The mechanism of STING autoinhibition and activation.

2',3'-cGAMP, produced by the DNA sensor cGAS, activates stimulator of interferon genes (STING) and triggers immune response during infection. Tremendous effort has been placed on unraveling the mechanism of STING activation. However, little is known about STING inhibition. Here, we found that apo-STING exhibits a bilayer with head-to-head as well as side-by-side packing, mediated by its ligand-binding domain (LBD). This type of assembly holds two endoplasmic reticulum (ER) membranes together not only to prevent STING ER exit but also to eliminate the recruitment of TBK1, representing the autoinhibited state of STING. Additionally, we obtained the filament structure of the STING/2',3'-cGAMP complex, which adopts a bent monolayer assembly mediated by LBD and transmembrane domain (TMD). The active, curved STING polymer could deform ER membrane to support its ER exit and anterograde transportation. Our data together provide a panoramic vision regarding STING autoinhibition and activation, which adds substantially to current understanding of the cGAS-STING pathway.

SNX27 suppresses SARS-CoV-2 infection by inhibiting viral lysosome/late endosome entry.

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27-retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.

Development of multiplex allele-specific RT-qPCR assays for differentiation of SARS-CoV-2 Omicron subvariants.

Quick differentiation of current circulating variants and the emerging recombinant variants of SARS-CoV-2 is essential to monitor their transmissions. However, the widely applied gene sequencing method is time-consuming and costly especially when facing recombinant variants, because a large part or whole genome sequencing is required. Allele-specific reverse transcriptase real time RT-PCR (RT-qPCR) represents a quick and cost-effective method for SNP (single nucleotide polymorphism) genotyping and has been successfully applied for SARS-CoV-2 variant screening. In the present study, we developed a panel of 5 multiplex allele-specific RT-qPCR assays targeting 20 key mutations for quick differentiation of the Omicron subvariants (BA.1 to BA.5 and their descendants) and the recombinant variants (XBB.1 and XBB.1.5). Two parallel multiplex RT-qPCR reactions were designed to separately target the prototype allele and the mutated allele of each mutation in the allele-specific RT-qPCR assay. Optimal annealing temperatures, primer and probe dosage, and time for annealing/extension for each reaction were determined by multi-factor and multi-level orthogonal test. The variation of Cp (crossing point) values (ΔCp) between the two multiplex RT-qPCR reactions was applied to determine if a mutation occurs or not. SARS-CoV-2 subvariants and related recombinant variants were differentiated by their unique mutation patterns. The developed multiplex allele-specific RT-qPCR assays exhibited excellent analytical sensitivities (with limits of detection (LoDs) of 1.47-18.52 copies per reaction), wide linear detection ranges (109-100 copies per reaction), good amplification efficiencies (88.25 to 110.68%), excellent reproducibility (coefficient of variations (CVs) < 5% in both intra-assay and inter-assay tests), and good clinical performances (99.5-100% consistencies with Sanger sequencing). The developed multiplex allele-specific RT-qPCR assays in the present study provide an alternative tool for quick differentiation of the SARS-CoV-2 Omicron subvariants and their recombinant variants. KEY POINTS: • A panel of five multiplex allele-specific RT-qPCR assays for quick differentiation of 11 SARS-CoV-2 Omicron subvariants (BA.1, BA.2, BA.4, BA.5, and their descendants) and 2 recombinant variants (XBB.1 and XBB.1.5). • The developed assays exhibited good analytical sensitivities and reproducibility, wide linear detection ranges, and good clinical performances, providing an alternative tool for quick differentiation of the SARS-CoV-2 Omicron subvariants and their recombinant variants.

Lipid droplet accumulation in Wdr45-deficient cells caused by impairment of chaperone-mediated autophagic degradation of Fasn.

BACKGROUND: ß-Propeller protein-associated neurodegeneration (BPAN) is a genetic neurodegenerative disease caused by mutations in WDR45. The impairment of autophagy caused by WDR45 deficiency contributes to the pathogenesis of BPAN; however, the pathomechanism of this disease is largely unknown. Lipid dyshomeostasis is involved in neurogenerative diseases, but whether lipid metabolism is affected by Wdr45 deficiency and whether lipid dyshomeostasis contributes to the progression of BPAN are unclear. METHODS: We generated Wdr45 knockout SN4741 cell lines using CRISPR‒Cas9-mediated genome editing, then lipid droplets (LDs) were stained using BODIPY 493/503. Chaperone-mediated autophagy was determined by RT-qPCR and western blotting. The expression of fatty acid synthase (Fasn) was detected by western blot in the presence or absence of the lysosomal inhibitor NH4Cl and the CMA activator AR7. The interaction between Fasn and HSC70 was analyzed using coimmunoprecipitation (Co-IP) assay. Cell viability was measured by a CCK-8 kit after treatment with the Fasn inhibitor C75 or the CMA activator AR7. RESULTS: Deletion of Wdr45 impaired chaperone-mediated autophagy (CMA), thus leading to lipid droplet (LD) accumulation. Moreover, Fasn can be degraded via CMA, and that defective CMA leads to elevated Fasn, which promotes LD formation. LD accumulation is toxic to cells; however, cell viability was not rescued by Fasn inhibition or CMA activation. Inhibition of Fasn with a low concentration of C75 did not affect cell viability but decreases LD density. CONCLUSIONS: These results suggested that Fasn is essential for cell survival but that excessive Fasn leads to LD accumulation in Wdr45 knockout cells.

Identification of Candidate Genes for Red-Eyed (Albinism) Domestic Guppies Using Genomic and Transcriptomic Analyses.

Guppies are small tropical fish with brightly colored bodies and variable tail shapes. There are two phenotypes of domestic guppy eye color: red and black. The wild type is black-eyed. The main object of this study was to identify candidate genes for the red-eyed phenotype in domestic guppies. We hope to provide molecular genetic information for the development of new domestic guppy strains. Additionally, the results also contribute to basic research concerning guppies. In this study, 121 domestic guppies were used for genomic analysis (GWAS), and 44 genes were identified. Furthermore, 21 domestic guppies were used for transcriptomic analysis, and 874 differentially expressed genes (DEGs) were identified, including 357 upregulated and 517 downregulated genes. Through GO and KEGG enrichment, we identified some important terms or pathways mainly related to melanin biosynthesis and ion transport. qRT-PCR was also performed to verify the differential expression levels of four important candidate genes (TYR, OCA2, SLC45A2, and SLC24A5) between red-eyed and black-eyed guppies. Based on the results of genomic and transcriptomic analyses, we propose that OCA2 is the most important candidate gene for the red-eyed phenotype in guppies.

Regulation of Follicular Development in Chickens: WIF1 Modulates Granulosa Cell Proliferation and Progesterone Synthesis via Wnt/ß-Catenin Signaling Pathway.

Proliferation, apoptosis, and steroid hormone secretion by granulosa cells (GCs) and theca cells (TCs) are essential for maintaining the fate of chicken follicles. Our previous study showed that the Wnt inhibitor factor 1 (WIF1) plays a role in follicle selection. However, the significance of WIF1 in GC- and TC-associated follicular development was not explicitly investigated. This study found that WIF1 expression was strongly downregulated during follicle selection (p < 0.05) and was significantly higher in GCs than in TCs (p < 0.05). WIF1 inhibits proliferation and promotes apoptosis in GCs. Additionally, it promotes progesterone secretion in prehierarchal GCs (pre-GCs, 1.16 ± 0.05 ng/mg vs. 1.58 ng/mg ± 0.12, p < 0.05) and hierarchal GCs (hie-GCs, 395.00 ng/mg ± 34.73 vs. 527.77 ng/mg ± 27.19, p < 0.05) with the participation of the follicle-stimulating hormone (FSH). WIF1 affected canonical Wnt pathways and phosphorylated ß-catenin expression in GCs. Furthermore, 604 upregulated differentially expressed genes (DEGs) and 360 downregulated DEGs in WIF1-overexpressed GCs were found through RNA-seq analysis (criteria: |log2⁡(FoldChange)| > 1 and p_adj < 0.05). Cytokine-cytokine receptor interaction and the steroid hormone biosynthesis pathway were identified. In addition, the transcript of estrogen receptor 2 (ESR2) increased significantly (log2⁡(FoldChange) = 1.27, p_adj < 0.05). Furthermore, we found that WIF1 regulated progesterone synthesis by upregulating ESR2 expression in GCs. Additionally, WIF1 suppressed proliferation and promoted apoptosis in TCs. Taken together, these results reveal that WIF1 stimulates follicle development by promoting GC differentiation and progesterone synthesis, which provides an insight into the molecular mechanism of follicle selection and egg-laying performance in poultry.

Varied hypoxia adaptation patterns of embryonic brain at different development stages between Tibetan and Dwarf laying chickens.

BACKGROUND: Tibetan chickens (Gallus gallus; TBCs), an indigenous breed distributed in the Qinghai-Tibet Plateau, are well adapted to the hypoxic environment. Currently, the molecular genetic basis of hypoxia adaptation in TBCs remains unclear. This study investigated hypoxia adaptation patterns of embryonic brain at different development stages by integrating analysis of the transcriptome with our previously published metabolome data in TBCs and Dwarf Laying Chickens (DLCs), a lowland chicken breed. RESULTS: During hypoxia, the results revealed that 1334, 578, and 417 differentially expressed genes (DEGs) (|log2 fold change|>1, p-value < 0.05) on days 8, 12, and 18 of development, respectively between TBCs and DLCs. Gene Ontology (GO) and pathway analyses revealed that DEGs are mainly related to metabolic pathways, vessel development, and immune response under hypoxia. This is consistent with our metabolome data that TBCs have higher energy metabolism than DLCs during hypoxia. Some vital DEGs between TBCs and DLCs, such as EPAS1, VEGFD, FBP1, FBLN5, LDHA, and IL-6 which are involved in the HIF pathway and hypoxia regulation. CONCLUSION: These results suggest varied adaptation patterns between TBCs and DLCs under hypoxia. Our study provides a basis for uncovering the molecular regulation mechanism of hypoxia adaptation in TBCs and a potential application of hypoxia adaptation research for other animals living on the Qinghai-Tibet Plateau, and may even contribute to the study of brain diseases caused by hypoxia.

Navigating mental health in a post-COVID world: the experience of colorectal cancer patients.

The impact of the post-COVID-19 pandemic on individuals with colorectal cancer (CRC) was evaluated in a study with 465 cases and controls. Participants completed a questionnaire, and data analysis was conducted using SPSS software. Results showed a higher prevalence of emotional stress symptoms such as fear, depression, and worry among cases compared to controls. Additionally, cases reported greater physiological stress symptoms, such as low energy levels. The results also indicated that cases had higher rates of racing thoughts, pessimism, and avoidance of responsibilities compared to controls. These findings highlight the significant negative impact of the pandemic on individuals with CRC and the need for prompt attention and support.

Evolutionary adaptation of bovine coronavirus (BCoV): Screening of natural recombinations across the complete genomes.

Bovine coronavirus (BCoV) is a member of pathogenic Betacoronaviruses that has been circulating for several decades in multiple host species. Given the similarity between BCoV and human coronaviruses, the current study aimed to review the complete genomes of 107 BCoV strains available on the GenBank database, collected between 1983 and 2017 from different countries. The maximum-likelihood based phylogenetic analysis revealed three main BCoV genogroups: GI, GII, and GIII. GI is further divided into nine subgenogroups: GI-a to GI-i. The GI-a to GI-d are restricted to Japan, and GI-e to GI-i to the USA. The evolutionary relationships were also inferred using phylogenetic network analysis, revealing two major distinct networks dominated by viruses identified in the USA and Japan, respectively. The USA strains-dominated Network Cluster includes two sub-branches: France/Germany and Japan/China in addition to the United States, while Japan strains-dominated Network Cluster is limited to Japan. Twelve recombination events were determined, including 11 intragenogroup (GI) and one intergenogroup (GII vs. GI-g). The breakpoints of the recombination events were mainly located in ORF1ab and the spike glycoprotein ORF. Interestingly, 10 of 12 recombination events occurred between Japan strains, one between the USA strains, and one from intercontinental recombination (Japan vs. USA). These findings suggest that geographical characteristics, and population density with closer contact, might significantly impact the BCoV infection and co-infection and boost the emergence of more complex virus lineages.

Bidirectional crosstalk between dysbiotic gut microbiota and systemic lupus erythematosus: What is new in therapeutic approaches?

Systemic lupus erythematosus is an autoimmune disease characterized by chronic inflammation and multiple organs damage. Its pathogenesis is complex and involves multiple factors including gut microbiota. Accumulating evidence indicates the interaction of microbial communities with the host immune system to maintain a state of homeostasis. Imbalances within the gut microbial composition and function may contribute to the development of many autoimmune diseases including SLE. In this review, we aim to highlight the dysregulation of commensal bacteria and their metabolites in the gastrointestinal tract and the resulting autoimmune responses in lupus and to decrypt the cross-link between the altered gut microbiota and the immune system in the SLE condition. We also provide new insights into targeting gut microbiota as a promising therapeutic approach to treat and manage SLE.

Evolutionary plasticity of zoonotic porcine Deltacoronavirus (PDCoV): genetic characteristics and geographic distribution.

The emergence and rapid spread of the acute respiratory syndrome coronavirus-2 have confirmed that animal coronaviruses represent a potential zoonotic source. Porcine deltacoronavirus is a worldwide evolving enteropathogen of swine, detected first in Hong Kong, China, before its global identification. Following the recent detection of PDCoV in humans, we attempted in this report to re-examine the status of PDCoV phylogenetic classification and evolutionary characteristics. A dataset of 166 complete PDCoV genomes was analyzed using the Maximum Likelihood method in IQ-TREE with the best-fitting model GTR + F + I + G4, revealing two major genogroups (GI and GII), with further seven and two sub-genogroups, (GI a-g) and (GII a-b), respectively. PDCoV strains collected in China exhibited the broadest genetic diversity, distributed in all subgenotypes. Thirty-one potential natural recombination events were identified, 19 of which occurred between China strains, and seven involved at least one China strain as a parental sequence. Importantly, we identified a human Haiti PDCoV strain as recombinant, alarming a possible future spillover that could become a critical threat to human health. The similarity and recombination analysis showed that PDCoV spike ORF is highly variable compared to ORFs encoding other structural proteins. Prediction of linear B cell epitopes of the spike glycoprotein and the 3D structural mapping of amino acid variations of two representative strains of GI and GII showed that the receptor-binding domain (RBD) of spike glycoprotein underwent a significant antigenic drift, suggesting its contribution in the genetic diversity and the wider spread of PDCoV.

Efficient TALEN-mediated gene knockin at the bovine Y chromosome and generation of a sex-reversal bovine.

Functional elucidation of bovine Y-chromosome genes requires available genome editing technologies. Meanwhile, it has yet to be proven whether the bovine Sry gene is the main or single factor involved in the development of the male phenotype in bovine. Here, we efficiently knocked out four Y-linked genes (Sry, ZFY, DDX3Y, and EIF2S3Y) in bovine fetal fibroblasts (BFFs) with transcription activator-like effector nucleases (TALENs) individually. Furthermore, we used TALEN-mediated gene knockin at the Sry gene and generated a sex-reversal bovine by somatic cell nuclear transfer (SCNT). The resulting bovine had only one ovary and was sterile. We demonstrate, for the first time, that the Sry gene is an important sex-determining gene in bovine. Our method lays a solid foundation for detecting the biology of the bovine Y chromosome, as it may provide an alternative biological model system for the study of mammalian sex determination, and new methods for the practical application in agricultural, especially for sex predetermination.

Genetic architectures and selection signatures of body height in Chinese indigenous donkeys revealed by next-generation sequencing.

Donkeys are widely distributed labour animals in the world. During the process of the domestication and artificial selection of domestic donkeys, body sizes show significant differences among different breeds of donkeys. Based on the genome resequencing data of 103 Chinese indigenous donkeys from 11 breeds (Biyang, Dezhou, Guangling, Hetian, Jiami, Kulun, Qingyang, Turfan, Tibetan, Xinjiang, and Yunnan), seven Spanish donkeys from two breeds (Zamorano~Leonés and Andalusian), and three wild donkeys, we investigated the population structures of Chinese domestic donkeys with different body sizes. We used FST and XP-EHH analyses to explore the selected regions related to body sizes. The results showed that Chinese indigenous donkeys have a closer relationship with African wild donkeys than with Asian wild donkeys. LCORL/NCAPG, FAM184B, TBX3, and IHH were identified as genes with strong signals in analysis of selection signature (FST and XP-EHH) in large and small donkeys. The seven identified variants can be served as candidate loci affecting the body size of Chinese donkeys. Five of seven loci were located in intron 9 of FAM184B and were in a haplotype block, and one of the identified variants (Chr03:112664848) located in the CDS region of the LCORL gene was found to cause stop-loss. These candidate genes and variants shed new light on the molecular basis of donkey body size and will facilitate the breeding activities of donkeys.

Identification and Functional Evaluation of a Novel TBX4 Mutation Underlies Small Patella Syndrome.

Small patella syndrome (SPS) is a rare autosomal dominant disorder caused by mutations in TBX4 gene which encodes a transcription factor of FGF10. However, how TBX4 mutations result in SPS is poorly understood. Here, a novel TBX4 mutation c.1241C>T (p.P414L) was identified in a SPS family and series of studies were performed to evaluate the influences of TBX4 mutations (including c.1241C>T and two known mutations c.256G>C and c.743G>T). Results showed that mesenchymal stem cells (MSCs) with stable overexpression of either TBX4 wild-type (TBX4wt) or mutants (TBX4mt) were successfully generated. Immunofluorescence study revealed that both the overexpressed TBX4 wild-type and mutants were evenly expressed in the nucleus suggesting that these mutations do not alter the translocation of TBX4 into the nucleus. Interestingly, MSCs overexpression of TBX4mt exhibited reduced differentiation activities and decreased FGF10 expression. Chromatin immunoprecipitation (ChIP) study demonstrated that TBX4 mutants still could bind to the promoter of FGF10. However, dual luciferase reporter assay clarified that the binding efficiencies of TBX4 mutants to FGF10 promoter were reduced. Taken together, MSCs were firstly used to study the function of TBX4 mutations in this study and the results indicate that the reduced binding efficiencies of TBX4 mutants (TBX4mt) to the promoter of FGF10 result in the abnormal biological processes which provide important information for the pathogenesis of SPS.

Functional Characterization of FH Mutation c.557G>A Underlies Uterine Leiomyomas.

The FH gene encodes the fumarate hydratase of the Krebs cycle and functions as a homotetramer to catalyze the hydration of fumarate to malate. Mutations in FH result in uterine leiomyomas, a rare autosomal dominant inherited metabolic disease. However, how FH mutations result in this disease is poorly understood. Here, the FH mutation c.557G>A (p.S186N) was identified in a family with uterine leiomyomas phenotype. A series of studies were performed to confirm the pathogenicity of this mutation. Results showed that the FH mutant exhibited significantly lower fumarase enzyme activity and increased the fumarates level compared with the wildtype, which might be due to the impaired homotetramer formation in the native gel electrophoresis. Interestingly, the immunofluorescence study revealed that the overexpressed FH mutant exhibited puncta structures compared with the evenly expressed FH wildtype in cytoplasm suggesting that the altered amino acid might result in dysfunctional proteins which were accumulated to reduce its cytotoxicity. Importantly, the cells overexpressing the FH mutant exhibited higher proliferation and extracellular acidification rate value (ECAR) which might be caused by the upregulated HIF-1α indicating the tumor phenotype. Notably, phospho-mTOR was significantly increased and autophagy was inhibited in the FH mutant overexpression cells compared with the wildtype. Our work provides new insight into the FH mutation c.557G>A (p.S186N) underlies uterine leiomyomas and important information for accurate genetic counseling and clinical diagnosis of the disease.

RNAseq and quantitative proteomic analysis of Dictyostelium knock-out cells lacking the core autophagy proteins ATG9 and/or ATG16.

BACKGROUND: Autophagy is an evolutionary ancient mechanism that sequesters substrates for degradation within autolysosomes. The process is driven by many autophagy-related (ATG) proteins, including the core members ATG9 and ATG16. However, the functions of these two core ATG proteins still need further elucidation. Here, we applied RNAseq and tandem mass tag (TMT) proteomic approaches to identify differentially expressed genes (DEGs) and proteins (DEPs) in Dictyostelium discoideum ATG9‾, ATG16‾ and ATG9‾/16‾ strains in comparison to AX2 wild-type cells. RESULT: In total, we identified 332 (279 up and 53 down), 639 (487 up and 152 down) and 260 (114 up and 146 down) DEGs and 124 (83 up and 41 down), 431 (238 up and 193 down) and 677 (347 up and 330 down) DEPs in ATG9‾, ATG16‾ and ATG9‾/16‾ strains, respectively. Thus, in the single knock-out strains, the number of DEGs was higher than the number of DEPs while in the double knock-out strain the number of DEPs was higher. Comparison of RNAseq and proteomic data further revealed, that only a small proportion of the transcriptional changes were reflected on the protein level. Gene ontology (GO) analysis revealed an enrichment of DEPs involved in lipid metabolism and oxidative phosphorylation. Furthermore, we found increased expression of the anti-oxidant enzymes glutathione reductase (gsr) and catalase A (catA) in ATG16‾ and ATG9‾/16‾ cells, respectively, indicating adaptation to excess reactive oxygen species (ROS). CONCLUSIONS: Our study provides the first combined transcriptome and proteome analysis of ATG9‾, ATG16‾ and ATG9‾/16‾ cells. Our results suggest, that most changes in protein abundance were not caused by transcriptional changes, but were rather due to changes in protein homeostasis. In particular, knock-out of atg9 and/or atg16 appears to cause dysregulation of lipid metabolism and oxidative phosphorylation.

HIF-1 regulates energy metabolism of the Tibetan chicken brain during embryo development under hypoxia.

The Tibetan chicken (Gallus gallus; TBC) is an indigenous breed found in the Qinghai-Tibet Plateau that are well adapted to a hypoxic environment. The energy metabolism of embryonic brains in TBCs under hypoxia has been little reported. This study investigated changes in energy metabolism of the TBC brain during embryo development under hypoxia. We found that TBCs exhibited a change of glycolysis and the tricarboxylic acid cycle during embryo development under hypoxia. Hypoxia-inducible factor (HIF)-1 was potentially involved in this by directly inducing overexpression of pyruvate dehydrogenase kinase 1 (PDK1) and the glycolytic genes hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) to increase glycolysis of TBCs to adapt to hypoxia. Although these may not be unique to TBCs, as we had also found similar results in Dwarf Laying Chickens, a lowland chicken breed, TBCs had a stronger regulating ability. In summary, our study revealed that HIF-1 induced energy metabolism changes in the TBC brain via upregulating expressions of PDK1 and other HIF-1 target genes like HK1 and LDHA to increase glycolysis for TBC hypoxic adaptations during embryo development. It indicates the potential application of TBC energy metabolism research for other animals living on the Qinghai-Tibet Plateau.

Low serum calcium and phosphorus and their clinical performance in detecting COVID-19 patients.

BACKGROUND: This study aimed to evaluate the clinical performance of low serum calcium and phosphorus in discriminative diagnosis of the severity of patients with coronavirus disease 2019 (COVID-19). We conducted a single-center hospital-based study and consecutively recruited 122 suspected and 104 confirmed patients with COVID-19 during January 24 to April 25, 2020. Clinical risk factors of COVID-19 were identified. The discriminative power of low calcium and phosphorus regarding the disease severity was evaluated. Low calcium and low phosphorus are more prevalent in severe or critical COVID-19 patients than moderate COVID-19 patients (odds ratio [OR], 15.07; 95% confidence interval [CI], 1.59-143.18 for calcium; OR, 6.90; 95% CI, 2.43-19.64 for phosphorus). The specificity in detecting the severe or critical patients among COVID-19 patients reached 98.5% (95% CI, 92.0%-99.7%) and 84.8% (95% CI, 74.3%-91.6%) by low calcium and low phosphorus, respectively, albeit with suboptimal sensitivity. Calcium and phosphorus combined with lymphocyte count could obtain the best discriminative performance for the severe COVID-19 patients (area under the curve [AUC] = 0.80), and combined with oxygenation index was promising (AUC = 0.71). Similar discriminative performances of low calcium and low phosphorus were found between suspected and confirmed COVID-19 patient. Low calcium and low phosphorus could indicate the severity of COVID-19 patients, and may be utilized as promising clinical biomarkers for discriminative diagnosis.

Two Novel Hydroxymethylbilane Synthase Splicing Mutations Predispose to Acute Intermittent Porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant genetic disease caused by a lack or decrease in hydroxymethylbilane synthase (HMBS) activity. It is characterized by acute nerve and visceral attacks caused by factors in the process of heme synthesis. The penetrance rate of this disease is low, and the heterogeneity is strong. Here, we reported two novel HMBS mutations from two unrelated Chinese AIP patients and confirmed the pathogenicity of these two mutations. We found the HMBS c.760-771+2delCTGAGGCACCTGGTinsGCTGCATCGCTGAA and HMBS c.88-1G>C mutations by second-generation sequencing and Sanger sequencing. The in vitro expression analysis showed that these mutations caused abnormal HMBS mRNA splicing and premature termination or partial missing of HMBS protein. Homologous modeling analysis showed that the HMBS mutants lacked the amino acids which are crucial for the enzyme activity or the protein stability. Consistently, enzyme activity analysis confirmed that the HMBS mutants' overexpression cells exhibited the reduced enzyme activity compared with the HMBS wildtype overexpression cells. Our study identified and confirmed two novel pathogenic HMBS mutations which will expand the molecular heterogeneity of AIP and provide further scientific basis for the clinical diagnosis of AIP.

[Clinical and genetic characteristics of Chinese patients with Waardenburg syndrome].

Waardenburg syndrome (WS), also known as auditorypigmentary syndrome, is characterized by non-progressive sensorineural hearing loss and anomalous pigmentation. Its mode of inheritance is either autosomal dominant or autosomal recessive. So far only PAX3, MITF, SOX10 and EDNRB mutations have been identified among Chinese patients with WS. This review has provided an update for WS-related genes, mutation databases, molecular and functional data, and a discussion over the molecular diagnosis of WS.