits were often observed. In the excessive group, the frequencies of mood disorders and binge eating were high, and a decrease in skeletal muscle mass due to low protein intake was observed. Furthermore, weight regain was shown 12 months after LSG in both groups. In the average group, there were fewer problems in weight loss outcomes, mental health, nutrition intake and body composition. CONCLUSIONS: Psychosocial and nutritional problems were often found not only in patients with insufficient weight loss, but also in those with seemingly "excellent" weight reduction. To improve long-term weight loss outcome and future health, a multidisciplinary approach focusing on mental health and nutrition is essential for patients undergoing bariatric surgery.

Numerical operations in living cells by programmable RNA devices.

Integrated bioengineering systems can make executable decisions according to the cell state. To sense the state, multiple biomarkers are detected and processed via logic gates with synthetic biological devices. However, numerical operations have not been achieved. Here, we show a design principle for messenger RNA (mRNA) devices that recapitulates intracellular information by multivariate calculations in single living cells. On the basis of this principle and the collected profiles of multiple microRNA activities, we demonstrate that rationally programmed mRNA sets classify living human cells and track their change during differentiation. Our mRNA devices automatically perform multivariate calculation and function as a decision-maker in response to dynamic intracellular changes in living cells.

Background characteristics and postoperative outcomes of insufficient weight loss after laparoscopic sleeve gastrectomy in Japanese patients.

AIM: Laparoscopic sleeve gastrectomy (LSG) is becoming popular in Japan, but insufficient weight loss is often observed in patients after LSG. We investigated the effect of LSG on obesity-related comorbidities and identified the background characteristics of Japanese patients with insufficient weight loss after LSG. METHODS: In this multi-institutional retrospective study at 10 certified bariatric institutions, 322 Japanese patients who underwent LSG with a follow-up period of more than 2 years were analyzed. Anthropometry, obesity-related comorbidities and psychosocial background data were collected. Weight loss was expressed as 2-year percent total weight loss (%TWL). RESULTS: Mean age, body weight, body mass index (BMI) and glycated hemoglobin were 46.9 years, 119.2 kg, 43.7 kg/m2 and 7.1%, respectively. Prevalence of mental disorders was 26.3%. Mean BMI declined to 30.3 kg/m2 at 2 years and %TWL was 29.9%. Improvements in the markers and prevalence of obesity-related comorbidities were observed. Remission rates of diabetes, dyslipidemia and hypertension were 75.6%, 59.7% and 41.8%, respectively. %TWL at the respective cut-off level of diabetes remission was 20.8%. Lower remission rates of diabetes in patients with %TWL <20%, and less calorie restriction and higher prevalence of mental disorders (46.9%) in patients with %TWL <15% were observed. Frequencies of %TWL <15% and <20% were 6.5% and 18.5%, respectively. CONCLUSION: %TWL 20% was a candidate cut-off point of insufficient weight loss for diabetes remission after LSG, and mental disorders might be relevant to intractable obesity in Japanese patients.

Protein-driven RNA nanostructured devices that function in vitro and control mammalian cell fate.

Nucleic acid nanotechnology has great potential for future therapeutic applications. However, the construction of nanostructured devices that control cell fate by detecting and amplifying protein signals has remained a challenge. Here we design and build protein-driven RNA-nanostructured devices that actuate in vitro by RNA-binding-protein-inducible conformational change and regulate mammalian cell fate by RNA-protein interaction-mediated protein assembly. The conformation and function of the RNA nanostructures are dynamically controlled by RNA-binding protein signals. The protein-responsive RNA nanodevices are constructed inside cells using RNA-only delivery, which may provide a safe tool for building functional RNA-protein nanostructures. Moreover, the designed RNA scaffolds that control the assembly and oligomerization of apoptosis-regulatory proteins on a nanometre scale selectively kill target cells via specific RNA-protein interactions. These findings suggest that synthetic RNA nanodevices could function as molecular robots that detect signals and localize target proteins, induce RNA conformational changes, and programme mammalian cellular behaviour.Nucleic acid nanotechnology has great potential for future therapeutic applications. Here the authors build protein-driven RNA nanostructures that can function within mammalian cells and regulate the cell fate.

Efficient, Selective Removal of Human Pluripotent Stem Cells via Ecto-Alkaline Phosphatase-Mediated Aggregation of Synthetic Peptides.

The incomplete differentiation of human induced pluripotent stem cells (iPSCs) poses a serious safety risk owing to their potential tumorigenicity, hindering their clinical application. Here, we explored the potential of phospho-D-peptides as novel iPSC-eliminating agents. Alkaline phosphatases overexpressed on iPSCs dephosphorylate phospho-D-peptides into hydrophobic peptides that aggregate and induce cell death. We isolated a peptide candidate, D-3, that selectively and rapidly induced toxicity in iPSCs within 1 hr but had little influence on various non-iPSCs, including primary hepatocytes and iPSC-derived cardiomyocytes. Two hours of D-3 treatment efficiently eliminated iPSCs from both single cultures and co-cultures spiked with increasing ratios of iPSCs. In addition, D-3 prevented residual iPSC-induced teratoma formation in a mouse tumorigenicity assay. These results suggest the enormous potential of D-3 as a low-cost and effective anti-iPSC agent for both laboratory use and for the safe clinical application of iPSC-derived cells in regenerative medicine.

Altered Neuronal Activity Topography Markers in the Elderly with Increased Atherosclerosis.

Background: Previously, we reported on vascular cognitive impairment (VCI) templates, consisting of patients with VCI associated with carotid stenosis (>60%) using a quantitative electroencephalographic (EEG) technique called neuronal activity topography (NAT). Here using the VCI templates, we investigated the hypothesis that internal carotid artery-intima-media thickness (ICA-IMT) is associated with EEG spectrum intensity (sNAT) and spectrum steepness (vNAT). Methods: A total of 221 community-dwelling elderly subjects were recruited. Four groups were classified according to quartiles of ICA-IMT as assessed by ultrasonography: control group A, normal (≤0.9 mm); group B, mild atherosclerosis (1-1.1 mm); group C, moderate atherosclerosis (1.2-1.8 mm); and group D, severe atherosclerosis (≥1.9 mm). EEG markers of power ratio index (PRI), and the binary likelihood of being in the VCI group vs. the that of being in control group A (sL x:VCI-A , vL x:VCI-A ) were assessed, respectively. Differences in mean total scores for PRI, sL x:VCI-A , vL x:VCI-A , between control group A and the other groups were compared using Dunnett's test, respectively. Results: The mean total scores of the PRI were 3.25, 3.00, 2.77, and 2.26 for groups A, B, C, and D, respectively. There was a significant decrease in the PRI in group D compared with group A (P = 0.0066). The mean total scores of the sL x:VCI-A were -0.14, -0.11, -0.1, and -0.03 for groups A, B, C, and D, respectively. The sL x:VCI-A in group D was significantly higher compared to that in group A (P < 0.0001). The mean total scores of the vL x:VCI-A were -0.04,-0.01, 0.01, and 0.06 for group A, B, C, and D, respectively. The vL x:VCI-A in group D and group C was significantly higher compared to that in group A, respectively (P < 0.0001, P = 0.02). Conclusion: Community-dwelling elderly subjects in the increased carotid atherosclerosis of ICA-IMT (≥1.9 mm) were at greatest risk of an EEG change as assessed by NAT.