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Abstract Genetically determined disorders are highly heterogenous in clinical presentation and underlying molecular mechanism. The evidence underpinning these conditions in the peer-reviewed literature requires robust critical evaluation for diagnostic use. Here, we present a structured curation process for Gene2Phenotype (G2P). This draws on multiple lines of clinical, bioinformatic and functional evidence. The process utilises and extends existing terminologies, allows for precise definition of the molecular basis of disease, and confidence levels to be attributed to a given gene-disease assertion. In-depth disease curation using this process will prove useful in applications including in diagnostics, research and development of targeted therapeutics. G2P: www.ebi.ac.uk/gene2phenotype .

Abstract Cancers are heterogeneous diseases with unifying features of abnormal and consuming cell growth, where the deregulation of normal cellular functions is initiated by the accumulation of genomic mutations in cells of - potentially - any organ. At diagnosis malignancies typically present with patterns of somatic genome variants on diverse levels of heterogeneity. Among the different types of genomic alterations, copy number variants (CNV) represent a distinct, near-ubiquitous class of structural variants. Cancer classifications are foundational for patient care and oncology research. Terminologies such as the National Cancer Institute Thesaurus provide large sets of hierarchical cancer classification vocabularies and promote data interoperability and ontology-driven computational analysis. To find out how categorical classifications correspond to genomic observations, we conducted a meta-analysis of inter-sample genomic heterogeneity for classification hierarchies on CNV profiles from 97,142 individual samples across 512 cancer entities, and evaluated recurring CNV signatures across diagnostic subsets. Our results highlight specific biological mechanisms across cancer entities with the potential for improvement of patient stratification and future enhancement of cancer classification systems and provide some indications for cooperative genomic events across distinct clinical entities.

Abstract Objectives Purple blotch, caused by the necrotrophic pathogen Alternaria porri, is one of the most economically significant diseases of onion and allied crops. While the virulent nature of many Alternaria spp. has been identified, the pathogenic repertoire of A. porri is still unknown. The objective of this work was to sequence the genome of A. porri using the PacBio SMRT sequencing strategy and analyse the repertoire of CAZymes, secondary metabolites, secretome and effectors in A. porri. Our research group is working to identify onion germplasm with purple blotch resistance and to understand the genetics of the pathogen. The reported de-novo assembly will contribute to the analysis of potential variants and the gene repertoire contributing to the virulence and pathogenicity of the purple blotch pathogen. Data description Long-read sequencing on a PacBio Sequel II system resulted in a 32.98 Mb (20 contigs) assembly with an N50 of 2, 657, 264 bp, the longest contig length of 5.05 Mb, and a GC content of 51.06%. The Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis resulted in 99.7% genome completeness at the Dothideomycetes lineage, representing a high-quality genome assembly. AUGUSTUS ab initio analysis resulted in 9875 protein-coding genes. Of the 6776 pathogenicity-related genes, 537 genes with effector functions were identified. Likewise, the glycoside hydrolases (434) were the most dominant group of the total 837 predicted CAZymes. The assembled genome of A. porri showed distinctive similarities to the genomes of A. alternata and A. brassicicola, the causal agents of leaf blight of onion and leaf spot of Brassica crops, respectively.

Abstract Objectives Cutibacterium acnes is a Gram-positive bacterium commonly found on human skin, particularly in sebaceous areas. While it is typically considered a commensal, specific strain types based on single locus sequence typing (SLST) have been associated with pathogenic conditions or healthy skin. Recently, SLST D1 strains, part of phylotype IA1, have received attention for their potential benefits related to skin health. However, their genetic characteristics remain underexplored. Therefore, the whole genome of C. acnes CN6, an SLST D1 strain isolated from the facial skin of a healthy individual, was sequenced to expand the understanding of SLST D1 strains and identify genomic features that may support skin health. Data description The whole genome sequencing of C. acnes CN6 was conducted using MinION reads based on de novo assembly, revealing a single circular complete chromosome. With the length of 2,550,458 bp and G + C content of 60.04%, the genome contains 2,492 genes, including 2,433 CDSs, 9 rRNAs, 46 tRNAs, 4 ncRNAs, and 134 pseudo genes. Previously predicted virulence proteins of C. ances were detected in the genome. Genome comparation with 200 C. acnes strains isolated from healthy facial skin revealed SLST D1 strain-specific genes and a unique variant of the znuC gene in D1 strains.

Abstract Background Efficient and scalable solutions are needed to identify patients who qualify for germline cancer genetic testing. We evaluated the clinical validity of a brief, patient-administered hereditary cancer risk assessment digital tool programmed to assess if patients meet criteria for germline genetic testing, based on personal and family history, and in line with national guidelines. Methods We applied the tool to cases seen in a nationwide telehealth genetic counseling practice. Validity of the tool was evaluated by comparing the tool’s assessment to that of the genetic counselor who saw the patient. Patients’ histories were extracted from genetic counselor-collected pedigrees and input into the tool by the research team to model how a patient would complete the tool. We also validated the tool’s assessment of which specific aspects of the personal and family history met criteria for genetic testing. Descriptive statistics were used. Results Of the 152 cases (80% female, mean age 52.3), 56% had a personal history of cancer and 66% met genetic testing criteria. The tool and genetic counselor agreed in 96% of cases. Most disagreements (4/6; 67%) occurred because the genetic counselor’s assessment relied on details the tool was not programmed to collect since patients typically don’t have access to the relevant information (pathology details, risk models). We also found complete agreement between the tool and research team on which specific aspects of the patient’s history met criteria for genetic testing. Conclusion We observed a high level of agreement with genetic counselor assessments, affirming the tool’s clinical validity in identifying individuals for hereditary cancer predisposition testing and its potential for increasing access to hereditary cancer risk assessment.

Abstract Background Lung adenocarcinoma (LUAD) accounts for the highest proportion of lung cancers; however, specific biomarkers are lacking for diagnosis, treatment, and prognostic assessment. Cell division cycle-associated 8 (CDCA8) is a cell cycle regulator with elevated expression in various cancers. However, the association between CDCA8 expression and LUAD prognosis remains unclear. Methods The association between CDCA8 and LUAD prognosis was evaluated based on the The Cancer Genome Atlas (TCGA) dataset, and CDCA8 related functions were determined using gene enrichment and gene ontology analyses. We also analyzed the association between CDCA8 expression and immune cell infiltration. Immunohistochemistry was used to determine the differential expression of CDCA8 in tumors and controls. Finally, we evaluated the differences in the sensitivity of different levels of CDCA8 to different anticancer drugs in LUAD. Results CDCA8 expression was significantly higher in primary LUAD tumors than in normal tissues (P

Abstract Background Weiss-Kruszka syndrome (WSKA) is a rare autosomal dominant syndrome characterized by multiple congenital anomalies caused by variants in the zinc finger protein 462 gene (ZNF462). About 40 cases of Weiss-Kruszka syndrome have been reported worldwide, and the aim of this study was to investigate the genetic causes of three patients from two Weiss-Kruszka syndrome family pedigrees with the aim of accumulating more data on the disease. Objective To explore the clinical and genetic characteristics of two pedigrees with Weiss–Kruszka syndrome. Methods The clinical data and family history of patients and family members of two pedigrees with Weiss–Kruszka syndrome were collected, and the pathogenic genes of the patients were analysed by whole-exon sequencing. Suspicious variants were verified by Sanger sequencing verification and bioinformatics prediction. Results Proband 1 has developmental delay, autistic behaviour, and abnormal electroencephalogram results. WES revealed a classical heterozygous c.6696–2 A > C splice variant of the ZNF462 gene, which was detected in neither parent. This position was conserved, and the variant was predicted to be deleterious. Minigene assays revealed that three types of aberrantly spliced mRNAs were produced. MRI of proband 2 suggested dysplasia of the corpus callosum with the formation of hemispheric cleft cysts, with a teardrop-like appearance in the lateral ventricle. WES revealed that a heterozygous c.4891 C > T:p. The Glu1631Ter nonsense variant of the ZNF462 gene was inherited from her mother. According to the guidelines of the American Society of Medical Genetics and combined with its clinical manifestations, c.6696–2 A > C and c.4891 C > T:p. Glu1631Ter was determined to be a possible pathogenic variant. Conclusion The c.6696-2 A>C and c.4891C > T:p.Glu1631Ter of the ZNF462 gene likely underlies Weiss–Kruszka syndrome in children (foetus), which enriches the variant spectrum of Chinese patients with Weiss–Kruszka syndrome and provides a basis for prenatal diagnosis and genetic counselling.

Abstract Background The copy number status (CNS) of the survival motor neuron (SMN) gene may influence the risk and prognosis of amyotrophic lateral sclerosis (ALS) and lower motor neuron diseases (LMND) other than spinal muscular atrophy (SMA). However, previous studies of this association, mainly from Europe, have yielded controversial results, suggesting possible regional differences. Here, we investigated the effect of the SMN gene in Japanese patients with ALS and LMND. Methods We examined the SMN copy numbers and clinical histories of 487 Japanese patients with sporadic ALS (281 men; mean age at onset 61.5 years), 50 with adult LMND (50 men; mean age at onset 58.4 years) and 399 Japanese controls (171 men; mean age 62.2 years). Patients with pathogenic mutations in ALS-causing genes were excluded. SMN1 and SMN2 copy numbers were determined using the droplet digital polymerase chain reaction. Results The frequency of a copy number of one for the SMN2 gene was higher in patients with ALS (38.0%) than in healthy controls (30.8%) (odds ratio (OR) = 1.37, 95% confidence interval (CI) = 1.04–1.82, p

Abstract Numerous studies have demonstrated the involvement of messenger RNAs (mRNAs) and non-coding RNAs, including long non-coding RNAs (lncRNA), circular RNAs (circRNAs) and microRNA (miRNAs), in gouty arthritis onset; however, the regulatory mechanism has not yet been elucidated. Here, we applied whole-transcriptome sequencing to identify the differentially expressed circRNAs, lncRNAs, miRNAs and mRNAs between the gout patients and normal people, and constructed co-regulated networks of circRNAs and lncRNAs according to the competitive endogenous RNA (ceRNA) theory for gouty arthritis onset to improve our understanding of the pathogenesis of this disease. The most significant finding of this study is the co-regulated ceRNA network of circRNAs and lncRNAs in gouty arthritis. The circRNA novel_circ_0030384 and the lncRNAs AAMP, TRIM16, PKN1, XLOC_184579 and XLOC_189826 were upstream genes in the co-regulated network. These upstream genes upregulated miR550a-5p and miR550a-3-5p, which downregulated PSME1 and FERMT3 expression. These mRNAs participated in proteasome dynamics, antigen processing and presentation, and platelet activation, which are associated with inflammation in gouty arthritis. In addition, the circRNA and lncRNAs upregulated miR550a-5p, which downregulated GRK2 and OS9 expression. Also, it proved that the down-regulated of PSME1, FERMT3, GRK2 and OS9 can aggravate gouty arthritis in vitro. In summary, these genes mediate inflammation in gouty arthritis through chemokine signaling to regulate neutrophil function.

Abstract Background Coffin-Siris syndrome is a clinically elusive and rare genetic disease characterized by a wide range of clinical manifestations. This study deeply analyzed and identified the clinical phenotype and genetic variant location in a pediatric patient with Coffin-Siris syndrome, aiming to enhance the understanding of this syndrome and assist in its screening and diagnosis. Methods A combination of advanced diagnostic tools, including high-throughput whole-exome sequencing (WES) and first-generation sequencing technologies, was employed to ascertain the etiology of the disease in the child. Results The clinical phenotype was characterized by stunted growth, reduced stature, spina bifida, enuresis, and a ventricular septal defect. WES revealed a de novo variant in the SOX11 gene locus (c.700G > T), identified as pathogenic. It is noteworthy that this variant has not been previously reported. Conclusions The combination of clinical presentation and genetic testing results supports that the patient suffers from Coffin-Siris syndrome due to a genetic variant in the SOX11 gene. This de novo variant expands our understanding of human gene variation, which is conducive to genetic counseling and screening for early diagnosis of Coffin-Siris syndrome.

Zhoushu Zheng,1 Lulu Yan,2,3 Lu Ding,1 Yinghui Zhang,1 Meihong Wang,1 Yihui Yang,1 Junhua Wu,3 Changshui Chen,3 Ming Tang,1 Haibo Li2,3 1Department of Otolaryngology Head and Neck Surgery, The Affiliated Women and Children’s Hospital of Ningbo University, Ningbo, People’s Republic of China; 2The Central Laboratory of Birth Defects Prevention and Control, The Affiliated Women and Children’s Hospital of Ningbo University, Ningbo, People’s Republic of China; 3Ningbo Key Laboratory for the Prevention and Treatment of Embryogenic Diseases, The Affiliated Women and Children’s Hospital of Ningbo University, Ningbo, People’s Republic of ChinaCorrespondence: Haibo Li, The Central Laboratory of Birth Defects Prevention and Control, Women and Children’s Hospital of Ningbo University, No. 339, Liuting Road, Haishu District, Ningbo City, Zhejiang Province, People’s Republic of China, Email lihaibo-775@163.com Ming Tang, Department of Otolaryngology head and neck surgery, Ningbo Women and Children’s Hospital, No. 339, Liuting Road, Haishu District, Ningbo City, Zhejiang Province, People’s Republic of China, Email tmtm-50@163.comIntroduction: Syndromic hearing loss (SHL) is characterized by distinctive clinical phenotypes as well as genetic and phenotypic heterogeneity. More than 400 species of SHL have been described, the majority of which are autosomal dominant.Methods: 11 forms of SHL were obtained from 14 unrelated families with probands ranging in age from 5 to 78 months. The results of whole exome sequencing(WES), audiological characteristics, middle and inner ear radiological findings, and additional clinical phenotype characteristics were retrospectively analyzed.Results: Fourteen people with SHL were found. Two of them had Waardenburg syndrome, two had Branchio-Oto-Renal syndrome, two had CHARGE syndrome, and one had Treacher Collins syndrome, Kleefstra syndrome, Muenke syndrome, Osteopathia Striata with Cranial Sclerosis, Ayme-Gripp syndrome, Tatton-Brown-Rahman syndrome, Stickler syndrome, or Stapes Ankylosis with Broad Thumbs and Toes. In this investigation, ten variants were first reported.Discussion: The combination of a neonatal hearing screening and WES can diagnose syndrome-type hearing loss in infancy and childhood, according to our findings, expansion of the gene variant spectrum and phenotype for various age groups of SHL is essential and can provide valuable guidelines for clinical intervention decisions. It is imperative for medical practitioners to conduct diligent and prolonged patient monitoring due to the inherent variability in both the auditory impairment and the comprehensive clinical manifestation of SHL.Keywords: syndromic hearing loss, whole exome sequencing, neonatal hearing screening, phenotype-genotype correlations

The complete chloroplast genome of a Chinese endemic species Hemiboea subacaulis var. jiangxiensis was reported and characterized. The length of chloroplast genome is 153,311 bp, with 37.6% GC content. It has a typical quadripartite structure, including a large single-copy region (LSC, 84,287 bp), a small single-copy region (SSC, 18,094 bp) and a pair of inverted repeats (IRs, 25,465 bp). Totally, 133 unique genes were predicted, including 88 protein-coding genes, 37 tRNA genes and eight rRNA genes. Phylogenetic analysis and genetic distance revealed this species is sister to its typic H. subacaulis var. subacaulis. This study offers novel genetic data for the molecular identification and phylogenetic analysis of the Hemiboea.

We sequenced the complete mitochondrial genome of the aphid Periphyllus koelreuteriae. It is 16,828 bp long and includes 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosome RNA genes (rRNAs), a control region, and a repeat region. The control region contains a 273-bp repeat motif repeated 2.3 times. The species-specific repeat region between trnE and trnF comprises two 340-bp repeat units. The maximum-likelihood tree based on all 37 mitochondrial genes indicated a close relationship between P. koelreuteriae and Periphyllus diacerivorus. This study provides a valuable mitogenomic resource for future research on Chaitophorinae phylogeny and P. koelreuteriae diversification.

Abstract Background The clinical presentation of ARG1‐D is characterised by elevated arginine levels leading to neurological and mobility impairments. Information about long‐term outcomes in adults is lacking, which prompted us to undertake a retrospective observational study. Methods We extracted ARG1‐D patient data spanning a 5‐year period from electronic health records. Ethical approval was not required for the study. Informed consent was obtained. Results We identified nine ARG1‐D patients from consanguineous backgrounds. Age of symptom onset ranged from infancy to age 7 years, age of diagnosis from infancy to 20 years. Patients had paraparesis or altered gait of varying degree and had experienced early ARG1‐D onset. Over 5 years, mobility declined in six (6/9, 67%) patients. Three patients (3/9, 33%) were fully dependent and hoisted. Two (2/9, 22%) reached adulthood before experiencing hyperammonaemia, another one (1/9, 11%) first experienced hyperammonaemia at age 15 years. One patient (1/9, 11%) started on ammonia scavenger therapy in adulthood, one (1/9, 11%) required a second scavenger to be added to their treatment regimen. Two patients (2/9, 22%) had gastrostomy tubes inserted for nutrition and supplements at age 9 years and 15 years. Six patients (6/9, 67%) had raised levels of ALT; of these, four (4/9, 44%) also had elevated AFP. Heterogeneity of ARG1‐D symptoms was evident, suggesting complex genetic and environmental interactions. Conclusion ARG1‐D presents significant lifelong challenges with deteriorating mobility and more frequent metabolic crises. Current management strategies are insufficient for preventing progression, highlighting the need for innovative treatments like enzyme replacement and gene therapy.

Abstract This study evaluates the effectiveness and safety of trientine dihydrochloride (TETA 2‐HCl) in patients with Wilson disease (WD) following a switch from trientine tetrahydrochloride (TETA 4‐HCl). A total of 30 WD patients with stable copper metabolism were identified for treatment with TETA 2‐HCl (Cufence™) after prior use of TETA 4‐HCl (Cuprior™). Biochemical markers including urinary copper, non‐ceruloplasmin bound copper (NCC) and liver function were analyzed at baseline and followed up over 12 months. Safety was assessed based on reported adverse events (AEs). Urinary copper levels and NCC remained stable across all follow‐ups, indicating adequate copper metabolism control. Reported AEs during TETA 2‐HCl treatment were mostly gastrointestinal discomfort (n = 6). In two patients, progressive elevation of transaminases occurred (despite stable copper metabolism). AEs led to discontinuation of treatment in five cases. Median baseline dose per day was 10.2 mg TETA 4‐HCl/kg bodyweight, whereas median baseline dose after therapeutic switch to TETA 2‐HCl was 12.8 mg/kg bodyweight. Median daily dose at 12 months did not differ significantly from TETA 2‐HCl dose at switching timepoint, with stable biochemical markers and markers of copper metabolism in most (25/30) of the patients. Transitioning from TETA 4‐HCl to TETA 2‐HCl maintained stable copper parameters and liver function in most of analyzed patients. TETA 2‐HCl treatment was generally well tolerated, suggesting that switching medications is safe and effective. In our real‐life cohort, adjustment factor of ~1.25× for the switch of TETA 4‐HCl to TETA 2‐HCl resulted in adequate copper metabolism control.

Abstract S‐adenosylhomocysteine hydrolase deficiency is an autosomal recessive inborn error of metabolism affecting methylation by disrupting the methionine cycle. Its clinical spectrum spans from severe perinatal encephalomyopathy and liver failure to asymptomatic course in patients with isolated hypermethioninemia. We present two new cases of S‐adenosylhomocysteine hydrolase deficiency from Pakistani origin clinically asymptomatic at presentation. Both siblings showed mild chronic liver failure and elevation of creatine kinase. The older patient presented at 6 years of age with isolated verbal processing difficulty and mild diffuse leukodystrophy, reversible 12 months after introduction of methionine dietary restriction. The patient showed subtle atrophy in the muscle MRI at the age of 7 years. S‐adenosylhomocysteine hydrolase deficiency was confirmed with homozygous missense variant c.146G>A (p.Arg49His) in the AHCY gene, a genotype previously reported in Pakistani patients with mild presentation. Dietary methionine restriction decreased plasma methionine but not plasma S‐adenosylhomocysteine and S‐adenosylmethionine. This work expands the mild spectrum of S‐adenosylhomocysteine hydrolase deficiency with no noticeable clinical symptoms in children, highlighting a specific hotspot variant from South Asia. This mild form of the disease is likely underdiagnosed and raises the question of therapeutic management to prevent long‐term complications documented in the literature, such as hepatocellular carcinoma and myopathy in early adulthood.

Abstract Glycerol kinase deficiency (GKD) is an X‐linked recessive disorder due to glycerol kinase (GK) gene mutations resulting in hyperglycerolermia, hyperglyceroluria, and “pseudohypertriglyceridemia.” In vivo glycerol metabolism has not been assessed in GKD. A 62‐year‐old man with suspected GKD and his extended family underwent whole exome sequencing and fasting blood work with two modes of lipid measurements: (1) standard lipase‐based methodology and (2) nuclear magnetic resonance (NMR). Two overnight fasted men with GKD and a heterozygote female carrier then underwent 13C3‐glycerol infusion. Affected family members had a novel two‐nucleotide deletion in exon 5 of the GK gene (c.373_374del). Compared to their family members (n = 14), men with GKD (n = 5) had significantly lower total cholesterol levels (3.72 ± 0.70 vs. 4.77 ± 0.85 mmol/L, p = 0.024). Compared to NMR, lipase‐based assays overreported triglycerides (5.28 ± 1.38 vs. 0.81 ± 0.32, mmol/L, p

Abstract Fructose‐1,6‐bisphosphatase (FBPase) deficiency is a rare, inborn error of metabolism, that causes hypoglycemia and lactic acidosis in response to inadequate glucose intake and/or high intakes of fructose, sucrose, or sorbitol. Pregnancy in women with FBPase deficiency puts them at high risk for metabolic decompensation due to increased glucose demands from the growing fetus. Here we report a 31‐year‐old primipara who was treated starting at 14 weeks gestation with a diet high in complex carbohydrates and low in fructose, sucrose, and sorbitol and close monitoring of glucose levels throughout her pregnancy. She delivered a healthy 2860 g baby at 37 weeks via vaginal delivery with no complications or hypoglycemia. At 5 months postpartum and 5 months of life, the patient and baby are doing well, although the patient experienced an episode of hypoglycemia and lactic acidosis at 4 months postpartum due to the increased metabolic demands of breastfeeding. This report adds to the limited case reports that discuss outcomes and proposed interventions during pregnancy in individuals with FBPase deficiency.

Abstract Glutaric aciduria type 1 (GA1) is an inherited neurometabolic disorder, in which deficiency of glutaryl‐CoA dehydrogenase leads to accumulation of glutaric acid (GA) and 3‐hydroxyglutaric acid (3‐HG). Some low excretors may exhibit only slight elevation of urinary 3‐HG, with normal urinary GA, yet are at significant risk of severe clinical disease. Accurate quantitation of urinary 3‐HG is crucial in diagnostic workup for GA1, but in this context, current gas chromatography–mass spectrometry (GC–MS) methods have inherent analytical challenges. Co‐elution and spectral similarities of the 3‐HG and 2‐HG structural isomers can cause difficulties in quantitation of slightly elevated 3‐HG. Our laboratory recently acquired a gas chromatography system coupled to a triple quadrupole mass spectrometer (GC–MS/MS), and we took advantage of its increased sensitivity and specificity to improve our existing GC–MS method. A stable isotope dilution process is used, with sample treatment consisting of a double liquid–liquid extraction followed by a trimethylsilyl derivatization. The transitions m/z 349 → 333 for 3‐HG and m/z 349 → 321 for 2‐HG were selected to differentiate these two isobaric molecules based on their characteristic fragments, thus minimizing interferences despite co‐elution. Method validation demonstrated satisfactory precision and accuracy. Using GC–MS/MS instead of GC–MS allowed us to decrease the required specimen volume, number of sample processing steps, chromatographic run time, and instrument maintenance. This enhanced assay facilitates clinical laboratory testing for GA1, both in confirmatory protocols following positive newborn screening and in diagnostic investigation of patients with suggestive signs or symptoms.

Abstract Patients with phenylketonuria (PKU) have a highly restrictive diet, which involves restriction of phenylalanine (Phe) intake and daily use of Phe‐free metabolic formula. However, little is known about the potential impact of this diet on chemical senses. The present study aimed to evaluate the olfactory and gustatory perceptions of patients with PKU. A cross‐sectional controlled study which included patients with PKU on dietary treatment and healthy controls was performed. Olfactory perception was assessed using the 12‐item Sniffin’ Sticks test, and taste perception using the Taste Strips test. Twenty‐five patients (mean age 19.3 ± 4.7 years; 13 females) and 25 controls (mean age 19.9 ± 4.9 years, p = 0.676; 13 females) were included. The mean age at treatment onset was 52.8 ± 29.7 days. The mean scores for olfactory and gustatory perceptions, and for bitter and salty flavors, were lower in patients than in controls (p = 0.039, p = 0.004, p = 0.008, and p = 0.020, respectively). Among patients, Phe levels at diagnosis correlated negatively with bitter taste (r = −0.493, p = 0.006). The lower olfaction and gustation scores found in patients may be related to understimulation caused by the highly restrictive PKU diet and the deprivation of flavors from breast milk.

Abstract SLC25A42 encodes the mitochondrial coenzyme A (CoA) transporter localized at the inner mitochondrial membrane. SLC25A42 deficiency leads to a congenital disease with a heterogeneous clinical presentation, including myopathy, developmental delay, lactic acidosis, and encephalopathy. Twenty‐one patients have been described so far. In the current study, we report on the identification of new biallelic variants in SLC25A42 in three siblings. Patients presented with symmetrical T2 hyperintensity of the putamen with minor volume depression at the brain MRI, elevated lactate, reduced oxygen consumption rates in muscle and fibroblasts, and reduced CoA levels in fibroblasts. Administration of pantothenic acid led to clinical stabilization and increased CoA levels in fibroblasts, thus confirming a role for SLC25A42 in energy metabolism and CoA homeostasis.

Abstract Glycosaminoglycan (GAG) and sialic acid (total and free) assays are used as first‐line screening tests for the diagnosis of mucopolysaccharidoses and glycoproteinoses, respectively. There is a pronounced age‐dependent variation in the urinary concentrations of these metabolites in the normal population, and the stratification of the reference values into discrete age ranges may lead to an undesirably high number of false‐positive or false‐negative results. The aim of this study was to design a method for calculating continuous reference intervals as a function of age and its application to the analysis of GAGs and sialic acid (total, free, and conjugated) in urine. In the postpubertal period, concentrations of urinary GAGs and sialic acid have reached a plateau, so a traditional calculation of the reference range in this specific age group was considered appropriate. In the prepubertal period, a nonlinear regression performed with the Excel add‐in Solver was used to fit the logarithmized concentrations of the controls to a curve that represents the mean values as a function of age. A uniform distribution of the residuals was obtained, which allowed the calculation of the reference intervals by adding the values of their 2.5 and 97.5 percentiles to the independent variable of the regression curve to calculate the upper and lower reference curves. The main advantages of the developed method are (1) a reduction in the number of control samples needed to obtain adequate reference intervals and (2) an improvement in the reliability of diagnostic screening by reducing the uncertainty generated by the gaps in the traditional age‐stratified method.

Abstract Huppke–Brendel syndrome (HBS) is an autosomal recessive disorder caused by SLC33A1 mutations, a gene coding for the acetyl‐CoA transporter‐1 (AT‐1). So far it has been described in nine pediatric and one adult patient. Therapeutic trials with copper histidinate failed to achieve any clinical improvement. Here, we describe the clinical characteristics of two novel patients, one of them diagnosed by gene analysis and his sib postmortally based on clinical characteristics. We demonstrate a therapeutic trial with acetylation therapy, consisting of N‐acetylcysteine and ketogenic diet, in one of them. We provide biochemical data on N‐acetylated amino acids in cerebrospinal fluid (CSF) and plasma before and after starting this treatment regimen. Our results indicate that ketogenic diet and N‐acetylcysteine do not seem to normalize the concentrations of N‐acetylated amino acids in CSF or plasma. The overall metabolic pattern shows a trend toward lowered levels of N‐acetylated amino acids in CSF and to a lesser extent in plasma. Although there are some assumptions, the function of AT‐1 is still not clear and further studies are needed to better understand mechanisms underlying this complex disorder.

Abstract Arginase 1 deficiency (ARG1‐D) is an ultrarare, metabolic disease which may cause spastic paraplegia, cognitive deficiency, seizures, and ultimately severe disability. The aim of this study was to assess disease burden in ARG1‐D by performing a cross‐sectional survey of patients with ARG1‐D and their caregivers in four European countries (France, Portugal, Spain, and the United Kingdom). Patients were enrolled at participating clinics and data were collected using a web‐based questionnaire. The findings indicate that there is a significant share of patients who experience severe cognitive and mobility impairment but also that there is a considerable variance in symptom severity among patients. Disease management was mostly in line with treatment guidelines and self‐reported adherence to treatment was reported to be high among a majority although following diet restrictions was perceived as difficult. However, despite this, since a large share of patients experienced severe cognitive and mobility impairment an unmet need among this patient population is suggested. The introduction of disease‐modifying therapies and early identification and diagnosis may help alleviate the disease burden associated with ARG1‐D in the future.

Bidens aurea (Asteraceae), a native of tropical America is now widespread in Asia and the Americas. We explored the B. aurea chloroplast genome and conducted a phylogenetic analysis. The chloroplast genome was circular, consisting of a large single copy (LSC) of 83,909 base pairs (bp), a small single copy (SSC) of 18,407 bp, and two inverted repeat regions (IR) of 24,729 bp each. Phylogenetic analysis showed that the 19 Bidens taxa were divided into five major clades, and B. aurea was most closely related to two species. Our findings offer a high-quality B. aurea chloroplast genome, aiding DNA barcode development and evolutionary history reconstruction.

Phalaenopsis deliciosa (Rchb. f.), an ornamental orchid known for vibrant flowers, has a 148,090 bp chloroplast genome with 36.78% GC content. It includes an 85,241 bp large single-copy (LSC) region, an 11,649 bp small single-copy (SSC) region, and two 13,800 bp inverted repeats (IRs), encoding 122 genes (76 protein-coding, 38 tRNA, and 8 rRNA). This genome data refines the Phalaenopsis gene database and supports research on phylogeny and molecular breeding.

The complete mitochondrial genome of Mylabris sibirica Fischer von Waldheim, 1823 was sequenced and characterized. The mitogenome is 15,794 bp long with 37 annotated genes, comprising 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. The phylogenetic analysis based on the mitochondrial genome sequences revealed that M. sibirica clustered with M. quadripunctata. This study presents the complete mitochondrial genome of M. sibirica for the first time, which could be beneficial for systematic studies on Mylabrini.

Abstract The paper presents Anatomy3DExplorer, a customized ChatGPT designed as a natural language dialogue interface for exploring 3D models of anatomical structures. It illustrates the significant potential of large language models (LLMs) as user-friendly interfaces for database access. Furthermore, it showcases the seamless integration of LLMs and database APIs, within the GPTS framework, offering a promising and straightforward approach.

Abstract Amphibians represent a diverse group of tetrapods, marked by deep divergence times between their three systematic orders and families. Studying amphibian biology through the genomics lens increases our understanding of the features of this animal class and that of other terrestrial vertebrates. The need for amphibian genomic resources is more urgent than ever due to the increasing threats to this group. Amphibians are one of the most imperiled taxonomic groups, with approximately 41% of species threatened with extinction due to habitat loss, changes in land use patterns, disease, climate change, and their synergistic effects. Amphibian genomic resources have provided a better understanding of ontogenetic diversity, tissue regeneration, diverse life history and reproductive modes, anti-predator strategies, and resilience and adaptive responses. They also serve as essential models for studying broad genomic traits, such as evolutionary genome expansions and contractions, as they exhibit the widest range of genome sizes among all animal taxa and possess multiple mechanisms of genetic sex determination. Despite these features, genome sequencing of amphibians has significantly lagged behind that of other vertebrates, primarily due to the challenges of assembling their large, repeat-rich genomes and the relative lack of societal support. The emergence of long-read sequencing technologies, combined with advanced molecular and computational techniques that improve scaffolding and reduce computational workloads, is now making it possible to address some of these challenges. To promote and accelerate the production and use of amphibian genomics research through international coordination and collaboration, we launched the Amphibian Genomics Consortium (AGC, https://mvs.unimelb.edu.au/amphibian-genomics-consortium ) in early 2023. This burgeoning community already has more than 282 members from 41 countries. The AGC aims to leverage the diverse capabilities of its members to advance genomic resources for amphibians and bridge the implementation gap between biologists, bioinformaticians, and conservation practitioners. Here we evaluate the state of the field of amphibian genomics, highlight previous studies, present challenges to overcome, and call on the research and conservation communities to unite as part of the AGC to enable amphibian genomics research to “leap” to the next level.

Abstract Background Cotton serves as a primary source of natural fibers crucial for the textile industry. However, environmental elements such as drought have posed challenges to cotton cultivation, resulting in adverse impacts on both production and fiber quality. Improving cotton’s resilience to drought could mitigate yield losses and foster the expansion of cotton farming. Rab7 protein, widely present in organisms, controls the degradation and recycling of cargo, and has a potential role in biotic and abiotic tolerance. However, comprehensive exploration of the Rab7 gene family in Gossypium remains scarce. Results Herein, we identified a total of 10, 10, 20, and 20 Rab7 genes through genome-wide analysis in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. Collinearity analysis unveiled the pivotal role of whole genome or segmental duplication events in the expansion of GhRab7s. Study of gene architecture, conserved protein motifs, and domains suggested the conservation of structure and function throughout evolution. Exploration of cis-regulatory elements revealed the responsiveness of GhRab7 genes to abiotic stress, corroborated by transcriptome analysis under diverse environmental stresses. Notably, the greatly induced expression of GhRab7B3-A under drought treatment prompted us to investigate its function through virus-induced gene silencing (VIGS) assays. Silencing GhRab7B3-A led to exacerbated dehydration and wilting compared with the control. Additionally, inhibition of stomatal closure, antioxidant enzyme activities and expression patterns of genes responsive to abiotic stress were observed in GhRab7B3-A silenced plants. Conclusions This study sheds light on Rab7 members in cotton, identifies a gene linked to drought stress, and paves the way for additional investigation of Rab7 genes associated with drought stress tolerance.

Abstract Colored leaves, a notable horticultural trait, have high research and ornamental value. The evergreen sweet olive (Osmanthus fragrans), one of the top ten traditional flowers in China, has been cultivated for more than two thousand years. However, in recent years, an increasing number of O. fragrans cultivars with colored leaves have been cultivated for their ornamental value. To study the molecular mechanism underlying the observed changes in leaf color, we selected O. fragrans ‘Yinbi Shuanghui’ (Y), which has yellow-white leaves, and O. fragrans ‘Sijigui’ (S), which has green leaves, as materials. Pigment content measurement showed that the chlorophyll, carotenoid and anthocyanin contents in Y were lower than in S. According to the SWATH-MS sequencing results, a total of 3,959 proteins were quantitatively identified, 1,300 of which were differentially expressed proteins (DEPs), including 782 up-regulated and 518 down-regulated proteins in Y compared to S. Functional enrichment analysis of DEPs revealed that down-regulated expression of photosynthesis related proteins may lead to the inhibition of chlorophyll synthesis in Y, this may be the main cause of leaf color change. Moreover, a protein interaction prediction model also showed that proteins such as PetC, PsbO, PsbP, and PsbQ were key proteins in the interaction network, and the up-regulated proteins participating in the anthocyanin and carotenoid pathways may be related to the formation of yellow-white leaves. Taken together, our findings represent the first SWATH-MS-based proteomic report on colored leaf O. fragrans and reveal that chlorophyll synthesis and secondary metabolism pathways contribute to the changes in leaf color.

Abstract Background This study aimed to assess the prevalence and genomic characteristics of Shiga-toxigenic (STEC) and Enteroaggregative E. coli (EAEC) strains in raw mussels and ready-to-eat (RTE)-stuffed mussels, focusing on potential public health implications for identifying virulence and antimicrobial resistance genes. Results The genome sequence analysis identified the E. coli strain named 23EM as serotype O111:H12, with adhesion (fimH-54) and fumarate hydratase (fumC-11) genes. The draft genome (4.9 Mb, 50.6% GC content, 111 contigs, 4,688 genes) is available in NCBI GenBank (accession JAWXVJ000000000). The strain, classified as ST292 and CC ST10, showed high similarity to nonpathogenic E. coli MG1655 but was distinct from pathogenic strains such as EAEC and ExPEC. In silico serotyping revealed the presence of O111-antigen flippase (wzx) and H12-antigen flagellin (fliC) genes. The strain harbors an IncFII (pCoo) plasmid with 96.95% identity. PathogenFinder predicted a 92% probability of being a human pathogen, supported by 720 pathogenic protein families. CRISPR analysis identified one high-evidence sequence with nine spacers and six low-evidence sequences. Phylogenetic analysis using RAxML positioned 23EM close to nonpathogenic E. coli but distant from other pathogenic strains. Antimicrobial resistance genes across multiple classes, including macrolides, fluoroquinolones, and aminoglycosides, were identified. The strain also contains several virulence factors, such as adhesins (e.g., ECP, ELF, TIF, type IV pili), and autotransporter genes (espP, pic), highlighting its significant pathogenic potential and public health risk. Conclusions This study highlights the ability of the detection of E. coli strains harboring virulence and antimicrobial resistance genes in mussels, thus emphasizing the importance of ongoing surveillance and careful consideration of the potential risks associated with the consumption of these shellfish.

Abstract Background Zoonotic human tuberculosis (TB) caused by Mycobacterium bovis (M. bovis) is as vital as Mycobacterium tuberculosis, however with scarce available information. We aimed to use whole-genome sequencing (WGS) technology to take a deep insight into the circulating genotypes of human M. bovis and the genomic characteristics underlying virulence and drug resistance. Methods The study included smear positive Ziehl-Neelsen samples from patients with suspected tuberculosis. Samples were cultured on Lowenstein-Jensen media and suspected colonies of M. bovis were selected to undergo DNA extraction and WGS. Data was analysed using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), and online bioinformatics tools. A phylogenetic tree was constructed for our sequenced strains, in addition to a set of 59 previously sequenced M. bovis genomes from different hosts and countries. Results Out of total 112 mycobacterial positive cultures, five M. bovis were isolated and underwent WGS. All sequenced strains belonged to Mycobacterium tuberculosis var bovis, spoligotype BOV_1; BOV_11. Resistance gene mutations were determined in 100% of strains to pyrazinamide (pncA and rpsA), isoniazid (KatG and ahpC), ethambutol (embB, embC, embR and ubiA), streptomycin (rpsl) and fluoroquinolones (gyrA and gyrB). Rifampin (rpoB and rpoC) and delamanid (fbiC) resistance genes were found in 80% of strains. The major represented virulence classes were the secretion system, cell surface components and regulation system. The phylogenetic analysis revealed close genetic relatedness of three sequenced M. bovis strains to previous reported cow strains from Egypt and human strains from France, as well as relatedness of one M. bovis strain to four human Algerian strains. One sequenced strain was related to one cow strain from Egypt and a human strain from South Africa. Conclusions All sequenced M. bovis isolates showed the same spoligotype, but diverse phylogeny. Resistance gene mutations were detected for anti-TB drugs including pyrazinamide, isoniazid, streptomycin, ethambutol, fluoroquinolones, cycloserine, rifampin and delamanid. The virulence profile comprised genes assigned mainly to secretion system, cell surface components and regulation system. Phylogenetic analysis revealed genetic relatedness between our isolates and previously sequenced bovine strains from Egypt as well as human strains from other nearby countries in the region.

Abstract Background Asthma, a complex respiratory disease, presents with inflammatory symptoms in the lungs, blood, and other tissues. We investigated the relationship between DNA methylation and 35 clinical markers of asthma. Methods The Illumina Infinium EPIC v1 methylation array was used to evaluate 742,442 CpGs in whole blood from 319 participants from 94 families. They were part of the Netherlands Twin Register from families with at least one member suffering from severe asthma. Repeat blood samples were taken after 10 years from 182 individuals. Principal component analysis on the clinical asthma markers yielded ten principal components (PCs) that explained 92.8% of the total variance. We performed epigenome-wide association studies (EWAS) for each of the ten PCs correcting for familial structure and other covariates. Results 221 unique CpGs reached genome-wide significance at timepoint 1 after Bonferroni correction. PC7, which correlated with loadings of eosinophil counts and immunoglobulin levels, accounted for the majority of associations (204). Enrichment analysis via the EWAS Atlas identified 190 of these CpGs to be previously identified in EWASs of asthma and asthma-related traits. Proximity assessment to previously identified SNPs associated with asthma identified 17 unique SNPs within 1 MB of two of the 221 CpGs. EWAS in 182 individuals with epigenetic data at a second timepoint identified 49 significant CpGs. EWAS Atlas enrichment analysis indicated that 4 of the 49 were previously associated with asthma or asthma-related traits. Comparing the estimates of all the significant associations identified across the two time points yielded a correlation of 0.81. Conclusion We identified 270 unique CpGs that were associated with PC scores generated from 35 clinical markers of asthma, either cross-sectionally or 10 years later. A strong correlation was present between effect sizes at the 2 timepoints. Most associations were identified for PC7, which captured blood eosinophil counts and immunoglobulin levels and many of these CpGs have previous associations in earlier studies of asthma and asthma-related traits. The results point to a robust DNA methylation profile as a new, stable biomarker for asthma.

Abstract Inflammation underlies many conditions causing excess morbidity and mortality among people with HIV (PWH). A handful of single-trait epigenome-wide association studies (EWAS) have suggested that inflammation is associated with DNA methylation (DNAm) among PWH. Multi-trait EWAS may further improve statistical power and reveal pathways in common between different inflammatory markers. We conducted single-trait EWAS of three inflammatory markers (soluble CD14, D-dimers and interleukin-6) in the Veterans Aging Cohort Study (n = 920). The study population was all male PWH with an average age of 51 years, and 82.3% self-reported as Black. We then applied two multi-trait EWAS methods—CPASSOC and OmniTest—to combine single-trait EWAS results. CPASSOC and OmniTest identified 189 and 157 inflammation-associated DNAm sites, respectively, of which 112 overlapped. Among the identified sites, 56% were not significant in any single-trait EWAS. Top sites were mapped to inflammation-related genes including IFITM1, PARP9 and STAT1. These genes were significantly enriched in pathways such as “type I interferon signaling” and “immune response to virus.” We demonstrate that multi-trait EWAS can improve the discovery of inflammation-associated DNAm sites, genes and pathways. These DNAm sites might hold the key to addressing persistent inflammation in PWH.

Abstract Whether DNA methylation changes follow human physical trauma is uncertain. We aimed to investigate if severe trauma was associated with DNA methylation changes. In a prospective, observational, clinical study, we included severely injured adults and adults undergoing elective surgery (controls). Blood was obtained from trauma patients (n = 60) immediately- and 30-45 days post-trauma, and from surgical patients (n = 57) pre-, post-, and 30-45 days post-surgery. Epigenome-wide DNA methylation profiling was performed and analyzed for significant differentially methylated CpGs and -regions (DMRs) within and between groups. Within the trauma group we identified 10,126 significant differentially methylated CpGs and 1169 DMRs. No significant differential methylation was found in the surgical group. In the trauma group, differentially methylated sites were enriched in genes and pathways involved in blood coagulation and inflammatory response. Severe trauma was associated with profound alterations in the DNA methylome of circulating leucocytes, and differential methylation was located in trauma-relevant genes.

Abstract The precise spatiotemporal expression of the hematopoietic ETS transcription factor PU.1, a key determinant of hematopoietic cell fates, is tightly regulated at the chromatin level. However, how chromatin signatures are linked to this dynamic expression pattern across different blood cell lineages remains uncharacterized. Here, we performed an in-depth analysis of the relationships between gene expression, chromatin structure, 3D architecture, and trans-acting factors at PU.1 cis-regulatory elements (PCREs). By identifying phylogenetically conserved DNA elements within chromatin-accessible regions in primary human blood lineages, we discovered multiple novel candidate PCREs within the upstream region of the human PU.1 locus. A subset of these elements localizes within an 8-kb-wide cluster exhibiting enhancer features, including open chromatin, demethylated DNA, enriched enhancer histone marks, present enhancer RNAs, and PU.1 occupation, presumably mediating PU.1 autoregulation. Importantly, we revealed the presence of a common 35-kb-wide CTCF-flanked insulated neighborhood that contains the PCRE cluster (PCREC), forming a chromatin territory for lineage-specific and PCRE-mediated chromatin interactions. These include functional PCRE-promoter interactions in myeloid and B cells that are absent in erythroid and T cells. By correlating chromatin structure and 3D architecture with PU.1 expression in various lineages, we were able to attribute enhancer versus silencer functions to individual elements. Our findings provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in the chromatin regulation of PU.1 expression. This study lays crucial groundwork for additional experimental studies that validate and dissect the role of PCREs in epigenetic regulation of normal and malignant hematopoiesis.

Abstract Canonical histone H3 and histone variant H3.3 are posttranslationally modified with the genomic distribution of these marks denoting different features and these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the amino acid H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent but exhibit a reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue within H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells.

Abstract Background Coronary artery disease (CAD) significantly contributes to global fatalities. Recent studies have demonstrated the crucial roles of sortilin1 (SORT1) and sestrin1 (SESN1) in lipid metabolism, as well as their involvement in the development of CAD. The aberrant expression or activity of SORT1 can consequently lead to metabolic and vascular diseases. Sestrins, including SESN1, play a crucial role in helping cells survive by maintaining metabolic balance while also reducing oxidative stress (OS). OS contributes to the progression of atherosclerosis-related diseases, such as CAD. The study aimed to compare the gene expression of SORT1 and SESN1 in peripheral blood mononuclear cells (PBMCs), alongside serum OS markers, in CAD patients and controls. Materials The case-control study included 49 CAD patients and 40 controls. The expression of the SORT1 and SESN1 genes was quantified using qRT-PCR, and the expression of the SORT1 protein was evaluated by western blotting. OS markers, including total oxidation status (TOS), total antioxidant capacity (TAC), and malondialdehyde (MDA), were measured using spectrophotometric and fluorometric methods. Results SORT1 gene and protein expressions were similar between groups. CAD patients had a non-significant decrease in SESN1 gene expression. MDA levels were significantly higher in CAD patients, whereas TOS and TAC levels did not differ significantly. Conclusion For atherosclerosis-related disorders like CAD, MDA shows potential as a non-invasive, easy-to-use, affordable, and stable biomarker. Further research is needed to elucidate the precise roles of SORT1 and SESN1 in CAD pathogenesis.

Polygonatum gracile P. Y. Li is a perennial rhizomatous medicinal herb endemic to China with recognized medicinal value. Here, the first complete plastid genome of P. gracile is reported and characterized, and the phylogenetic relationships of P. gracile with other Polygonatum species are clarified by phylogenetic inferences based on complete plastid genome sequences. The complete plastid genome of P. gracile is 155,734 bp in length and exhibits the typical quadripartite circular structure consisting of a large single-copy (LSC, 84,385 bp) region, a small single-copy (SSC, 18,519 bp) region, and a pair of inverted repeat regions (IRa/b, 26,415 bp). A total of 112 unique genes, including 78 protein-coding genes, 30 tRNA genes, and four rRNA genes were identified. Seven protein-coding genes, eight tRNA genes, and four rRNA genes were duplicated in the IR regions. Phylogenetic reconstructions indicated that P. gracile belongs to sect. Verticillata and is most closely related to P. zanlanscianense Pamp. This study provides a basis for further phylogenetic reconstructions, conservation and utilization of Polygonatum species.

The Diospyros genus (Ebenaceae) has significant economic value. During field surveys, we discovered a Diospyros specimen showing morphological overlap with both D. oleifera and D. kaki var. silvestris, provisionally named Diospyros aff. oleifera. To resolve its taxonomy, we sequenced and analyzed its chloroplast genome. The complete chloroplast genome is 157,732 bp with a quadripartite structure. mVISTA analysis revealed unique sequence variations compared to related species. Phylogenetic analysis using 75 protein-coding genes grouped it with D. oleifera, indicating their close relationship. Our findings suggest this specimen likely represents a novel, undescribed species. This study provides insights into Diospyros diversity and a foundation for future research.

Diglycerol (DAG) is a structural lipid with the functions to lower body fat accumulation and decrease serum triglyceride level. However, the enzymatic synthesis of DAG is limited by the high-efficient and economic lipases. In this paper, the immobilized lipase PS@LXTE-1000 was self-made by immobilizing the Pseudomomas cepacian lipase on to the hydrophobic microporous resin LXTE-1000. The results indicate that LXTE-1000 was a uniform mesoporous sphere with the mean diameter of 400 ​μm, pore size of 14.6 ​nm, pore volume of 0.5 ​cm3/g and surface area of 126.0 ​m2/g, showing superior structural properties for lipase immobilization. Under the optimal reaction conditions with the molar ratio of rapeseed oil to glycerol being 1:1, adding amount of immobilized lipase being 4%, reaction at 50 ​°C, the highest DAG content of 46.7% was achieved in 3 ​h via enzymatic glycerolysis catalyzed by LXTE-1000. After 7 cycles of reuse, the self-made LXTE-1000 could still retain 78.3% of its initial catalytic ability. Besides, LXTE-1000 was observed to facilitate the DAG production via glycerolysis reaction between glycerol with other seven edible oils including corn oil, sesame oil, peony seed oil, rice bran oil, peanut oil, soybean oil and flaxseed oil. Specifically, the glycerolysis reaction with sesame oil, peony seed oil and rice bran oil even led to the DAG content of 52.1%, 53.3% and 51.2%, respectively, Hence, this paper provide a novel strategy to produce high-efficient and economic immobilized lipases, which shows great potential in the green synthesis of functional lipids such as DAG.

Oil content is a crucial indicator for evaluating the quality of peanuts. A rapid and non-destructive method to determine oil content of individual peanut seed can provide robust technical support for breeding high-oil-content peanut varieties. In this study, we established a rapid determination method using near-infrared hyperspectral imaging and chemometrics to assess the oil content of single peanut seed. After selecting key wavelengths through competitive adaptive reweighted sampling (CARS), uninformative variable elimination (UVE), and random frog (RF), we constructed an oil content calibration model based on partial least squares regression for single peanut seed. Validation results demonstrated that the correlation coefficient was 0.8393 with a root mean square error of 1.7771 in the calibration set, while it was 0.7915 with a root mean square error of 2.2943 in the independent prediction set. Most samples exhibited relative errors below 5%, confirming the reliability of this model in predicting oil content of single peanut seed.

Flaxseed milk coproduct (FMC) is a by-product of flaxseed milk. Okara is a by-product of processed soybean products. In this study, we investigated the quality of dough and Chinese steamed bread (CSB) with the addition of FMC and okara. We also examined the in vitro starch digestibility, expected glycemic index (eGI), starch crystallinity, and short-range order structure of CSB. The results showed that FMC and okara decreased the dough fluidity, formed a dense structure, and enhanced the mechanical properties of the dough. FMC and okara increased the hardness, gumminess, and chewiness of the CSB, while decreasing its cohesion and elasticity. The addition of FMC and okara improved the nutrient content of CSB and reduced the eGI from 75.86 to 51.56. FMC and okara altered the multiscale structure of starch, effectively shielding the amylase site of action, and limited the interaction between amylase and starch. This study provides a reference for the high-value utilization of oilseed processing by-products.

Abstract Glycosaminoglycans are often deprioritized as targets for synthetic immunotherapy due to the complexity of glyco-epitopes and limited options for obtaining specific subtype binding. Solid tumors express proteoglycans that are modified with oncofetal chondroitin sulfate (CS), a modification normally restricted to the placenta. Here, we report the design and functionality of transient chimeric antigen receptor (CAR) T cells with selectivity to oncofetal CS. Following expression in T cells, the CAR could be “armed” with recombinant VAR2CSA lectins (rVAR2) to target tumor cells expressing oncofetal CS. While unarmed CAR T cells remained inactive in the presence of target cells, VAR2-armed CAR T cells displayed robust activation and the ability to eliminate diverse tumor cell types in vitro. Cytotoxicity of the CAR T cells was proportional to the concentration of rVAR2 available to the CAR, offering a potential molecular handle to finetune CAR T cell activity. In vivo, armed CAR T cells rapidly targeted bladder tumors and increased the survival of tumor-bearing mice. Thus, our work indicates that cancer-restricted glycosaminoglycans may be exploited as potential targets for CAR T cell therapy.

Abstract Alzheimer’s Disease (AD), as the most common neurodegenerative disease worldwide, severely impairs patients’ cognitive functions. Although its exact etiology remains unclear, the abnormal aggregations of misfolded β-amyloid peptide and tau protein are considered pivotal in its pathological progression. Recent studies identify ubiquitin-specific protease 11 (USP11) as the key regulator of tau deubiquitination, exacerbating tau aggregation and AD pathology. Thereby, inhibiting USP11 function, via either blocking USP11 activity or lowering USP11 protein level, may serve as an effective therapeutic strategy against AD. Our research introduces IsoLiPro, a unique lithium isobutyrate-L-proline coordination compound, effectively lowers USP11 protein level and enhances tau ubiquitination in vitro. Additionally, long-term oral administration of IsoLiPro dramatically reduces total and phosphorylated tau levels in AD transgenic mice. Moreover, IsoLiPro also significantly lessens β-amyloid deposition and synaptic damage, improving cognitive functions in these animal models. These results indicate that IsoLiPro, as a novel small-molecule USP11 inhibitor, can effectively alleviate AD-like pathologies and improve cognitive functions, offering promise as a potential multi-targeting therapeutic agent against AD.

Abstract Fibrosis contributes to tissue repair, but excessive fibrosis disrupts organ function. Alagille syndrome (ALGS, caused by mutations in JAGGED1) results in liver disease and characteristic fibrosis. Here, we show that Jag1 Ndr/Ndr mice, a model for ALGS, recapitulate ALGS-like fibrosis. Single-cell RNA-seq and multi-color flow cytometry of the liver revealed immature hepatocytes and paradoxically low intrahepatic T cell infiltration despite cholestasis in Jag1 Ndr/Ndr mice. Thymic and splenic regulatory T cells (Tregs) were enriched and Jag1 Ndr/Ndr lymphocyte immune and fibrotic capacity was tested with adoptive transfer into Rag1 −/− mice, challenged with dextran sulfate sodium (DSS) or bile duct ligation (BDL). Transplanted Jag1 Ndr/Ndr lymphocytes were less inflammatory with fewer activated T cells than Jag1 +/+ lymphocytes in response to DSS. Cholestasis induced by BDL in Rag1 −/− mice with Jag1 Ndr/Ndr lymphocytes resulted in periportal Treg accumulation and three-fold less periportal fibrosis than in Rag1 −/− mice with Jag1 +/+ lymphocytes. Finally, the Jag1 Ndr/Ndr hepatocyte expression profile and Treg overrepresentation were corroborated in patients’ liver samples. Jag1-dependent hepatic and immune defects thus interact to determine the fibrotic process in ALGS.

Abstract Sepsis is a heterogeneous syndrome resulting from a dysregulated host response to infection. It is considered as a global major health priority. Sepsis is characterized by significant metabolic perturbations, leading to increased circulating metabolites such as lactate. In mammals, pyruvate is the primary substrate for lactate production. It plays a critical role in metabolism by linking glycolysis, where it is produced, with the mitochondrial oxidative phosphorylation pathway, where it is oxidized. Here, we provide an overview of all cytosolic and mitochondrial enzymes involved in pyruvate metabolism and how their activities are disrupted in sepsis. Based on the available data, we also discuss potential therapeutic strategies targeting these pyruvate-related enzymes leading to enhanced survival.

Abstract Cerebral cavernous malformations (CCMs) are anomalies of the cerebral vasculature. Loss of the CCM proteins CCM1/KRIT1, CCM2, or CCM3/PDCD10 trigger a MAPK-Krüppel-like factor 2 (KLF2) signaling cascade, which induces a pathophysiological pattern of gene expression. The downstream target genes that are activated by KLF2 are mostly unknown. Here we show that Chromobox Protein Homolog 7 (CBX7), component of the Polycomb Repressive Complex 1, contributes to pathophysiological KLF2 signaling during zebrafish cardiovascular development. CBX7/cbx7a mRNA is strongly upregulated in lesions of CCM patients, and in human, mouse, and zebrafish CCM-deficient endothelial cells. The silencing or pharmacological inhibition of CBX7/Cbx7a suppresses pathological CCM phenotypes in ccm2 zebrafish, CCM2-deficient HUVECs, and in a pre-clinical murine CCM3 disease model. Whole-transcriptome datasets from zebrafish cardiovascular tissues and human endothelial cells reveal a role of CBX7/Cbx7a in the activation of KLF2 target genes including TEK, ANGPT1, WNT9, and endoMT-associated genes. Our findings uncover an intricate interplay in the regulation of Klf2-dependent biomechanical signaling by CBX7 in CCM. This work also provides insights for therapeutic strategies in the pathogenesis of CCM.

Abstract Spastic paraplegia 47 (SPG47) is a neurological disorder caused by mutations in the adaptor protein complex 4 β1 subunit (AP4B1) gene leading to AP-4 complex deficiency. SPG47 is characterised by progressive spastic paraplegia, global developmental delay, intellectual disability and epilepsy. Gene therapy aimed at restoring functional AP4B1 protein levels is a rational therapeutic strategy to ameliorate the disease phenotype. Here we report that a single delivery of adeno-associated virus serotype 9 expressing hAP4B1 (AAV9/hAP4B1) into the cisterna magna leads to widespread gene transfer and restoration of various hallmarks of disease, including AP-4 cargo (ATG9A) mislocalisation, calbindin-positive spheroids in the deep cerebellar nuclei, anatomical brain defects and motor dysfunction, in an SPG47 mouse model. Furthermore, AAV9/hAP4B1-based gene therapy demonstrated a restoration of plasma neurofilament light (NfL) levels of treated mice. Encouraged by these preclinical proof-of-concept data, we conducted IND-enabling studies, including immunogenicity and GLP non-human primate (NHP) toxicology studies. Importantly, NHP safety and biodistribution study revealed no significant adverse events associated with the therapeutic intervention. These findings provide evidence of both therapeutic efficacy and safety, establishing a robust basis for the pursuit of an IND application for clinical trials targeting SPG47 patients.