[Analysis of liver function injury associated with 2019-nCoV Omicron mutant strains].

Objective: To investigate the clinical features and influencing factors of liver function injury in patients with 2019-nCoV/SARS-CoV-2 Omicron mutant strains. Methods: 1 183 confirmed imported cases of SARS-CoV-2 who were admitted at Shanghai Public Health Clinical Center (affiliated to Fudan University) from July 1, 2021 to January 15, 2022 were collected. Clinical data, viral genotyping and laboratory test results were collected to retrospectively analyze the basic condition and clinical characteristics of liver function injury. Statistical analysis was performed using t-test or Wilcoxon rank-sum test, χ2 test or Fisher's exact test, Pearson correlation test and logistic regression analysis. Results: 125 (10.6%) cases had raised baseline ALT level and 60 (5.1%) cases had abnormal baseline AST level. Among them, 33 cases (2.8%) had received hepatoprotective drugs. Liver function injury was generally mild in SARS-CoV-2 infection and minimal in Omicron mutant strains. Leukocyte count was increased in patients with raised alanine aminotransferase (ALT) [(6.96±1.78)×109/L vs. (6.41±1.96)×109/L, P=0.005 2], CT scan showed the proportion of liver hypodensity was significantly increased (2.4% vs. 0.3%, P=0.018 0). High-sensitivity C-reactive protein [(7.83±22.36) mg/L vs. (2.68±6.21) mg/L, P=0.007 8] and D-dimer [(0.34±0.39) µg/ml vs. (0.31±0.75) µg/ml, P=0.047 5] levels were higher in patients with raised AST than normal group. 26 cases had normal liver function at hospital admission; however, abnormal liver function was occurred during the course of the disease. Another 8 patients had abnormal liver function at hospital admission, and reduced liver function further during the course of treatment. Recovery time and length of hospital stay was significantly affected in patients with worsened liver function. Baseline body mass index value [odds ratio (OR)]=1.80, P=0.047), non-Omicron strains (OR=12.63, P=0.046), D-dimer (OR=2.36, P=0.047) and interleukin-6 levels (OR=1.03, P=0.009), and those who used glucocorticoids and/or ulinastatin after hospital admission (OR=6.89, P=0.034) had a higher risk of worsening liver function. Conclusions: Liver dysfunction could be observed among COVID-19 patients. Patients infected with omicron variant generally showed mild liver injury. Dynamic monitoring of liver function is necessary, especially among those with baseline elevated IL-6, D-Dimer level and use of antiinflammation medication during treatment.

The synergistic antibacterial effect and inhibition of biofilm formation of nisin in combination with terpenes against Listeria monocytogenes.

This study was to investigate the synergistic antibacterial effect and inhibition of biofilm formation of nisin in combination with terpenes (carvacrol, cinnamaldehyde, citral and thymol) against Listeria monocytogenes. The bactericidal ranking of terpenes combined with nisin was carvacrol > cinnamaldehyde, citral > thymol. The minimum inhibitory concentration (MIC) of nisin and carvacrol when used together was determined to be 0·1563 mg ml-1 + 0·0195 mg ml-1 (nisin at MIC/2 + carvacrol at MIC/16). The addition of nisin at MIC/2 + carvacrol at MIC/2 caused more decrease in membrane potential than carvacrol or nisin at MIC individually. The decrease rates of hlyA and plcA gene expressions caused by nisin at MIC/2 + carvacrol at MIC/2 were significantly higher than those caused by carvacrol or nisin at MIC individually (P < 0·05). Nisin combined with carvacrol showed the highest inhibition activity to the formation of L. monocytogenes biofilm on stainless steel and lettuce. The inhibition effect of nisin at MIC/2 + carvacrol at MIC/16 was significantly higher than that of nisin at MIC/2 and carvacrol at MIC/16 (P < 0·05).

Identification of the mammalian Not gene via a phylogenomic approach.

Despite the great morphological diversity of early embryos, the underlying mechanisms of gastrulation are known to be broadly conserved in vertebrates. However, a number of genes characterized as fulfilling an essential function in this process in several model organisms display no clear ortholog in mammalian genomes. We have devised an in silico phylogenomic approach, based on exhaustive similarity searches in vertebrate genomes and subsequent bayesian phylogenetic analyses, to identify such missing genes, presumed to be highly divergent. This approach has been used to identify mammalian orthologs of Not, an homeodomain containing gene previously characterized in Xenopus, chick and zebrafish as playing a critical role in the formation of the notochord. This attempt led to the identification of a highly divergent mammalian Not-related gene in the mouse, human and rat. The results from phylogenetic reconstructions, synteny analyses, expression pattern analyses in wild-type and mutant mouse embryos, and overexpression experiments in Xenopus embryos converge to confirm these genes as representatives of the Not family in mammals. The identification of the mammalian Not gene delivers an important component for the understanding of the genetics underlying notochord formation in mammals and its evolution among vertebrates. The phylogenomic method used to retrieve this gene thus provides a tool, which can complement or validate genome annotations in situations when they are weakly supported.

Expression patterns of an Otx2 and an Otx5 orthologue in the urodele Pleurodeles waltl: implications on the evolutionary relationships between the balancers and cement gland in amphibians.

We report the characterization of an Otx2 and an Otx5 orthologue in the urodele Pleurodeles waltl. These two genes, termed PwOtx2 and PwOtx5, share highly conserved expression domains with their gnathostome counterparts at tailbud stages, like the developing forebrain ( PwOtx2), or the embryonic eye and epiphysis ( PwOtx5). As in Xenopus laevis, both are also transcribed in the dorsal lip of the blastopore during gastrulation and in anterior parts of the neural plate during neurulation. In addition, PwOtx5 displays a prominent expression in the developing balancers and the lateral non-neural ectoderm during neurulation, from which they derive. By contrast, PwOtx2 expression remains undetectable in the balancers and their presumptive territory. These data suggest that PwOtx5, but not PwOtx2, may be involved in the differentiation and early specification of balancers. Comparisons of Otx5 expression patterns in P. waltland X. laevis embryos suggest that, as previously shown for Otx2, changes in the regulatory mechanisms controlling Otx5 early expression in the non-neural ectoderm may occur frequently among amphibians. These changes may be related to the rise of cement glands in anurans and of balancers in urodeles. This hypothesis could account for some similarities between the two organs, but does not support a homology relationship between them.

Repression of XMyoD expression and myogenesis by Xhairy-1 in Xenopus early embryo.

Activated Notch-Delta signalling was shown to inhibit myogenesis, but whether and how it regulates myogenic gene expression is not clear. We analyzed the implication of Xenopus hairy-1 (Xhairy-1), a member of the hairy and enhancer-of-split (E(spl)) family that may function as nuclear effector of Notch signalling pathway, in regulating XMyoD gene expression at the initial step of myogenesis. Xhairy-1 transcripts are expressed soon after mid-blastula transition and exhibits overlapping expression with Notch pathway genes such as Delta-1 in the posterior somitic mesoderm. We show that overexpression of Xhairy-1 blocks the expression of XMyoD in early gastrula ectodermal cells treated with the mesoderm-inducing factor activin, and in the mesoderm tissues of early embryos. It inhibits myogenesis and produces trunk defects at later stages. Xhairy-1 also inhibits the expression of the pan-mesodermal marker Xbra, but expression of other early mesoderm markers such as goosecoid and chordin is not affected. These effects require the basic helix-loop-helix (bHLH) domain, as well as a synergy between the central Orange domain and the C-terminus WRPW-Groucho-interacting domain. Furthermore, overexpression in ectodermal cells of Xhairy-1/VP16, in which Xhairy-1 repressor domain is replaced by the activator domain of the viral protein VP16, induces the expression of XMyoD in the absence of protein synthesis. Interestingly, Xhairy-1/VP16 does not induce the expression of Xbra and XMyf5 in the same condition. During neurulation, the expression of XMyoD induced by Xhairy-1/VP16 declines and the expression of muscle actin gene was never detected. These results suggest that Notch signalling through hairy-related genes may specifically regulate XMyoD expression at the initial step of myogenesis in vertebrates.

The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/beta-catenin signalling.

Frizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/beta-catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C-terminal cytoplasmic regions transduce the Wnt/beta-catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/beta-catenin signalling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/beta-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/beta-catenin signalling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5 in which the tryptophan is substituted by a tyrosine.

Xenopus frizzled 4 is a maternal mRNA and its zygotic expression is localized to the neuroectoderm and trunk lateral plate mesoderm.

We describe the identification and expression pattern of Xenopus frizzled 4 (Xfz4) gene during early development. Xfz4 protein presents characteristic features of a frizzled family member. The mature protein sequence of Xfz4 is 93% identical to murine Mfz4. Xfz4 is a maternal mRNA, its expression level remains constant during early development. The mRNA is first localized during gastrulation to the dorsal presumptive neuroectoderm. At the end of gastrulation, Xfz4 mRNA is detected in the dorso-anterior neuroectoderm. During neurulation, Xfz4 mRNA is expressed as a band on both side of the forebrain, and in the trunk lateral plate mesoderm. As development proceeds, expression of Xfz4 mRNA in the trunk lateral plate mesoderm decreases but persists in the forebrain. It is also expressed in the posterior unsegmented somitic mesoderm from late tail-bud stage onward.

Expression of Xfz3, a Xenopus frizzled family member, is restricted to the early nervous system.

Recent advances in analyzing wnt signaling have provided evidence that frizzled proteins can function as wnt receptors. We have identified Xfz3, a Xenopus frizzled family member. The amino acid sequence is 89% identical to the product of the murine gene Mfz3, and is predicted to be a serpentine receptor with seven transmembrane domains. Xfz3 is a maternal mRNA with low levels of expression until the end of gastrulation. The expression level increases significantly from neurulation onward. Whole-mount in situ hybridization analysis shows that expression of Xfz3 is highly restricted to the central nervous system. High levels of expression are detected in the anterior neural folds. Low levels of expression are also detected in the optic and otic vesicles, as well as in the pronephros anlage. In addition, Xfz3 mRNA is concentrated in a large band in the midbrain. Overexpression of Xfz3 blocks neural tube closure, resulting in embryos with either bent and strongly reduced anteroposterior axis in a dose-dependent manner. However, it does not affect gastrulation, the expression and localization of organizer-specific genes such as goosecoid, chordin and noggin. Therefore, Xfz3 is not involved in early mesodermal patterning. Injection of RNA encoding GFP-tagged Xfz3 shows that overexpressed proteins can be detected on the cell surface until at least late neurula stage, suggesting that they can exert an effect after gastrulation. Our expression data and functional analyses suggest that the Xfz3 gene product has an antagonizing activity in the morphogenesis during Xenopus development.

Identification and developmental expression of cyclin-dependent kinase 4 gene in Xenopus laevis.

Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases which play a pivotal role in the eucaryote cell cycle regulation. We have identified the Xenopus homologue of mammalian CDK4 (XCDK4). The protein sequence of XCDK4 has 78 and 77% overall identity to human and mouse CDK4, respectively. Northern blot analysis revealed a single transcript of approximately 4.5 kb present at various stages. XCDK4 transcripts show very dynamic expression during early development. The level of expression is higher during cleavage and gastrulation. In situ hybridization analysis revealed that the transcripts are enriched in the dorsal mesoderm at the beginning of gastrulation, then extend to both the lateral and ventral mesoderm. At the end of gastrulation, XCDK4 transcripts are mainly distributed in the blastoporal region and in the anterior neural fold. During neurulation they become restricted to optic vesicles and to neural crest cells organizing the branchial arches. As development proceeds, XCDK4 transcripts are highly expressed in the branchial arches. At late tail-bud stages, XCDK4 transcripts are also detected in ventral hematopoietic precursor cells. Therefore, this analysis clearly shows that XCDK4 has a regionalized expression during Xenopus embryogenesis.

Reactivation and graded axial expression pattern of Wnt-10a gene during early regeneration stages of adult tail in amphibian urodele Pleurodeles waltl.

Adult urodele amphibians such as Pleurodeles waltl are able to regenerate their amputated limbs or tail. The mechanisms implicated in growth control and formation of the blastema are unknown but it has been proposed that regeneration in newts may proceed through reactivation of genes involved in embryonic development. Knowing the role of Wnt genes in the patterning of the primary and secondary axes of the vertebrate embryo, we suspected that some of these genes could be involved in axial pattern during newt tail regeneration. Pwnt-10a gene, cloned from a newt tail regenerate cDNA library, showed an expression pattern compatible with such a role in tail regenerates. Pwnt-10a, which is highly expressed during embryonic development (from gastrula to tailbud-stage) and weakly expressed in the adult tail, is strongly re-expressed during tail regeneration. In the blastemal mesenchyme Pwnt-10a transcripts exhibited a graded distribution along the antero-posterior axis, the mRNA accumulation being maximal in the caudal most part corresponding to the growing zone. These findings strongly support the view that Pwnt-10a may act in cooperation with other factors to control growth and patterning in newt tail regeneration. Until now Wnt-10a was only known to be involved in central nervous system development; our results suggest that this gene may also play a role in other developmental processes.

What mechanisms drive cell migration and cell interactions in Pleurodeles?

Embryogenesis implies a strict control of cell interaction and cell migration. The spatial and temporal regulation of morphogenetic movements occurring during gastrulation is directly dependent on the early cell interactions that take place in the blastula. The newt Pleurodeles waltl is a favorable model for the study of these early morphogenetic events. The combination of orthotopic grafting and fluorescent lineage tracers has led to precise early gastrula mesoderm fate maps. It is now clear that there are no sharp boundaries between germ layers at the onset of gastrulation but rather diffuse transition zones. The coordination of cell movements during gastrulation is closely related to the establishment of dorsoventral polarity. Ventralization by U.V. irradiation or dorsalization by lithium treatment modifies the capacity for autonomous migration on the fibronectin coated substratum of marginal zone cells accordingly. It is now firmly established that mesodermal cells need to adhere to a fibrillar extracellular matrix (ECM) to undergo migration during gastrulation. Extracellular fibrils contain laminin and fibronectin (FN). Interaction of cells with ECM involves receptors of the beta 1 integrin family. A Pleurodeles homolog of the alpha v integrin subunit has been recently identified. Protein alpha v expression is restricted to the surface of mesodermal cells during gastrulation. Integrin-mediated interactions of cells with FN are essential for ECM assembly and mesodermal cell migration. Intracellular injection of antibodies to the cytoplasmic domain of beta 1 into early cleavage embryos causes inhibition of FN fibril formation. Intrablastocoelic injections of several probes including antibodies to FN or integrin alpha 5 beta 1, competitive peptides to the major cell binding site of FN or the antiadhesive protein tenascin all block mesodermal cell migration. This results in a complete arrest of gastrulation indicating that mesodermal cell migration is a major driving force in urodele gastrulation. It is now possible to approach the role of fibroblast growth factor (FGF) during cell interactions taking place in urodele embryos. Four different FGF receptors (FGFR) have been cloned in Pleurodeles. Each of them has a unique mRNA expression pattern. FGFR-1, FGFR-3, and the variant of FGFR-2 containing the IIIb exon are maternally expressed and might be involved in mesodermal induction. During gastrulation, FGFR-3 and FGFR-4 have a restricted pattern of expression, whereas FGFR-1 mRNA is nearly uniformly distributed. Splicing variants FGFR-2IIIb and FGFR-2IIIc have exclusive expression patterns during neurulation. IIIb is expressed in epidermis and IIIc in neural tissue, suggesting a function in the differentiation of ectodermal derivatives.

A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers.

We have examined the role of fibroblast growth factor (FGF) signalling in neural induction. The approach takes advantage of the fact that both noggin and the dominant negative mutant activin receptor (delta1XAR1) directly induce neural tissues in the absence of dorsal mesoderm. A truncated FGF receptor (XFD) is co-expressed with noggin or delta1XAR1 in both whole embryos and isolated animal caps. We demonstrate that inhibition of FGF signalling prevents neural induction by both factors. Furthermore, neural induction by organizers (the dorsal lip of blastopore and Hensen's node) is also blocked by inhibiting FGF signalling in ectoderm. It has been proposed that the specification of anterior neuroectoderm, including the cement gland, occurs in a sequential manner as gastrulation proceeds. We show that the specification of the most anterior neuroectoderm by noggin may occur before gastrulation and does not require FGF signalling, since both the cement gland marker XCG-1 and the anterior neural marker Otx-2 are normally expressed in ectodermal explants co-injected with noggin and XFD RNAs, but the cement gland cells are poorly differentiated. In contrast, the expression of both genes induced by CSKA.noggin, which is expressed after the mid-blastula transition, is strongly inhibited by the presence of XFD. Therefore the noggin-mediated neural induction that takes place at gastrula stages is abolished in the absence of FGF signalling. Since inhibition of FGF signalling blocks the neuralizing effect of different neural inducers that function through independent mechanisms, we propose that FGF receptor-related-signalling is required for the response to inducing signals of ectodermal cells from gastrula.

Comparative analysis of the tissue distribution of three fibroblast growth factor receptor mRNAs during amphibian morphogenesis.

We have used in situ hybridization to survey the expression pattern of three fibroblast growth factor receptor (FGFR) mRNAs (PFR-1, PFR-3 and PFR-4, which we previously identified as the amphibian Pleurodeles waltl homologs of human FGFR-1, FGFR-3 and FGFR-4, respectively) during morphogenesis. Previous work suggests that these FGFR mRNAs exhibit a distinct pattern of expression at early developmental stages. In the present study we have tested the functional activity of these receptors and shown that both FGF-1 (acidic FGF) and FGF-2 (basic FGF), but not FGF-7 (keratinocyte growth factor), can lead to their activation, suggesting that the three cDNAs encode functional receptors. Results from in situ hybridization indicate that various FGFRs are involved in various developmental events. Their involvement in these processes is both overlapping and distinct. During the differentiation of the central nervous system (CNS), PFR-1 and PFR-4 mRNAs show high levels of redundant expression, while the sites of expression of PFR-3 mRNA correlate with regions, such as the diencephalon and the rhombencephalon, undergoing important anatomic changes. The three FGFR mRNAs are distinctly expressed in the cranial ganglia, the pigmented epithelia of retina and the otic vesicles. Most significantly, we found that they are strongly expressed at cranial and branchial mesenchymal condensation sites. PFR-3 mRNA is expressed earlier in this process than PFR-1 and PFR-4 mRNAs. Furthermore PFR-3 mRNA is detected in the mesenchyme of the limb bud, while PFR-1 and PFR-4 mRNAs are found in the primordia of the skeletal elements. In addition, PFR-1 mRNA is expressed in axial mesenchyme and PFR-4 mRNA is detected in the melanophores, xanthophores and in the pronephros. These results suggest that various FGFRs may be involved in distinct developmental events including cell proliferation and differentiation. We also discuss the functional redundancy of the FGFR system during amphibian morphogenesis.

Expression of fibroblast growth factor receptor-2 splice variants is developmentally and tissue-specifically regulated in the amphibian embryo.

There is increasing evidence that distinct fibroblast growth factor receptor (FGFR) genes are involved in embryonic development and that unique expression patterns of individual FGFRs correlate with tissue-specific functions. In addition, alternative splicing of mRNA transcripts from at least two of these genes (FGFR-1 and FGFR-2) can generate receptor variants with different ligand-binding specificity. By polymerase chain reaction methods and by screening a cDNA library, we have isolated five amphibian FGFR-2 splice variants which share a high degree of identity to their human counterparts. These mRNAs are developmentally regulated and are expressed in a tissue-specific manner. In particular, two alternative exons (termed IIIb and IIIc) in the second half of the third immunoglobulin-like loop are remarkably conserved and have a distinct pattern of regulation during development. Either aFGF or bFGF can activate IIIb- or IIIc-containing receptors. In contrast, KGF only activates IIIb-containing receptors. Exon IIIb-containing receptors are maternally derived mRNAs, whereas exon IIIc-containing receptors are zygotically expressed. Furthermore, their tissue distribution pattern was mutually exclusive. From the beginning of the neurula stage onward, IIIb transcripts are expressed in the epidermis, while IIIc transcripts are activated in the neuroectoderm by neural induction. At the late tail-bud stage, in situ hybridization revealed expression of IIIc mRNA in the telencephalon and the diencephalon, as well as in the head mesenchyme condensation sites originating from the proliferating neural crest cells. IIIb mRNA was detected in the epidermis and in the epithelium of the pharynx. Our data suggest that exon IIIb-containing receptors may play a role in the development of epithelial tissues, while exon IIIc-containing receptors may play a role during neural tissue formation.