A splice-switching oligonucleotide treatment ameliorates glycogen storage disease type 1a in mice with G6PC c.648G>T.

Glycogen storage disease type 1a (GSD1a) is caused by a congenital deficiency of glucose-6-phosphatase-α (G6Pase-α, encoded by G6PC), which is primarily associated with life-threatening hypoglycemia. Although strict dietary management substantially improves life expectancy, patients still experience intermittent hypoglycemia and develop hepatic complications. Emerging therapies utilizing new modalities such as adeno-associated virus and mRNA with lipid nanoparticles are under development for GSD1a but potentially require complicated glycemic management throughout life. Here, we present an oligonucleotide-based therapy to produce intact G6Pase-α from a pathogenic human variant, G6PC c.648G>T, the most prevalent variant in East Asia causing aberrant splicing of G6PC. DS-4108b, a splice-switching oligonucleotide, was designed to correct this aberrant splicing, especially in liver. We generated a mouse strain with homozygous knockin of this variant that well reflected the pathophysiology of patients with GSD1a. DS-4108b recovered hepatic G6Pase activity through splicing correction and prevented hypoglycemia and various hepatic abnormalities in the mice. Moreover, DS-4108b had long-lasting efficacy of more than 12 weeks in mice that received a single dose and had favorable pharmacokinetics and tolerability in mice and monkeys. These findings together indicate that this oligonucleotide-based therapy could provide a sustainable and curative therapeutic option under easy disease management for GSD1a patients with G6PC c.648G>T.

Possible contribution of phosphate to the pathogenesis of chronic kidney disease in dolphins.

This study aimed to investigate whether phosphate contributes to the pathogenesis of chronic kidney disease (CKD) in dolphins. Renal necropsy tissue of an aged captive dolphin was analyzed and in vitro experiments using cultured immortalized dolphin proximal tubular (DolKT-1) cells were performed. An older dolphin in captivity died of myocarditis, but its renal function was within the normal range until shortly before death. In renal necropsy tissue, obvious glomerular and tubulointerstitial changes were not observed except for renal infarction resulting from myocarditis. However, a computed tomography scan showed medullary calcification in reniculi. Micro area X-ray diffractometry and infrared absorption spectrometry showed that the calcified areas were primarily composed of hydroxyapatite. In vitro experiments showed that treatment with both phosphate and calciprotein particles (CPPs) resulted in cell viability loss and lactate dehydrogenase release in DolKT-1 cells. However, treatment with magnesium markedly attenuated this cellular injury induced by phosphate, but not by CPPs. Magnesium dose-dependently decreased CPP formation. These data support the hypothesis that continuous exposure to high phosphate contributes to the progression of CKD in captive-aged dolphins. Our data also suggest that phosphate-induced renal injury is mediated by CPP formation in dolphins, and it is attenuated by magnesium administration.

Tyrosinase-Related Protein2 Peptide with Replacement of N-Terminus Residue by Cysteine Binds to H-2Kb and Induces Antigen-Specific Cytotoxic T Lymphocytes after Conjugation with CpG-DNA.

Recent studies have shown the potent efficacy of peptide-based vaccines for cancer immunotherapy. Immunological performance is optimized through the co-delivery of adjuvant and antigenic peptide molecules to antigen-presenting cells simultaneously. In our previous study, we showed that a conjugate consisting of 40-mer CpG-DNA and an antigenic ovalbumin peptide through disulfide bonding could efficiently induce ovalbumin-specific cytotoxic T lymphocyte (CTL) responses in vivo. In this study, based on the conjugation design, we prepared a conjugate consisting of 30-mer CpG-DNA (CpG30) and a cancer antigenic peptide of Tyrosinase-related protein 2 (TRP2180-188) using a cysteine residue attached at the N-terminus of TRP2180-188. However, the immunization of mice with this conjugate did not induce efficient TRP2180-188-specific immune responses. It was thought that the resultant peptide (10-mer) cleaved from the conjugate might be too long to fit into the H-2Kb molecule because the optimal length for binding to it is 8-9 amino acids. We newly designed a conjugate consisting of CpG30 and the C-TRP2181-188 peptide (9-mer), in which the N-terminal serine residue of TRP2180-188 is replaced by a cysteine. By adjusting the peptide length, we succeeded in inducing strong TRP2180-188 peptide-specific CTL activity upon immunization with the CpG30-C-TRP2181-188 conjugate. Furthermore, various CpG30-C-TRP2181-188 conjugates having other CpG-DNA sequences or cysteine analogues also induced the same level of CTL activity. Therefore, CpG-C-peptide conjugates prepared by replacement of the amino acid residue at the N-terminus with a cysteine residue could be a new and effective platform for peptide vaccines for targeting specific antigens of cancers and infectious diseases.

Novel deterministic epidemic model considering mass vaccination and lockdown against coronavirus disease 2019 spread in Israel: a numerical study.

Why public health intervention by the Israeli government against coronavirus disease 2019 spread has been successful while the majority of other countries are still coping with it? To give a quantitative answer, a simple numerical epidemic model is prepared to simulate the entire trend of various infection-related variables considering the first and second vaccination campaigns against the alpha variant and simultaneous lockdown. This model is an extension of our previously published deterministic physical model, that is Apparent Time Lag Model, which aims at predicting an entire trend of variables in a single epidemic. The time series data of both vaccine dose ratio and lockdown period are employed in the model. Predictions have been compared with observed data in terms of daily new cases, isolated people, infections at large and effective reproductive number, and, further, the model is verified. Moreover, parameter survey calculations for several scenarios have clarified the synergy effects of vaccination and lockdown. In particular, the key element of Israel's success has been suggested to lie in a high-dose vaccination rate that prevents the onset of a rebound in daily new cases on the rescission of the lockdown.

Infection spread simulation technology in a mixed state of multi variant viruses.

ATLM (Apparent Time Lag Model) was extended to simulate the spread of infection in a mixed state of the variant virus and original wild type. It is applied to the 4th wave of infection spread in Tokyo, and (1) the 4th wave bottoms out near the end of the state of emergency, and the number of infected people increases again. (2) The rate of increase will be mainly by d strain (L452R) virus, while the increase by a strain (N501Y) virus will be suppressed. (3) It is anticipated that the infection will spread during the Olympic Games. (4) When variant viruses compete, the infection of highly infectious virus rises sharply while the infection by weakly infectious ones has converged. (5) It is effective as an infection control measure to find an infected person early and shorten the period from infection to quarantine by PCR test or antigen test as a measure other than the vaccine.

Renadirsen, a Novel 2'OMeRNA/ENA® Chimera Antisense Oligonucleotide, Induces Robust Exon 45 Skipping for Dystrophin In Vivo.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients' DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2'-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2'OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.

A novel model of ischemia in rats with middle cerebral artery occlusion using a microcatheter and zirconia ball under fluoroscopy.

The failure of neuroprotective treatment-related clinical trials may be partially caused by unestablished animal models. Existing animal models are less likely to provide occlusion confined to the middle cerebral artery (MCA), making transarterial intervention difficult. We aimed to develop a novel focal stroke model using a microcatheter and zirconium dioxide that is non-magnetic under fluoroscopic guidance, which can monitor MCA occlusion and can improve hemorrhagic complications. Using male Sprague Dawley rats (n = 10), a microcatheter was navigated from the caudal ventral artery to the left internal carotid artery using an X-ray fluoroscopy to establish local occlusion. All rat cerebral angiographies were successful. No rats had hemorrhagic complications. Eight (80%) rats underwent occlusion of the MCA bifurcation by zirconium dioxide. Accidentally, the left posterior cerebral artery was failure embolized in 2 rats (20%). The median operating time was 8 min. All rats of occlusion MCA revealed an incomplete hemiparesis on the right side with neurological deficit score ranging from 1 to 3 (median 1, interquartile range 1-3) at 24 h after the induction of ischemia. Moreover, 2% 2,3,5-triphenyl tetrazolium chloride staining showed that the median infarct volume (mm3) was 280 (interquartile range 267-333) 24 h after the left MCA bifurcation occlusion. We present a novel rat model for focal stroke using a microcatheter and zirconium dioxide which does not affect the MRI. The model is predictable which is well confined within the territory supplied by the MCA, and reproducibility of this model is 80%. Fluoroscopy was able to identify which the MCA occlusion and model success while creating the model. It permitted exclusion of animals with complications from the experiment.

RNA interference activity of single-stranded oligonucleotides linked between the passenger strand and the guide strand with an aryl phosphate linker.

Recently, we demonstrated that asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined 2'-O-methyl RNA and DNA, termed MED-siRNAs, show high RNase resistance, efficient cleavage of target mRNA, and the subsequent reduction of target protein expression. The 5'-terminal phosphate group and the 3'-overhang of the guide strand were required to fully activate the RNAi activity of MED-siRNAs. Here, we evaluated MED-siRNAs modified with aryl phosphate groups at the 5'-end of the guide strand. The 5'-aryl phosphorylated MED-siRNAs showed highly efficient reduction of target protein expression comparable to 5'-phosphorylated MED-siRNAs. Moreover, 5'-aryl phosphorylated MED-siRNAs linked between the aryl phosphate group at the 5'-end of the guide strand and the hydroxyl group at the 3'-end of the passenger strand with alkyl amide linkers or peptides (e.g., DL-Ser-L-Ala-L-Tyr), resulted in single-stranded MED-siRNAs with a highly efficient cleavage activity of target mRNA with binding to Argonaute 2 via an RNA interference mechanism. These linker techniques could also be used to create siRNAs composed of naturally-occurring molecules such as amino acids. These findings suggest the possibility of using these single-stranded MED-siRNAs as siRNA reagents.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1927077 .

Marmoset angiography just by percutaneous puncture of the caudal ventral artery.

Surgery in humans is continuously evolving and promoted minimally invasive treatment. On the other hand, despite the importance of the 3Rs principles for experimental animals is well documented, no reports describe specific methodologies for implementing "refinement" in practice. Here, we describe a new technique, the "Ohta Method" for caudal arthrocentesis in the pursuit of the 3Rs for animal experiments and the development of innovative methods for investigating systemic organ arteries through minimally invasive procedures. This procedure requires only a percutaneous puncture of the caudal artery without any injury to the limb or body trunk. In addition, it does not cut down the artery, making hemostasis easier and recovering arterial damage easier. We will show multiple organ artery angiographies in marmoset for the first time in the world. The principle described in this paper could also be applied to many other small animals, such as rats. Moreover, using this method, multiple doses of the drug or cells can be administered to the target organ at the time of therapeutic intervention, thereby enabling the establishment of more sophisticated and complex therapeutic intervention studies as translational research.

A reproducible swine model of a surgically created saccular thoracic aortic aneurysm.

A reproducible swine thoracic aortic aneurysm (TAA) model is useful for investigating new therapeutic interventions. We report a surgical method for creating a reproducible swine saccular TAA model. We used eight female swine weighing 20-25 kg (LWD; ternary species). All procedures were performed under general anesthesia and involved left thoracotomy. Following aortic cross-clamping, the thoracic aorta was surgically dissected and the media and intima were resected, and the dissection plane was extended by spreading the outer layer for aneurysmal space. Subsequently, only the adventitial layer of the aorta was sutured. At 2 weeks after these procedures, angiography and computed tomography were performed. After follow-up imaging, the model animals were euthanized. Macroscopic, histological, and immunohistological examinations were performed. All model animals survived, and a saccular TAA was confirmed by follow-up imaging in all cases. The mean length of the shorter and the longer aortic diameter after the procedure were 14.01 ± 1.0 mm and 18.35 ± 1.4 mm, respectively (P<0.001). The rate of increase in the aortic diameter was 131.7 ± 13.8%, and the mean length of aneurysmal change at thoracic aorta was 22.4 ± 1.9 mm. Histological examination revealed intimal tears and defects of elastic fibers in the media. Immunostaining revealed MMP-2 and MMP-9 expressions at the aneurysm site. We report our surgical method for creating a swine saccular TAA model. Our model animal may be useful to investigate new therapeutic interventions for aortic disease.

Are lifestyle pattern changes associated to poor subjective sleep quality?: a cross-sectional study by gender among the general Japanese population underwent specified medical check-ups in 2014 and 2015.

OBJECTIVES: Subjective sleep quality (SSQ) is defined by the satisfaction of one's overall sleep experience and is composed of sleep depth and restfulness. It has not been clarified how poor SSQ is associated to changes in lifestyles. The purpose is to reveal the association of lifestyle pattern changes and poor SSQ. DESIGN: A cross-sectional study. SETTING: The data on basic attributes, SSQ and lifestyle such as presence/absence of smoking, exercise, physical activity, supper time close to bedtime, drinking habits and alcohol intake amount per day were obtained from database and questionnaire of specified medical check-ups in fiscal year 2014-2015 in Japan. The analysis was conducted in 2019. PARTICIPANTS: The subjects comprised 49 483 residents (26 087 men and 23 396 women), aged 40-74 years who had undergone an annual specified medical check-up from 2014 to 2015 in Fukushima Prefecture, Japan. OUTCOME MEASURE: Status of SSQ in 2015 was assessed using a question asking whether or not the subjects usually got enough sleep. Poor SSQ in 2015 and lifestyle pattern changes in 2014-2015 were compared between those who were in healthy status both in 2014 and 2015 (referent) and non-referent, using binary logistic regression analysis. RESULTS: Unhealthy lifestyle pattern for 2014-2015 was significantly associated to poor SSQ in 2015: 'absent to absent' in exercise for men (OR=1.472; 95% CI 1.316 to 1.647) and women (OR=1.428; 95% CI 1.285 to 1.587), physical activity for men (OR=1.420; 95% CI 1.270 to 1.588) and women (OR=1.471; 95% CI 1.322 to 1.638) and 'present to present' in supper time for men (OR=1.149; 95% CI 1.020 to 1.294) and women (OR=1.288; 95% CI 1.102 to 1.505). CONCLUSIONS: Healthcare workers may be able to contribute to the improvement of SSQ, focusing on changeable lifestyles.

An Epidemiological Model Considering Isolation to Predict COVID-19 Trends in Tokyo, Japan: Numerical Analysis.

BACKGROUND: COVID-19 currently poses a global public health threat. Although Tokyo, Japan, is no exception to this, it was initially affected by only a small-level epidemic. Nevertheless, medical collapse nearly happened since no predictive methods were available to assess infection counts. A standard susceptible-infectious-removed (SIR) epidemiological model has been widely used, but its applicability is limited often to the early phase of an epidemic in the case of a large collective population. A full numerical simulation of the entire period from beginning until end would be helpful for understanding COVID-19 trends in (separate) counts of inpatient and infectious cases and can also aid the preparation of hospital beds and development of quarantine strategies. OBJECTIVE: This study aimed to develop an epidemiological model that considers the isolation period to simulate a comprehensive trend of the initial epidemic in Tokyo that yields separate counts of inpatient and infectious cases. It was also intended to induce important corollaries of governing equations (ie, effective reproductive number) and equations for the final count. METHODS: Time-series data related to SARS-CoV-2 from February 28 to May 23, 2020, from Tokyo and antibody testing conducted by the Japanese government were adopted for this study. A novel epidemiological model based on a discrete delay differential equation (apparent time-lag model [ATLM]) was introduced. The model can predict trends in inpatient and infectious cases in the field. Various data such as daily new confirmed cases, cumulative infections, inpatients, and PCR (polymerase chain reaction) test positivity ratios were used to verify the model. This approach also derived an alternative formulation equivalent to the standard SIR model. RESULTS: In a typical parameter setting, the present ATLM provided 20% less infectious cases in the field compared to the standard SIR model prediction owing to isolation. The basic reproductive number was inferred as 2.30 under the condition that the time lag T from infection to detection and isolation is 14 days. Based on this, an adequate vaccine ratio to avoid an outbreak was evaluated for 57% of the population. We assessed the date (May 23) that the government declared a rescission of the state of emergency. Taking into consideration the number of infectious cases in the field, a date of 1 week later (May 30) would have been most effective. Furthermore, simulation results with a shorter time lag of T=7 and a larger transmission rate of α=1.43α0 suggest that infections at large should reduce by half and inpatient numbers should be similar to those of the first wave of COVID-19. CONCLUSIONS: A novel mathematical model was proposed and examined using SARS-CoV-2 data for Tokyo. The simulation agreed with data from the beginning of the pandemic. Shortening the period from infection to hospitalization is effective against outbreaks without rigorous public health interventions and control.

Immune Responses and Antitumor Effect through Delivering to Antigen Presenting Cells by Optimized Conjugates Consisting of CpG-DNA and Antigenic Peptide.

Immunotherapy using antigen-specific cytotoxic T lymphocytes (CTLs) has become one of the most attractive strategies for cancer treatment. For the induction of antigen-specific CTLs in vivo, the co-delivery of CpG-DNAs and antigens to the same antigen-presenting cells (APCs) is a promising strategy. In this study, we prepared conjugates consisting of 40mer of CpG-DNA (CpG40) and antigenic peptide (OVA257-264), which have the following distinctive features: (1) multiple CpG motifs in a molecule; (2) cleavage in the cytosol because of the disulfide bonding via cysteine residue between peptide and CpG-DNA; (3) conjugation designed to induce antigen presentation on MHC class I molecules. Immunization with the conjugate CpG40-C-OVA257-264 at the mouse tail base induced strong CTL activity at a very low peptide dose of 20 ng/head. It was found that the conjugates were internalized into C-type mannose receptor 1 (MRC1)-expressing cells in inguinal lymph nodes, indicating that the CpG portion in the conjugate acts as not only an adjuvant for the activation of TLR9 but also a carrier to APCs expressing MRC1. In a tumor-bearing mice model, mice immunized with CpG40-C-OVA257-264 conjugates exhibited long delays in tumor growth compared with those treated with PBS, OVA257-264 alone, or a mixture of CpG40 and OVA257-264. Therefore, CpG-C-peptide conjugates could be a new and effective platform for peptide vaccine for the treatment of cancers and infectious diseases.

Evaluating the Knockdown Activity of MALAT1 ENA Gapmers In Vitro.

Antisense oligonucleotides (ASOs) are widely used for the identification of gene functions and regulation of genes involved in different diseases for therapeutic purposes. For in vitro evaluation of the knockdown activity of gapmer ASOs, we often use lipofection or electroporation to deliver gapmer ASOs into the cells. Here, we describe a method for evaluating the knockdown activity of gapmer ASOs by a cell-free uptake mechanism, termed as gymnosis, using MALAT1 gapmer ASOs modified with 2'-O-methoxyethyl RNA (2'-MOE) or 2'-O,4'-C-ethylene-bridged nucleic acid (ENA). This method is robust because it does not involve the use of any transfection reagent and has minimal effects on cell growth. Further, we describe a convenient technique for performing one-step reverse transcription and real-time qPCR using cell lysates without RNA extraction. Data for up to 96 samples can be obtained following these methods.

The profile of lipid metabolites in urine of marmoset wasting syndrome.

Marmoset wasting syndrome (MWS) is clinically characterized by progressive weight loss. Although morbidity and mortality of MWS are relatively high in captive marmosets, its causes remain unknown. Lipid mediators are bioactive metabolites which are produced from polyunsaturated fatty acids, such as arachidonic acid (AA) and eicosapentaenoic acid. These lipid metabolites regulate a wide range of inflammatory responses and they are excreted into the urine. As urinary lipid profiles reflect systemic inflammatory conditions, we comprehensively measured the levels of 141 types of lipid metabolites in the urines obtained from healthy common marmoset (Callithrix jacchus) (N = 7) or marmosets with MWS (N = 7). We found that 41 types of metabolites were detected in all urine samples of both groups. Among them, AA-derived metabolites accounted for 63% (26/41 types) of all detected metabolites. Notably, the levels of AA-derived prostaglandin (PG) E2, PGF2α, thromboxane (TX) B2 and F2-isoprostanes significantly increased in the urine samples of marmosets with MWS. In this study, we found some urinary lipid metabolites which may be involved in the development of MWS. Although the cause of MWS remains unclear, our findings may provide some insight into understanding the mechanisms of development of MWS.

Development of an exon skipping therapy for X-linked Alport syndrome with truncating variants in COL4A5.

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.

An improved synthesis of 2'-O,4'-C-ethylene nucleic acid (ENA) and thermodynamic studies of duplex formation containing the guanosine ENA unit.

Oligonucleotides containing 2'-O,4'-C-ethylene nucleic acids (ENA) have been proven highly effective for antisense therapeutics. 2'-O,4'-C-Ethyleneguanosine and its phosphoramidite were previously obtained from 3,5-di-O-benzy1-4-C-(p-tolulenesulfonyloxyethyl)-1,2-di-O-acetyl-α-D-erythropentofuranose by glycosylation, but with limited efficiency. Using 3,5-di-O-benzy1-4-C-(2-t-butyldiphenylsilyloxyethyl)-1,2-di-O-acetyl-α-D-erythropentofuranose as an alternative substrate, we developed several methods to obtain 2'-O,4'-C-ethyleneguanosine derivatives with much higher yields than previously reported. These methods were also applicable for the synthesis of 2'-O,4'-C-ethyleneadenosine and 2'-O,4'-C-ethylene-5-methyluridine derivatives. Moreover, we investigated the thermodynamic benefit of DNA strands containing 2'-O,4'-C-ethyleneguanosines during duplex formation with complementary RNA. Only a single modification by the nucleoside resulted in a 10-fold greater binding constant of the DNA/RNA duplex.

Design of 2'-O-methyl RNA and DNA double-stranded oligonucleotides: naturally-occurring nucleotide components with strong RNA interference gene expression inhibitory activity.

Double-stranded RNAs consisting of 21-nucleotide passenger and guide strands, known as small interfering RNAs (siRNAs), can be used for the identification of gene functions and the regulation of genes involved in disease for therapeutics. The difficulty with unmodified siRNAs lies in the chemical synthesis of RNA, its degradation by RNase, the immune response derived from natural RNA, and the off-target effects mediated by the passenger strand. In this study, asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined DNAs and 2'-O-methyl RNAs, denoted as MED-siRNA, were evaluated. These modified oligonucleotides showed high RNase resistance, a reduced immune response, a highly efficient cleavage of target mRNA with binding to Argonaute 2 (Ago2) via RNA interference, and the subsequent reduction of target protein expression. These findings suggest the possibility of alternatives to unmodified siRNAs with potential use in therapeutics.

Evolution of the avian digital pattern.

Variation in digit number has occurred multiple times in the history of archosaur evolution. The five digits of dinosaur limbs were reduced to three in bird forelimbs, and were further reduced in the vestigial forelimbs of the emu. Regulation of digit number has been investigated previously by examining genes involved in anterior-posterior patterning in forelimb buds among emu (Dromaius novaehollandiae), chicken (Gallus gallus) and zebra finch (Taeniopygia guttata). It was described that the expression of posterior genes are conserved among these three birds, whereas expression of anterior genes Gli3 and Alx4 varied significantly. Here we re-examined the expression pattern of Gli3 and Alx4 in the forelimb of emu, chicken and zebra finch. We found that Gli3 is expressed in the anterior region, although its range varied among species, and that the expression pattern of Alx4 in forelimb buds is broadly conserved in a stage-specific manner. We also found that the dynamic expression pattern of the BMP antagonist Gremlin1 (Grem1) in limb buds, which is critical for autopodial expansion, was consistent with the digital pattern of emu, chicken and zebra finch. Furthermore, in emu, variation among individuals was observed in the width of Grem1 expression in forelimb buds, as well as in the adult skeletal pattern. Our results support the view that the signalling system that regulates the dynamic expression of Grem1 in the limb bud contributes substantially to variations in avian digital patterns.

Unhealthy changes in eating habits cause acute onset hypertension in the normotensive community-dwelling elderly-3 years cohort study.

The aim of this study was to determine what lifestyle changes can predict acute onset hypertension in the normotensive community-dwelling elderly.This study targeted elderly people enrolled in National Health Insurance in Fukushima Prefecture, Japan. The subjects were 24,490 people who took all of the specific health examination conducted by National Health Insurance in fiscal years 2013, 2014, and 2015 continuously and had a recorded systolic blood pressure (BP) <130 mm Hg and diastolic BP <85 mm Hg in the first 2 fiscal years. We examined their lifestyle changes for the first 2 fiscal years using the questionnaires given at the health examination. Multivariate Poisson regression analysis was conducted to examine the relationship between new-onset hypertension observed at the last examination and unhealthy lifestyle changes.The mean age of the subjects was 61.5 ±â€Š8.2 years old at baseline. We observed new-onset hypertension in 1.062 subjects at the last examination. Of the study subjects, 12,027 (49.1%) answered to having at least one of the items of unhealthy lifestyle change in the questionnaire. In the multivariate logistic regression, eating supper before bedtime showed a significant increase in the risk ratio for acute onset hypertension (risk ratio 1.27, 95% confidence interval, 1.01-1.58).This study indicated that eating before bedtime is a risk factor of new-onset hypertension in the normotensive community-dwelling elderly. Adequate health guidance to avoid unhealthy lifestyle changes is required even in normotensive people as this hypertension is preventable.