HNF4α is required for Tkfc promoter activation by ChREBP.

Triokinase/FMN cyclase (Tkfc) is involved in fructose metabolism and is responsible for the phosphorylation of glyceraldehyde to glyceraldehyde-3-phosphate. In this study, we show that refeeding induced hepatic expression of Tkfc in mice. Luciferase reporter gene assays using the Tkfc promoter revealed the existence of two hepatocyte nuclear factor 4α (HNF4α)-responsive elements (HNF4RE1 and HNF4RE2) and one carbohydrate-responsive element-binding protein (ChREBP)-responsive element (ChoRE1). Deletion and mutation of HNF4RE1 and HNF4RE2 or ChoRE1 abolished HNF4α and ChREBP responsiveness, respectively. HNF4α and ChREBP synergistically stimulated Tkfc promoter activity. ChoRE1 mutation attenuated but maintained HNF4α responsiveness, whereas HNF4RE1 and HNF4RE2 mutations abolished ChREBP responsiveness. Moreover, Tkfc promoter activity stimulation by ChREBP was attenuated upon HNF4α knockdown. Furthermore, Tkfc expression was decreased in livers of ChREBP-/- and liver-specific HNF4-/- (Hnf4αΔHep) mice. Altogether, our data indicate that Tkfc is a target gene of ChREBP and HNF4α, and Tkfc promoter activity stimulation by ChREBP requires HNF4α.

Description and Pathogenicity of Colletotrichum kapreanum sp. nov, a Cherelle Wilt Pathogen Belonging to the Gigasporum Species Complex.

Similar to cacao pod rot, cherelle wilt decreases production from cacao fields. Among all known fungal pathogens of the cacao, Colletotrichum spp. are common infectious agents that affect the cherelles and pods of cacao; thus, cacao diseases are often classified by stage. Therefore, knowing whether these pathogens are common in both fruit stages is necessary for implementing disease control measures. Symptoms of cherelle wilt were found in cacao plants in Pangasinan, Philippines, in 2022. The fungal strain obtained from the lesion was found to be pathogenic towards cherelles, but not towards pods. The strain was classified as an unknown species belonging to the gigasporum species complex, based on the morphological and molecular phylogenetic analyses of ITS, GAPDH, CHS1, ACT, and TUB2. We propose Colletotrichum kapreanum sp. nov. as a causal agent of cacao cherelle wilt, but not pod rot.

Divergent neurodevelopmental profiles of very-low-birth-weight infants.

BACKGROUND: Advanced perinatal medicine has decreased the mortality rate of preterm infants. Long-term neurodevelopmental outcomes of very-low-birth-weight infants (VLBWIs) remain to be investigated. METHODS: Participants were 124 VLBWIs who had in-hospital birth from 2007 to 2015. Perinatal information, developmental or intelligence quotient (DQ/IQ), and neurological comorbidities at ages 3 and 6 years were analyzed. RESULTS: Fifty-eight (47%) VLBWIs received neurodevelopmental assessments at ages 3 and 6 years. Among them, 15 (26%) showed DQ/IQ <75 at age 6 years. From age 3 to 6 years, 21 (36%) patients showed a decrease (≤-10), while 5 (9%) showed an increase (≥+10) in DQ/IQ scores. Eight (17%) with autism spectrum disorder or attention-deficit hyperactivity disorder (ASD/ADHD) showed split courses of DQ/IQ, including two with ≤-10 and one with +31 to their scores. On the other hand, all 7 VLBWIs with cerebral palsy showed DQ ≤35 at these ages. Magnetic resonance imaging detected severe brain lesions in 7 (47%) of those with DQ <75 and 1 (18%) with ASD/ADHD. CONCLUSIONS: VLBWIs show a broad spectrum of neurodevelopmental outcomes after 6 years. These divergent profiles also indicate that different risks contribute to the development of ASD/ADHD from those of cerebral palsy and epilepsy in VLBWIs. IMPACT: Very-low-birth-weight infants (VLBWIs) show divergent neurodevelopmental outcomes from age 3 to 6 years. A deep longitudinal study depicts the dynamic change in neurodevelopmental profiles of VLBWIs from age 3 to 6 years. Perinatal brain injury is associated with developmental delay, cerebral palsy and epilepsy, but not with ASD or ADHD at age 6 years.

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.

Rare coding variants in CHRNB2 reduce the likelihood of smoking.

Human genetic studies of smoking behavior have been thus far largely limited to common variants. Studying rare coding variants has the potential to identify drug targets. We performed an exome-wide association study of smoking phenotypes in up to 749,459 individuals and discovered a protective association in CHRNB2, encoding the ß2 subunit of the α4ß2 nicotine acetylcholine receptor. Rare predicted loss-of-function and likely deleterious missense variants in CHRNB2 in aggregate were associated with a 35% decreased odds for smoking heavily (odds ratio (OR) = 0.65, confidence interval (CI) = 0.56-0.76, P = 1.9 × 10-8). An independent common variant association in the protective direction ( rs2072659 ; OR = 0.96; CI = 0.94-0.98; P = 5.3 × 10-6) was also evident, suggesting an allelic series. Our findings in humans align with decades-old experimental observations in mice that ß2 loss abolishes nicotine-mediated neuronal responses and attenuates nicotine self-administration. Our genetic discovery will inspire future drug designs targeting CHRNB2 in the brain for the treatment of nicotine addiction.

Fusarium mindanaoense sp. nov., a New Fusarium Wilt Pathogen of Cavendish Banana from the Philippines Belonging to the F. fujikuroi Species Complex.

The pathogen causing Fusarium wilt in banana is reported to be Fusarium oxysporum f. sp. cubense (FOC). In 2019, wilt symptoms in banana plants (cultivar: Cavendish) in the Philippines were detected, i.e., the yellowing of the leaves and discoloration of the pseudostem and vascular tissue. The fungus isolated from the vascular tissue was found to be pathogenic to Cavendish bananas and was identified as a new species, F. mindanaoense, belonging to the F. fujikuroi species complex (FFSC); species classification was assessed using molecular phylogenetic analyses based on the tef1, tub2, cmdA, rpb1, and rpb2 genes and morphological analyses. A reciprocal blast search using genomic data revealed that this fungus exclusively included the Secreted in Xylem 6 (SIX6) gene among the SIX homologs related to pathogenicity; it exhibited a highly conserved amino acid sequence compared with that of species in the FFSC, but not with that of FOC. This was the first report of Fusarium wilt in Cavendish bananas caused by a species of the genus Fusarium other than those in the F. oxysporum species complex.

Utility of Positional Instillation of Contrast Cystography for Diagnosing Occult Vesicoureteral Reflux in Children: A Report of Two Cases.

Positional instillation of contrast (PIC) cystography is effective for detecting occult vesicoureteral reflux (VUR), which can not be revealed by standard voiding cystourethrography (VCUG). We experienced two cases of young female patients; one had repeated urinary tract infection with a negative VUR on standard VCUG, and the other had findings suggestive of reflux hydronephrosis and intolerance of standard VCUG. They underwent PIC cystography, and occult VUR was detected in both cases. Both were successfully treated with simultaneous endoscopic injection therapy with dextranomer/hyaluronic acid. PIC cystography is useful for detecting occult VUR in children with negative VUR findings on standard VCUG or who are unable to tolerate standard VCUG.

Changing risk factors for postpartum depression in mothers admitted to a perinatal center.

BACKGROUND: The Edinburgh postnatal depression scale (EPDS) is commonly used in screening for major postpartum depression (PPD). We explored the clinical factors associated with score changes. METHODS: Mothers (n=1,287) who delivered a single live-born infant in Kokura Medical Center in Japan during 2018-2019 were analyzed. The EPDS-Japanese version was conducted at the first and fourth weeks after childbirth. Scores of ≥9 were considered to indicate an increased risk of PPD. RESULTS: The scores improved during the four-week period (5.03±0.12 to 3.79±0.10). Primiparity, Cesarean section (CS), and a low Apgar score were identified as initial risk factors, however, primiparity remained in the multivariate analysis (aOR 2.02, 95% CI 1.37-2.97). Age ≥35 years was associated with worsened scores (aOR 1.88, 95%CI 1.01-3.51), but CS improved (aOR 0.38, 95%CI 0.21-0.70). Primiparity, CS, and neonatal respiratory support were the initial risk factors, while infant anomaly was a late risk factor in mothers whose infants were admitted to the neonatal intensive care unit (NICU) (aOR 3.35, 95%CI 1.31-8.56). In mothers of infants with an NICU stay of ≥4 weeks, infant anomaly was associated with worsened scores (aOR 6.61, 95%CI 1.11-39.3), while respiratory support was associated with improved scores (aOR 0.09, 95%CI 0.01-0.65). Twenty-six mothrs with worsened scores received psychiatric support; three developed PPD. Two of the three were ≥35 years of age, neither of their infants had anomalies. CONCLUSION: Maternal aging and infant anomaly were risk factors for PPD. PPD occurred in mothers with worsened EPDS scores after mental care. Puerperants with worsening risk factors should be targeted to control PPD.

Biallelic loss of LDB3 leads to a lethal pediatric dilated cardiomyopathy.

Autosomal dominant variants in LDB3 (also known as ZASP), encoding the PDZ-LIM domain-binding factor, have been linked to a late onset phenotype of cardiomyopathy and myofibrillar myopathy in humans. However, despite knockout mice displaying a much more severe phenotype with premature death, bi-allelic variants in LDB3 have not yet been reported. Here we identify biallelic loss-of-function variants in five unrelated cardiomyopathy families by next-generation sequencing. In the first family, we identified compound heterozygous LOF variants in LDB3 in a fetus with bilateral talipes and mild left cardiac ventricular enlargement. Ultra-structural examination revealed highly irregular Z-disc formation, and RNA analysis demonstrated little/no expression of LDB3 protein with a functional C-terminal LIM domain in muscle tissue from the affected fetus. In a second family, a homozygous LDB3 nonsense variant was identified in a young girl with severe early-onset dilated cardiomyopathy with left ventricular non-compaction; the same homozygous nonsense variant was identified in a third unrelated female infant with dilated cardiomyopathy. We further identified homozygous LDB3 frameshift variants in two unrelated probands diagnosed with cardiomegaly and severely reduced left ventricular ejection fraction. Our findings demonstrate that recessive LDB3 variants can lead to an early-onset severe human phenotype of cardiomyopathy and myopathy, reminiscent of the knockout mouse phenotype, and supporting a loss of function mechanism.

Common and rare variant associations with clonal haematopoiesis phenotypes.

Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1-5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications.

Germline Mutations in CIDEB and Protection against Liver Disease.

BACKGROUND: Exome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets. METHODS: We performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations. RESULTS: The multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, -1.24 U per liter; 95% confidence interval [CI], -1.66 to -0.83; P = 4.8×10-9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P = 9.9×10-7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, -0.98; 95% CI, -1.54 to -0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets. CONCLUSIONS: Rare germline mutations in CIDEB conferred substantial protection from liver disease. (Funded by Regeneron Pharmaceuticals.).

Molecular signatures and cellular diversity during mouse habenula development.

The habenula plays a key role in various motivated and pathological behaviors and is composed of molecularly distinct neuron subtypes. Despite progress in identifying mature habenula neuron subtypes, how these subtypes develop and organize into functional brain circuits remains largely unknown. Here, we performed single-cell transcriptional profiling of mouse habenular neurons at critical developmental stages, instructed by detailed three-dimensional anatomical data. Our data reveal cellular and molecular trajectories during embryonic and postnatal development, leading to different habenular subtypes. Further, based on this analysis, our work establishes the distinctive functional properties and projection target of a subtype of Cartpt+ habenula neurons. Finally, we show how comparison of single-cell transcriptional profiles and GWAS data links specific developing habenular subtypes to psychiatric disease. Together, our study begins to dissect the mechanisms underlying habenula neuron subtype-specific development and creates a framework for further interrogation of habenular development in normal and disease states.

Genome-wide meta-analysis of insomnia prioritizes genes associated with metabolic and psychiatric pathways.

Insomnia is a heritable, highly prevalent sleep disorder for which no sufficient treatment currently exists. Previous genome-wide association studies with up to 1.3 million subjects identified over 200 associated loci. This extreme polygenicity suggested that many more loci remain to be discovered. The current study almost doubled the sample size to 593,724 cases and 1,771,286 controls, thereby increasing statistical power, and identified 554 risk loci (including 364 novel loci). To capitalize on this large number of loci, we propose a novel strategy to prioritize genes using external biological resources and functional interactions between genes across risk loci. Of all 3,898 genes naively implicated from the risk loci, we prioritize 289 and find brain-tissue expression specificity and enrichment in specific gene sets of synaptic signaling functions and neuronal differentiation. We show that this novel gene prioritization strategy yields specific hypotheses on underlying mechanisms of insomnia that would have been missed by traditional approaches.

Mapping genomic loci implicates genes and synaptic biology in schizophrenia.

Schizophrenia has a heritability of 60-80%1, much of which is attributable to common risk alleles. Here, in a two-stage genome-wide association study of up to 76,755 individuals with schizophrenia and 243,649 control individuals, we report common variant associations at 287 distinct genomic loci. Associations were concentrated in genes that are expressed in excitatory and inhibitory neurons of the central nervous system, but not in other tissues or cell types. Using fine-mapping and functional genomic data, we identify 120 genes (106 protein-coding) that are likely to underpin associations at some of these loci, including 16 genes with credible causal non-synonymous or untranslated region variation. We also implicate fundamental processes related to neuronal function, including synaptic organization, differentiation and transmission. Fine-mapped candidates were enriched for genes associated with rare disruptive coding variants in people with schizophrenia, including the glutamate receptor subunit GRIN2A and transcription factor SP4, and were also enriched for genes implicated by such variants in neurodevelopmental disorders. We identify biological processes relevant to schizophrenia pathophysiology; show convergence of common and rare variant associations in schizophrenia and neurodevelopmental disorders; and provide a resource of prioritized genes and variants to advance mechanistic studies.

Genome-wide analysis provides genetic evidence that ACE2 influences COVID-19 risk and yields risk scores associated with severe disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human host cells via angiotensin-converting enzyme 2 (ACE2) and causes coronavirus disease 2019 (COVID-19). Here, through a genome-wide association study, we identify a variant (rs190509934, minor allele frequency 0.2-2%) that downregulates ACE2 expression by 37% (P = 2.7 × 10-8) and reduces the risk of SARS-CoV-2 infection by 40% (odds ratio = 0.60, P = 4.5 × 10-13), providing human genetic evidence that ACE2 expression levels influence COVID-19 risk. We also replicate the associations of six previously reported risk variants, of which four were further associated with worse outcomes in individuals infected with the virus (in/near LZTFL1, MHC, DPP9 and IFNAR2). Lastly, we show that common variants define a risk score that is strongly associated with severe disease among cases and modestly improves the prediction of disease severity relative to demographic and clinical factors alone.

Additional Effect of Luseogliflozin on Semaglutide in Nonalcoholic Steatohepatitis Complicated by Type 2 Diabetes Mellitus: An Open-Label, Randomized, Parallel-Group Study.

INTRODUCTION: Untreated nonalcoholic fatty liver may progress to nonalcoholic steatohepatitis (NASH) and cirrhosis and induce hepatocellular carcinoma and liver failure. Type 2 diabetes mellitus (T2DM), often complicated with nonalcoholic fatty liver disease (NAFLD), is a driver of NAFLD progression. Thus, efficacious treatment strategies for patients with coexisting NAFLD and T2DM are important for preventing NAFLD progression. Although previous studies have demonstrated that either sodium-glucose transporter 2 inhibitors (SGLT2is) or glucagon-like peptide 1 receptor agonists (GLP-1 RAs) benefit NASH patients with T2DM, the rate of NASH resolution has not sufficiently improved. Therefore, we developed a protocol for a randomized controlled trial to examine whether the addition of an SGLT2i to the treatment regimen of patients receving a GLP-1 RA (combination therapy), within the therapeutic dose range for T2DM, increases the rate of NASH resolution in patients with coexisting NASH and T2DM. METHODS: This open-label, randomized, parallel-group study commenced in June 2021, will conclude recruitment in May 2023, and will end by March 2025. Sixty patients with NASH complicated by T2DM are enrolled at the Ehime University Hospital in Toon, Japan. Participants will be randomized into: (1) an intervention group receiving combination therapy with the SGLT2i luseogliflozin 2.5 mg, once daily (Taisho Pharmaceutical, Tokyo, Japan) and the GLP-1 RA semaglutide 0.5 mg, once per week (Novonordisk, Copenhagen, Denmark); and (2) a control group receiving monotherapy with the GLP-1 analog semaglutide. The primary endpoints, which will be ascertained by liver biopsy, are: (1) NASH resolution rate from baseline without worsening of liver fibrosis after 52 weeks of intervention; (2) rate of improvement from baseline of at least 1 point in the NAFLD activity score without worsening of liver fibrosis after 52 weeks of intervention; and (3) rate of improvement from baseline of at least one fibrosis stage without worsening of NASH after 52 weeks of intervention. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trial Registry (UMIN-CTR) number: UMIN000045003. Japan Registry of Clinical Trials registration number: jRCTs061210009.

Development of an Automatic Measurement Method for CD8 and PD-1 Positive T Cells Using Image Analysis Software.

BACKGROUND/AIM: With the progress in cancer immunotherapy using immune checkpoint blockade (ICB) therapy, histological observations of tumor-infiltrating lymphocyte (TIL) status are needed to evaluate the antitumor effect of ICB using imaging analysis software. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded sections obtained from colorectal cancer and gastric cancer patients with more than 500 single nucleotide variants were stained with anti-CD8 and anti-PD-1 antibodies. Based on our own algorithm and imaging analysis software, an automatic TIL measurement method was established and compared to the manual counting methods. RESULTS: In the CD8+ T cell number measurement, there was a good correlation (r=0.738 by Pearson test) between the manual and automated counting methods. However, in the PD-1+ T cell measurement, there was a large difference in TIL numbers in both groups. After adjustment of the parameter settings, the correlation between the manual and automated methods in the PD-1+ T cell measurements improved (r=0.668 by Pearson test). CONCLUSION: An imaging software-based automatic measurement could be a simple and useful tool for evaluating the therapeutic effect of cancer immunotherapies in terms of TIL status.

Statistical testing in transcriptomic-neuroimaging studies: A how-to and evaluation of methods assessing spatial and gene specificity.

Multiscale integration of gene transcriptomic and neuroimaging data is becoming a widely used approach for exploring the molecular underpinnings of large-scale brain organization in health and disease. Proper statistical evaluation of determined associations between imaging-based phenotypic and transcriptomic data is key in these explorations, in particular to establish whether observed associations exceed "chance level" of random, nonspecific effects. Recent approaches have shown the importance of statistical models that can correct for spatial autocorrelation effects in the data to avoid inflation of reported statistics. Here, we discuss the need for examination of a second category of statistical models in transcriptomic-neuroimaging analyses, namely those that can provide "gene specificity." By means of a couple of simple examples of commonly performed transcriptomic-neuroimaging analyses, we illustrate some of the potentials and challenges of transcriptomic-imaging analyses, showing that providing gene specificity on observed transcriptomic-neuroimaging effects is of high importance to avoid reports of nonspecific effects. Through means of simulations we show that the rate of reported nonspecific effects (i.e., effects that cannot be specifically linked to a specific gene or gene-set) can run as high as 60%, with only less than 5% of transcriptomic-neuroimaging associations observed through ordinary linear regression analyses showing both spatial and gene specificity. We provide a discussion, a tutorial, and an easy-to-use toolbox for the different options of null models in transcriptomic-neuroimaging analyses.