Interferon subverts an AHR-JUN axis to promote CXCL13+ T cells in lupus.

Systemic lupus erythematosus (SLE) is prototypical autoimmune disease driven by pathological T cell-B cell interactions1,2. Expansion of T follicular helper (TFH) and T peripheral helper (TPH) cells, two T cell populations that provide help to B cells, is a prominent feature of SLE3,4. Human TFH and TPH cells characteristically produce high levels of the B cell chemoattractant CXCL13 (refs. 5,6), yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4+ T cell phenotypes in patients with SLE, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4+ T cells. Transcriptomic, epigenetic and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ TPH/TFH cell differentiation and promote an IL-22+ phenotype. Type I interferon, a pathogenic driver of SLE7, opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ TPH/TFH cells on a polarization axis opposite from T helper 22 (TH22) cells and reveal AHR, JUN and interferon as key regulators of these divergent T cell states.

TEDD: a database of temporal gene expression patterns during multiple developmental periods in human and model organisms.

Characterization of the specific expression and chromatin profiles of genes enables understanding how they contribute to tissue/organ development and the mechanisms leading to diseases. Whilst the number of single-cell sequencing studies is increasing dramatically; however, data mining and reanalysis remains challenging. Herein, we systematically curated the up-to-date and most comprehensive datasets of sequencing data originating from 2760 bulk samples and over 5.1 million single-cells from multiple developmental periods from humans and multiple model organisms. With unified and systematic analysis, we profiled the gene expression and chromatin accessibility among 481 cell-types, 79 tissue-types and 92 timepoints, and pinpointed cells with the co-expression of target genes. We also enabled the detection of gene(s) with a temporal and cell-type specific expression profile that is similar to or distinct from that of a target gene. Additionally, we illustrated the potential upstream and downstream gene-gene regulation interactions, particularly under the same biological process(es) or KEGG pathway(s). Thus, TEDD (Temporal Expression during Development Database), a value-added database with a user-friendly interface, not only enables researchers to identify cell-type/tissue-type specific and temporal gene expression and chromatin profiles but also facilitates the association of genes with undefined biological functions in development and diseases. The database URL is https://TEDD.obg.cuhk.edu.hk/.

Gasdermin B, an asthma-susceptibility gene, promotes MAVS-TBK1 signalling and airway inflammation.

RATIONALE: Respiratory virus-induced inflammation is the leading cause of asthma exacerbation, frequently accompanied by induction of interferon-stimulated genes (ISGs). How asthma-susceptibility genes modulate cellular response upon viral infection by fine-tuning ISG induction and subsequent airway inflammation in genetically susceptible asthma patients remains largely unknown. OBJECTIVES: To decipher the functions of gasdermin B (encoded by GSDMB) in respiratory virus-induced lung inflammation. METHODS: In two independent cohorts, we analysed expression correlation between GSDMB and ISG s. In human bronchial epithelial cell line or primary bronchial epithelial cells, we generated GSDMB-overexpressing and GSDMB-deficient cells. A series of quantitative PCR, ELISA and co-immunoprecipitation assays were performed to determine the function and mechanism of GSDMB for ISG induction. We also generated a novel transgenic mouse line with inducible expression of human unique GSDMB gene in airway epithelial cells and infected the mice with respiratory syncytial virus to determine the role of GSDMB in respiratory syncytial virus-induced lung inflammation in vivo. RESULTS: GSDMB is one of the most significant asthma-susceptibility genes at 17q21 and acts as a novel RNA sensor, promoting mitochondrial antiviral-signalling protein (MAVS)-TANK binding kinase 1 (TBK1) signalling and subsequent inflammation. In airway epithelium, GSDMB is induced by respiratory viral infections. Expression of GSDMB and ISGs significantly correlated in respiratory epithelium from two independent asthma cohorts. Notably, inducible expression of human GSDMB in mouse airway epithelium led to enhanced ISGs induction and increased airway inflammation with mucus hypersecretion upon respiratory syncytial virus infection. CONCLUSIONS: GSDMB promotes ISGs expression and airway inflammation upon respiratory virus infection, thereby conferring asthma risk in risk allele carriers.

Lineage Tracing of RGS5-CreER-Labeled Cells in Long Bones During Homeostasis and Injury.

Regulator of G protein signaling 5 (RGS5) is a GTPase activator for heterotrimeric G-protein α-subunits, shown to be a marker of pericytes. Bone marrow stromal cell population (BMSCs) is heterogeneous. Populations of mesenchymal progenitors, cells supportive of hematopoiesis, and stromal cells regulating bone remodeling have been recently identified. Periosteal and bone marrow mesenchymal stem cells (MSCs) are participating in fracture healing, but it is difficult to distinguish the source of cells within the callus. Considering that perivascular cells exert osteoprogenitor potential, we generated an RGS5 transgenic mouse model (Rgs5-CreER) which when crossed with Ai9 reporter animals (Rgs5/Tomato), is suitable for lineage tracing during growth and post-injury. Flow cytometry analysis and histology confirmed the presence of Rgs5/Tomato+ cells within CD31+ endothelial, CD45+ hematopoietic, and CD31-CD45- mesenchymal/perivascular cells. A tamoxifen chase showed expansion of Rgs5/Tomato+ cells expressing osterix within the trabeculae positioned between mineralized matrix and vasculature. Long-term chase showed proportion of Rgs5/Tomato+ cells contributes to mature osteoblasts expressing osteocalcin. Following femoral fracture, Rgs5/Tomato+ cells are observed around newly formed bone within the BM cavity and expressed osterix and osteocalcin, while contribution within periosteum was low and limited to fibroblastic callus with very few positive chondrocytes. In addition, BM injury model confirmed that RGS5-Cre labels population of BMSCs expands during injury and participates in osteogenesis. Under homeostatic conditions, lineage-traced RGS5 cells within the trabecular area demonstrate osteoprogenitor capacity that in an injury model contributes to new bone formation primarily within the BM niche.

A nanocarrier immersion vaccine encoding surface immunogenic protein confers cross-immunoprotection against Streptococcus agalactiae and Streptococcus iniae infection in tilapia.

Streptococcosis is a highly contagious aquatic bacterial disease that poses a significant threat to tilapia. Vaccination is a well-known effective measure to prevent and control fish bacterial diseases. Among the various immunization methods, immersion vaccination is simple and can be widely used in aquaculture. Besides, nanocarrier delivery technology has been reported as an effective solution to improve the immune effect of immersion vaccine. In this study, the surface immunogenic protein (Sip) was proved to be conserved and potential to provide cross-immunoprotection for both Streptococcus agalactiae (S. agalactiae) and Streptococcus iniae (S. iniae) by multiple sequences alignment and Western blotting analysis. On this basis, we expressed and obtained the recombinant protein rSip and connected it with functionalized carbon nanotubes (CNT) to construct the nanocarrier vaccine system CNT-rSip. After immersion immunization, the immune effect of CNT-rSip against above two streptococcus infections was evaluated in tilapia based on some aspects including the serum specific antibody level, non-specific enzyme activities, immune-related genes expression and relative percent survival (RPS) after bacteria challenge. The results showed that compared with control group, CNT-rSip significantly (P < 0.05) increased the serum antibody levels, related enzyme activities including acid phosphatase, alkaline phosphatase, lysozyme and total antioxidant capacity activities, as well as the expression levels of immune-related genes from 2 to 4 weeks post immunization (wpi), and all these indexes peaked at 3 wpi. Besides, the above indexes of CNT-rSip were higher than those of rSip group with different extend during the experiment. Furthermore, the challenge test indicated that CNT-rSip provided cross-immunoprotection against S. agalactiae and S. iniae infection with RPS of 75 % and 72.41 %, respectively, which were much higher than those of other groups. Our study indicated that the nanocarrier immersion vaccine CNT-rSip could significantly improve the antibody titer and confer cross-immuneprotection against S. agalactiae and S. iniae infection in tilapia.

Theoretical study of the structural and thermodynamic properties of U-He compounds under high pressure.

Uranium is considered as a very important nuclear energy material because of the huge amount of energy it releases. As the main product of the spontaneous decay of uranium, it is difficult for helium to react with uranium because of its chemical inertness. Therefore, bubbles will be formed inside uranium, which could greatly reduce the performance of uranium or cause safety problems. Additionally, nuclear materials are usually operated in an environment of high-temperature and high-pressure, so it is necessary to figure out the exact state of helium inside uranium under extreme conditions. Here, we explored the structural stability of the U-He system under high pressure and high temperature by using density functional theory calculations. Two metastable phases are found between 50 and 400 GPa: U4He with space group Fmmm and U6He with space group P1̄. Both are metallic and adopt layered structures. Electron localization function calculation combined with charge density difference analysis indicates that there are covalent bonds between U and U atoms in both Fmmm-U4He and P1̄-U6He. Regarding the elastic modulus of α-U, the addition of helium has certain influence on the mechanical properties of uranium. Besides, first-principles molecular dynamics simulations were carried out to study the dynamical behavior of Fmmm-U4He and P1̄-U6He at high-temperature. It was found that Fmmm-U4He and P1̄-U6He undergo one-dimensional superionic phase transitions at 150 GPa. Our study revealed the exotic structure of U-He compounds beyond the formation of bubbles under high-pressure and high-temperature, which might be relevant to the performance and safety issues of nuclear materials under extreme conditions.

Renal and extra-renal phenotypes in a fetus with a de novo pathogenic variant in the HNF1B gene.

We report a fetus with prenatal ultrasound at 21 gestational weeks showing left cystic renal dysplasia with subcapsular cysts and echogenic parenchyma, right echogenic kidney with absent corticomedullary differentiation, and left congenital diaphragmatic hernia (CDH) with bowel herniation, with intestinal atresia (IA) found on postmortem examination. Whole genome sequencing of fetal blood DNA revealed a heterozygous pathogenic variant c.344 + 2 T>G in the HNF1B gene (NM_000458). Sanger sequencing of the parental samples suggested that it arose de novo in the fetus. HNF1B-associated disorders affect multiple organs with significant phenotypic heterogeneity. In pediatric and adult patients, renal cystic disease and cystic dysplasia are the dominant phenotypes. In prenatal settings, renal anomaly is also the most common presentation, typically with bilateral hyperechogenic kidneys. Our case presented with two uncommon extra-renal phenotypes of CDH and IA besides the typical bilateral cystic renal dysplasia. This association has been reported in fetuses with 17q12 microdeletion but not with HNF1B point mutation. Our case is the first prenatal report of such an association and highlights the possible causal relationship of HNF1B defects with CDH and IA in addition to the typical renal anomalies.

Prenatal diagnosis of polycystic kidney caused by biallelic hypomorphic variants in the PKD1 gene.

Heterozygous loss-of-function variants in the PKD1 gene are commonly associated with adult-onset autosomal dominant polycystic kidney disease (ADPKD), where the formation of renal cysts depends on the dosage of the PKD1 gene. Biallelic null PKD1 variants are not viable, but biallelic hypomorphic variants could lead to early-onset PKD. We report a non-consanguineous Chinese family with recurrent fetal polycystic kidney and negative findings in the coding region of the PKHD1 gene or chromosomal microarray analysis. Trio exome analysis revealed compound heterozygous variants of uncertain significance in the PKD1 gene in the index pregnancy: a novel paternally inherited c.7863 + 5G > C and a maternally inherited c.9739C > T, p.(Arg3247Cys). Segregation analysis through long-range PCR followed by nested PCR and Sanger sequencing confirmed another affected fetus had both variants, while the other two normal siblings and the parents carried either variant. Thus, these two variants, both of which were hypomorphic as opposed to null variants, co-segregated with prenatal onset polycystic kidney disease in this family. Functional studies are needed to further determine the impact of these two variants. Our findings highlight the biallelic inheritance of hypomorphic PKD1 variants causing prenatal onset polycystic kidney disease, which provides a better understanding of phenotype-genotype correlation and valuable information for reproductive counseling.

Cold exposure-induced plasma exosomes impair bone mass by inhibiting autophagy.

Recently, environmental temperature has been shown to regulate bone homeostasis. However, the mechanisms by which cold exposure affects bone mass remain unclear. In our present study, we observed that exposure to cold temperature (CT) decreased bone mass and quality in mice. Furthermore, a transplant of exosomes derived from the plasma of mice exposed to cold temperature (CT-EXO) can also impair the osteogenic differentiation of BMSCs and decrease bone mass by inhibiting autophagic activity. Rapamycin, a potent inducer of autophagy, can reverse cold exposure or CT-EXO-induced bone loss. Microarray sequencing revealed that cold exposure increases the miR-25-3p level in CT-EXO. Mechanistic studies showed that miR-25-3p can inhibit the osteogenic differentiation and autophagic activity of BMSCs. It is shown that inhibition of exosomes release or downregulation of miR-25-3p level can suppress CT-induced bone loss. This study identifies that CT-EXO mediates CT-induced osteoporotic effects through miR-25-3p by inhibiting autophagy via targeting SATB2, presenting a novel mechanism underlying the effect of cold temperature on bone mass.

Quantitative Acetylomics Reveals Dynamics of Protein Lysine Acetylation in Mouse Livers During Aging and Upon the Treatment of Nicotinamide Mononucleotide.

Lysine acetylation is a reversible and dynamic post-translational modification that plays vital roles in regulating multiple cellular processes including aging. However, acetylome-wide analysis in the aging process remains poorly studied in mammalian tissues. Nicotinamide adenine dinucleotide (NAD+), a hub metabolite, benefits health span at least in part due to the activation of Sirtuins, a family of NAD+-consuming deacetylases, indicating changes in acetylome. Here, we combine two antibodies for the enrichment of acetylated peptides and perform label-free quantitative acetylomic analysis of mouse livers during natural aging and upon the treatment of beta-nicotinamide mononucleotide (NMN), a NAD+ booster. Our study describes previously unknown acetylation sites and reveals the acetylome-wide dynamics with age as well as upon the treatment of NMN. We discover protein acetylation events as potential aging biomarkers. We demonstrate that the life-beneficial effect of NMN could be partially reflected by the changes in age-related protein acetylation. Our quantitative assessment indicates that NMN has mild effects on acetylation sites previously reported as substrates of Sirtuins. Collectively, our data analyze protein acetylation with age, laying critical foundation for the functional study of protein post-translational modification essential for healthy aging and perhaps disease conditions.

Porphyromonas gingivalis OMVs promoting endothelial dysfunction via the STING pathway in periodontitis.

OBJECTIVE: This study aimed to assess the effects of Porphyromonas gingivalis outer membrane vesicles (Pg-OMVs) in chronic periodontitis and explore the underlying mechanism involved. METHODS: In vitro, Pg-OMVs were incubated with Ea.hy926 (vessel endothelial cells, ECs) to evaluate their effects on endothelial functions and to investigate the underlying mechanism. The effects of endothelial dysfunction on MG63 osteoblast-like cells were verified using an indirect co-culture method. For in vivo studies, micro-CT was conducted to identify alveolar bone mass. Immunofluorescence staining was conducted to confirm the levels of stimulator of interferon genes (STING) in the blood vessel and the number of Runx2+ cells around the alveolar bone. RESULTS: Pg-OMVs were endocytosed by ECs, leading to endothelial dysfunction. The cGAS-STING-TBK1 pathway was activated in ECs, which subsequently inhibited MG63 migration and early osteogenesis differentiation. In vivo, Pg-OMVs promoted alveolar bone resorption, increased STING levels in the blood vessel, and decreased Runx2+ cells around the alveolar bone. CONCLUSIONS: Pg-OMVs caused endothelial dysfunction and activated the cGAS-STING-TBK1 signal cascade in ECs, thereby impairing ECs-mediated osteogenesis. Furthermore, Pg-OMVs aggregated alveolar bone loss and altered the blood vessel-mediated osteogenesis with elevated STING.

Destabilization of ultra-instantaneous UHT sterilization milk stored at different temperatures.

Ultra-instantaneous UHT (UI-UHT, > 155°C, < 0.1 s) treated milk exhibits higher retention of active protein than regular UHT milk. However, UI-UHT products demonstrate increased susceptibility to destabilization during storage. This study aimed at monitoring the destabilizing process of UI-UHT milk across different storage temperatures and uncovering its potential mechanisms. Compared with regular UHT treatment, ultra-instantaneous treatment markedly accelerated the milk's destabilization process. Aged gel formation occurred after 45 d of storage at 25°C, while creaming and sedimentation were observed after 15 d at 37°C. To elucidate the instability mechanism, measurements of plasmin activity, protein hydrolysis levels, and proteomics of the aged gel were conducted. In UI-UHT milk, plasmin activity, and protein hydrolysis levels significantly increased during storage. Excessive protein hydrolysis at 37°C resulted in sedimentation, while moderate hydrolysis and an increase in protein particle size at 25°C resulted in aged gel formation. Proteomics analysis results indicated that the aged gel from UI-UHT milk contained intact caseins, major whey proteins, and their derived peptides. Furthermore, specific whey proteins including albumin, lactotransferrin, enterotoxin-binding glycoprotein PP20K, and MFGM proteins were identified in the gel. Additionally, MFGM proteins in UI-UHT milk experienced considerable hydrolysis during storage, contributing to fat instability. This study lays a theoretical foundation for optimizing UI-UHT milk storage conditions to enhance the quality of liquid milk products.

Investigating oral microbiome profiles in patients with cleft lip and palate compared with the healthy control.

BACKGROUND: Patients with cleft lip and palate (CLP) have an oronasal communication differed from the closed state in healthy individuals, leading to a unique oral microbiome. This study aimed to determine if variances in the oral microbiota persist among CLP patients who have received treatments for the closure of these fistulas compared to the microbiota of healthy individuals. METHODS: Saliva samples were collected from a cohort comprising 28 CLP patients (CLP group) and 30 healthy controls (HC group). Utilizing 16S rRNA sequencing on the Illumina NovaSeq platform, we conducted a comprehensive analysis of the diversity and composition of the oral microbiota. RESULTS: The analysis of the microbiota in the saliva samples revealed a total of 23 microbial phyla, 38 classes, 111 orders, 184 families, 327 genera and 612 species. The alpha diversity with microbial abundance and evenness indicated the significant difference between the CLP and HC groups. Principal coordinate analysis (PCoA) and the ADONIS test further supported the presence of distinct microorganisms between the two groups. The CLP group displayed elevated abundances of Neisseria, Haemophilus, Porphyromonas, and Granulicatella, as indicated by LefSe analysis. Conversely, Rothia, Veillonella, and Pauljensenia exhibited significant reductions in abundance in the CLP group. The results of the PICRUSt analysis indicated significant differences in the relative abundance of 25 KEGG pathways within the CLP group. Through Spearman correlation analysis, strong associations between Rothia, Veillonella, and Pauljensenia and 25 functional pathways linked to CLP were identified. CONCLUSION: Findings of this study offer a thorough comprehension of the microbiome profiles of CLP patients after the restoration of oronasal structure and are anticipated to present innovative concepts for the treatment of CLP.

Quantitative Acetylomics Reveals Substrates of Lysine Acetyltransferase GCN5 in Adult and Aging Drosophila.

Protein lysine acetylation is a dynamic post-translational modification (PTM) that regulates a wide spectrum of cellular events including aging. General control nonderepressible 5 (GCN5) is a highly conserved lysine acetyltransferase (KAT). However, the acetylation substrates of GCN5 in vivo remain poorly studied, and moreover, how lysine acetylation changes with age and the contribution of KATs to aging remain to be addressed. Here, using Drosophila, we perform label-free quantitative acetylomic analysis, identifying new substrates of GCN5 in the adult and aging process. We further characterize the dynamics of protein acetylation with age, which exhibits a trend of increase. Since the expression of endogenous fly Gcn5 progressively increases during aging, we reason that, by combining the substrate analysis, the increase in acetylation with age is triggered, at least in part, by GCN5. Collectively, our study substantially expands the atlas of GCN5 substrates in vivo, provides a resource of protein acetylation that naturally occurs with age, and demonstrates how individual KAT contributes to the aging acetylome.

Mate-pair genome sequencing reveals structural variants for idiopathic male infertility.

Currently, routine genetic investigation for male infertility includes karyotyping analysis and PCR for Y chromosomal microdeletions to provide prognostic information such as sperm retrieval success rate. However, over 85% of male infertility remain idiopathic. We assessed 101 male patients with primary infertility in a retrospective cohort analysis who have previously received negative results from standard-of-care tests. Mate-pair genome sequencing (large-insert size library), an alternative long-DNA sequencing method, was performed to detect clinically significant structural variants (SVs) and copy-number neutral absence of heterozygosity (AOH). Candidate SVs were filtered against our in-house cohort of 1077 fertile men. Genes disrupted by potentially clinically significant variants were correlated with single-cell gene expression profiles of human fetal and postnatal testicular developmental lineages and adult germ cells. Follow-up studies were conducted for each patient with clinically relevant finding(s). Molecular diagnoses were made in 11.1% (7/63) of patients with non-obstructive azoospermia and 13.2% (5/38) of patients with severe oligozoospermia. Among them, 12 clinically significant SVs were identified in 12 cases, including five known syndromes, one inversion, and six SVs with direct disruption of genes by intragenic rearrangements or complex insertions. Importantly, a genetic defect related to intracytoplasmic sperm injection (ICSI) failure was identified in a patient with non-obstructive azoospermia, illustrating the additional value of an etiologic diagnosis in addition to determining sperm retrieval rate. Our study reveals a landscape of various genomic variants in 101 males with idiopathic infertility, not only advancing understanding of the underlying mechanisms of male infertility, but also impacting clinical management.

Bi-allelic Mutations in NADSYN1 Cause Multiple Organ Defects and Expand the Genotypic Spectrum of Congenital NAD Deficiency Disorders.

Birth defects occur in up to 3% of all live births and are the leading cause of infant death. Here we present five individuals from four unrelated families, individuals who share similar phenotypes with disease-causal bi-allelic variants in NADSYN1, encoding NAD synthetase 1, the final enzyme of the nicotinamide adenine dinucleotide (NAD) de novo synthesis pathway. Defects range from the isolated absence of both kidneys to multiple malformations of the vertebrae, heart, limbs, and kidney, and no affected individual survived for more than three months postnatally. NAD is an essential coenzyme for numerous cellular processes. Bi-allelic loss-of-function mutations in genes required for the de novo synthesis of NAD were previously identified in individuals with multiple congenital abnormalities affecting the heart, kidney, vertebrae, and limbs. Functional assessments of NADSYN1 missense variants, through a combination of yeast complementation and enzymatic assays, show impaired enzymatic activity and severely reduced NAD levels. Thus, NADSYN1 represents an additional gene required for NAD synthesis during embryogenesis, and NADSYN1 has bi-allelic missense variants that cause NAD deficiency-dependent malformations. Our findings expand the genotypic spectrum of congenital NAD deficiency disorders and further implicate mutation of additional genes involved in de novo NAD synthesis as potential causes of complex birth defects.

Pyridine Functionalized Carbon Nanotubes: Unveiling the Role of External Pyridinic Nitrogen Sites for Oxygen Reduction Reaction.

Pyridinic nitrogen has been recognized as the primary active site in nitrogen-doped carbon electrocatalysts for the oxygen reduction reaction (ORR), which is a critical process in many renewable energy devices. However, the preparation of nitrogen-doped carbon catalysts comprised of exclusively pyridinic nitrogen remains challenging, as well as understanding the precise ORR mechanisms on the catalyst. Herein, a novel process is developed using pyridyne reactive intermediates to functionalize carbon nanotubes (CNTs) exclusively with pyridine rings for ORR electrocatalysis. The relationship between the structure and ORR performance of the prepared materials is studied in combination with density functional theory calculations to probe the ORR mechanism on the catalyst. Pyridinic nitrogen can contribute to a more efficient 4-electron reaction pathway, while high level of pyridyne functionalization result in negative structural effects, such as poor electrical conductivity, reduced surface area, and small pore diameters, that suppressed the ORR performance. This study provides insights into pyridine-doped CNTs-functionalized for the first time via pyridyne intermediates-as applied in the ORR and is expected to serve as valuable inspiration in designing high-performance electrocatalysts for energy applications.

Tyrosine phosphorylation of band 3 impairs the storage quality of suspended red blood cells in the Tibetan high-altitude polycythemia population.

Due to environmental hypoxia on the Tibetan Plateau, local residents often exhibit a compensative increase in hemoglobin concentration to maintain the body's oxygen supply. However, increases in hemoglobin and hematocrit (Hct) pose a serious challenge to the quality of stored suspended red blood cells (SRBCs) prepared from the blood of high-hemoglobin populations, especially populations at high altitude with polycythemia in Tibet. To explore the difference in storage quality of SRBCs prepared from plateau residents with a high hemoglobin concentration, blood donors were recruited from Tibet (> 3600 m) and Chengdu (≈ 500 m) and divided into a high-altitude control (HAC) group, high-altitude polycythemia (HAPC) group and lowland control (LLC) group according to their hemoglobin concentration and altitude of residence. The extracellular acidification rate (ECAR), pyruvate kinase (PK) activity and band 3 tyrosine phosphorylation were analyzed on the day of blood collection. Then, whole-blood samples were processed into SRBCs, and storage quality parameters were analyzed aseptically on days 1, 14, 21 and 35 of storage. Overall, we found that tyrosine 21 phosphorylation activated glycolysis by releasing glycolytic enzymes from the cytosolic domain of band 3, thus increasing glucose consumption and lactate accumulation during storage, in the HAPC group. In addition, band 3 tyrosine phosphorylation impaired erythrocyte deformability, accompanied by the highest hemolysis rate in the HAPC group, during storage. We believe that these results will stimulate new ideas to further optimize current additive solutions for the high-hemoglobin population in Tibet and reveal new therapeutic targets for the treatment of HAPC populations.

A pilot investigation of low-pass genome sequencing identifying site-specific variation in chromosomal mosaicisms by a multiple site sampling approach in first-trimester miscarriages.

STUDY QUESTION: Can multiple-site low-pass genome sequencing (GS) of products of conception (POCs) improve the detection of genetic abnormalities, especially heterogeneously distributed mosaicism and homogeneously distributed mosaicism in first-trimester miscarriage? SUMMARY ANSWER: Multiple-site sampling combined with low-pass GS significantly increased genetic diagnostic yield (77.0%, 127/165) of first-trimester miscarriages, with mosaicisms accounting for 17.0% (28/165), especially heterogeneously distributed mosaicisms (75%, 21/28) that are currently underappreciated. WHAT IS KNOWN ALREADY: Aneuploidies are well known to cause first-trimester miscarriage, which are detectable by conventional karyotyping and next-generation sequencing (NGS) on a single-site sampling basis. However, there are limited studies demonstrating the implications of mosaic genetic abnormalities in first-trimester miscarriages, especially when genetic heterogeneity is present in POCs. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional cohort study carried out at a university-affiliated public hospital. One hundred seventy-four patients diagnosed with first-trimester miscarriage from December 2018 to November 2021 were offered ultrasound-guided manual vacuum aspiration (USG-MVA) treatment. Products of conception were subjected to multiple-site low-pass GS for the detection of chromosomal imbalances. PARTICIPANTS/MATERIALS, SETTING, METHODS: For each POC, multiple sites of villi (three sites on average) were biopsied for low-pass GS. Samples with maternal cell contamination (MCC) and polyploidy were excluded based on the quantitative fluorescence polymerase chain reaction (QF-PCR) results. The spectrum of chromosomal abnormalities, including mosaicism (heterogeneously distributed and homogeneously distributed) and constitutional abnormalities was investigated. Chromosomal microarray analysis and additional DNA fingerprinting were used for validation and MCC exclusion. A cross-platform comparison between conventional karyotyping and our multiple-site approach was also performed. MAIN RESULTS AND THE ROLE OF CHANCE: One hundred sixty-five POCs (corresponding to 490 DNA samples) were subjected to low-pass GS. Genetic abnormalities were detected in 77.0% (127/165) of POCs by our novel approach. Specifically, 17.0% (28/165) of cases had either heterogeneously distributed mosaicism (12.7%, 21/165) or homogeneously distributed mosaicism (6.1%, 10/165) (three cases had both types of mosaicism). The remaining 60.0% (99/165) of cases had constitutional abnormalities. In addition, in the 71 cases with karyotyping performed in parallel, 26.8% (19/71) of the results could be revised by our approach. LIMITATIONS, REASONS FOR CAUTION: Lack of a normal gestational week-matched cohort might hinder the establishment of a causative link between mosaicisms and first-trimester miscarriage. WIDER IMPLICATIONS OF THE FINDINGS: Low-pass GS with multiple-site sampling increased the detection of chromosomal mosaicisms in first-trimester miscarriage POCs. This innovative multiple-site low-pass GS approach enabled the novel discovery of heterogeneously distributed mosaicism, which was prevalent in first-trimester miscarriage POCs and frequently observed in preimplantation embryos, but is currently unappreciated by conventional single-site cytogenetic investigations. STUDY FUNDING/COMPETING INTEREST(S): This work was supported partly by Research Grant Council Collaborative Research Fund (C4062-21GF to K.W.C), Science and Technology Projects in Guangzhou (202102010005 to K.W.C), Guangdong-Hong Kong Technology Cooperation Funding Scheme (TCFS), Innovation and Technology Fund (GHP/117/19GD to K.W.C), HKOG Direct Grant (2019.050 to J.P.W.C), and Hong Kong Health and Medical Research Fund (05160406 to J.P.W.C). The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Expansion of the clinical and molecular spectrum of WWOX-related epileptic encephalopathy.

WWOX biallelic loss-of-function pathogenic single nucleotide variants (SNVs) and copy number variants (CNVs) including exonic deletions and duplications cause WWOX-related epileptic encephalopathy (WOREE) syndrome. This disorder is characterized by refractory epilepsy, axial hypotonia, peripheral hypertonia, progressive microcephaly, and premature death. Here we report five patients with WWOX biallelic predicted null variants identified by exome sequencing (ES), genome sequencing (GS), and/or chromosomal microarray analysis (CMA). SNVs and intragenic deletions of one or more exons were commonly reported in WOREE syndrome patients which made the genetic diagnosis challenging and required a combination of different diagnostic technologies. These patients presented with severe, developmental and epileptic encephalopathy (DEE), and other cardinal features consistent with WOREE syndrome. This report expands the clinical phenotype associated with this condition, including failure to thrive in most patients and epilepsy that responded to a ketogenic diet in three patients. Dysmorphic features and abnormal prenatal findings were not commonly observed. Additionally, recurrent pancreatitis and sensorineural hearing loss each were observed in single patients. In summary, these phenotypic features broaden the clinical spectrum of WOREE syndrome.