35Background: To explore the cellular immunity and cytokines status of NCP patients and to predict 36 the correlation between the cellular immunity levels, cytokines and the severity of patients. 37Methods: 123 NCP patients were divided into mild and severe groups. Peripheral blood was 38 collected, lymphocyte subsets and cytokines were detected. Correlation analysis was performed on 39 the lymphocyte subsets and cytokines, and the differences between the indexes of the two groups 40 were analyzed. 41Results: 102 mild and 21 severe patients were included. Lymphocyte subsets were reduced in two 42 groups. The proportion of CD8 + T reduction in the mild and severe group was 28.43% and 61.9%, 43 respectively; The proportion of B cell reduction was 25.49% and 28.57%; The proportion of NK 44 cell reduction was 34.31% and 47.62%; The detection value of IL-6 was 0 in 55.88% of the mild 45 group, mild group has a significantly lower proportion of patients with IL-6 higher than normal than 46 severe group; There was no significant linear correlation between the lymphocyte subsets and 47 cytokines, while significant differences were noticed between the two groups in CD4 + T, CD8 + T, 48 IL-6 and IL-10. 49Conclusions: Low levels of CD4+T and CD8+T are common in severe NCP. IL-6 and IL-10 levels 50were higher in severe patients. T cell subsets and cytokines can be used as one of the basis for 51 predicting the transition from mild to severe. Large number of samples are still needed to confirm 52 the "warning value" of CD4 + T, CD8 + T IL-6 and IL-10. 53

We explored the relationships between lymphocyte subsets, cytokines, pulmonary inflammation index (PII) and disease evolution in patients with (corona virus disease 2019) COVID-19. A total of 123 patients with COVID-19 were divided into mild and severe groups. Lymphocyte subsets and cytokines were detected on the first day of hospital admission and lung computed tomography results were quantified by PII. Difference analysis and correlation analysis were performed on the two groups. A total of 102 mild and 21 severe patients were included in the analysis. There were significant differences in cluster of differentiation 4 (CD4 + T), cluster of differentiation 8 (CD8 + T), interleukin 6 (IL-6), interleukin 10 (IL-10) and PII between the two groups. There were significant positive correlations between CD4 + T and CD8 + T, IL-6 and IL-10 in the mild group (r 2 = 0Á694, r 2 = 0Á633, respectively; P < 0Á01). After 'five-in-one' treatment, all patients were discharged with the exception of the four who died. Higher survival rates occurred in the mild group and in those with IL-6 within normal values. CD4 + T, CD8 + T, IL-6, IL-10 and PII can be used as indicators of disease evolution, and the PII can be used as an independent indicator for disease progression of COVID-19.

Tissue factor (TF) is the cellular receptor for coagulation factor VII/VIla and is the membrane-bound glycoprotein that is generally viewed as the primary physiological initiator of blood coagulation. To define in greater detail the physiological role of TF in development and hemostasis, the TF gene was disrupted in mice. Mice heterozygous for the inactivated TF allele expressed approximately half the TF activity of wild-type mice but were phenotypically normal. However, homozygous TF-/-pups were never born in crosses between heterozygous mice. Analysis of mid-gestation embryos showed that TF-/-embryos die in utero between days 8.5 and 10.5. TF-/-embryos were morphologically distinct from their TF+/+ and TF+/-littermates after day 9.5 in that they were pale, edematous, and growth retarded. Histological studies showed that early organogenesis was normal. The initial failure in TF-/-embryos appeared to be hemorrhaging, leading to the leakage of embryonic red cells from both extraembryonic and embryonic vessels. These studies indicate that TF plays an indispensable role in establishing and/or maintaining vascular integrity in the developing embryo at a time when embryonic and extraembryonic vasculatures are fusing and blood circulation begins.Tissue factor (TF) is a 47-kDa membrane-bound glycoprotein that functions as the cellular receptor for coagulation factor VII/VIIa. TF is thought to be the primary physiological initiator of blood coagulation following vascular damage (1). Unlike other coagulation factors, TF need not be activated and is delivered to the cell surface as a functional VII/VIla receptor. Factor VIla bound to TF at the cell surface efficiently catalyzes the proteolytic activation of coagulation factors IX and X. This ultimately leads to local thrombin generation and thrombin-catalyzed events such as fibrin formation and the activation of factors V, VIII, XIII, thrombin receptor, and protein C (1).The central role of TF in blood coagulation in vivo is supported by several compelling, but indirect, observations.

The conversion of prothrombin (FII) to the serine protease, thrombin (FIIa), is a key step in the coagulation cascade because FIIa triggers platelet activation, converts fibrinogen to fibrin, and activates regulatory pathways that both promote and ultimately suppress coagulation. However, several observations suggest that FII may serve a broader physiological role than simply stemming blood loss, including the identification of multiple G protein-coupled, thrombin-activated receptors, and the well-documented mitogenic activity of FIIa in in vitro test systems. To explore in greater detail the physiological roles of FII in vivo, FIIdeficient (FII ؊/؊ ) mice were generated. Inactivation of the FII gene leads to partial embryonic lethality with more than one-half of the FII ؊/؊ embryos dying between embryonic days 9.5 and 11.5. Bleeding into the yolk sac cavity and varying degrees of tissue necrosis were observed in many FII ؊/؊ embryos within this gestational time frame. However, at least one-quarter of the FII ؊/؊ mice survived to term, but ultimately they, too, developed fatal hemorrhagic events and died within a few days of birth. This study directly demonstrates that FII is important in maintaining vascular integrity during development as well as postnatal life.Prothrombin (FII), a vitamin K-dependent zymogen synthesized by hepatocytes, is activated to form thrombin (FIIa) by factor Xa in the presence of factor Va (FVa), calcium, and a phospholipid surface. FIIa plays a central role in the blood coagulation system by triggering the activation of platelets, converting soluble fibrinogen into insoluble fibrin polymer, and activating regulatory pathways that control the rate of further thrombin formation (1). In the presence of thrombomodulin, FIIa functions as an anticoagulant by activating protein C and protein S, which in turn inactivates factors Va and VIIIa. FIIa is thought to serve a broader biological role than merely controlling blood loss, based on the fact that there are at least two G protein-coupled receptors (i.e., PAR-1 and PAR-3) that are proteolytically activated by thrombin, and these receptors are present on a variety of cell types (2-4). FIIa has been proposed to influence a variety of physiological and pathological processes, including inflammation, tissue repair, neurite outgrowth, atherosclerosis, and tumor cell metastasis (5-9). The expression of both thrombin receptor and FII during organogenesis in the mouse suggests that FIIa may play an important role in development (3), a hypothesis that is supported further by the finding of partial embryonic lethality in mice deficient in tissue factor (TF), factor V (FV), and PAR-1 (10-16). To understand in greater detail the diverse biological roles of FII in vivo, and to directly establish the importance of FII in development, the FII gene was disrupted in mice. We report that FII deficiency results in a loss of vascular integrity and death around the tenth day of gestation in a high percentage of FII Ϫ/Ϫ embryos. Partial embryonic lethali...

The Wnt/β-catenin pathway comprises a family of proteins that play critical roles in embryonic development and adult tissue homeostasis. The deregulation of Wnt/β-catenin signalling often leads to various serious diseases, including cancer and non-cancer diseases. Although many articles have reviewed Wnt/β-catenin from various aspects, a systematic review encompassing the origin, composition, function, and clinical trials of the Wnt/β-catenin signalling pathway in tumour and diseases is lacking. In this article, we comprehensively review the Wnt/β-catenin pathway from the above five aspects in combination with the latest research. Finally, we propose challenges and opportunities for the development of small-molecular compounds targeting the Wnt signalling pathway in disease treatment.

Methionine aminopeptidase (MetAP) catalyzes the hydrolytic cleavage of the N-terminal methionine from newly synthesized polypeptides. The extent of methionyl removal from a protein is dictated by its N-terminal peptide sequence. Earlier studies revealed that MetAPs require amino acids containing small side chains (e.g., Gly, Ala, Ser, Cys, Pro, Thr, and Val) as the P1' residue, but their specificity at positions P2' and beyond remains incompletely defined. In this work, the substrate specificities of Escherichia coli MetAP1, human MetAP1, and human MetAP2 were systematically profiled by screening against a combinatorial peptide library and kinetic analysis of individually synthesized peptide substrates. Our results show that although all three enzymes require small residues at the P1' position, they have differential tolerance for Val and Thr at this position. The catalytic activity of human MetAP2 toward Met-Val peptides is consistently two orders of magnitude higher than that of MetAP1, suggesting that MetAP2 is responsible for processing proteins containing N-terminal Met-Val and Met-Thr sequences in vivo. At positions P2' to P5', all three MetAPs have broad specificity, but are poorly active toward peptides containing a proline at the P2' position. In addition, the human MetAPs disfavor acidic residues at the P2' to P5' positions. The specificity data have allowed us to formulate a simple set of rules that can reliably predict the N-terminal processing of E. coli and human proteins. KeywordsMethionine aminopeptidase; N-terminal processing; substrate specificity; kinetics; peptide library Ribosomal protein synthesis is universally initiated with methionine (in eukaryotic cytoplasm) or N-formylmethionine (in prokaryotes, mitochondria, and chloroplasts). During protein maturation, the N-formyl group is removed by peptide deformylase, leaving methionine with a free NH 2 group (1). Subsequently, the initiator methionine is removed from many but not all of the proteins by methionine aminopeptidase(s) (MetAPs). For example, in a cytosolic extract of Escherichia coli, only 40% of the polypeptides retain the initiator methionine, whereas the majority of the polypeptides display alanine, serine, or threonine at their N-termini (2). There are two types of MetAPs, MetAP1 and MetAP2.

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.

A critical link between hemostatic factors and atherosclerosis has been inferred from a variety of indirect observations, including the expression of procoagulant and fibrinolytic factors within atherosclerotic vessels, the presence of fibrin in intimal lesions, and the cellular infiltration of mural thrombi leading to their incorporation into developing plaques. To directly examine the role of the key fibrinolytic factor, plasminogen, in atherogenesis, plasminogen-deficient mice were crossed to hypercholesterolemic, apolipoprotein E-deficient mice predisposed to atherosclerosis. We report that the loss of plasminogen greatly accelerates the formation of intimal lesions in apolipoprotein E-deficient animals, whereas plasminogen deficiency alone does not cause appreciable atherosclerosis. These studies provide direct evidence that circulating hemostatic factors strongly inf luence vessel wall disease in the context of a disorder in lipid metabolism.