Glia Maturation Factor-ß Supports Liver Regeneration by Remodeling Actin Network to Enhance STAT3 Proliferative Signals.

BACKGROUND & AIMS: Glia maturation factor-ß (GMFB) is a bona fide member of the actin depolymerizing factor homology family. Recently, emerging evidence suggested its implication in liver diseases, but data on its role in liver remain limited. METHODS: Assessment of GMFB in liver histology, impact on liver regeneration and hepatocyte proliferation, and the underlying molecular pathways were conducted using mouse models with acute liver injury. RESULTS: GMFB is widely distributed in normal liver. Its expression increases within 24 hours after partial hepatectomy (PHx). Adult Gmfb knockout mice and wild-type littermates are similar in gross appearance, body weight, liver function, and histology. However, compared with wild-type control, Gmfb knockout mice post-PHx develop more serious liver damage and steatosis and have delayed liver regeneration; the dominant change in liver transcriptome at 24 hours after PHx is the significantly suppressed acute inflammation pathways; the top down-regulated gene sets relate to interleukin (IL)6/Janus kinase/signal transducer and activator of transcription 3 (STAT3) signaling. Another mouse model intoxicated with carbon tetrachloride replicated these findings. Furthermore, Gmfb knockout and wild-type groups have the similar numbers of Kupffer cells, but Gmfb knockout Kupffer cells once stimulated produce less IL6, tumor necrosis factor, and IL1ß. In hepatocytes treated with IL6, GMFB associates positively with cell proliferation and STAT3/cyclin D1 activation, but without any direct interaction with STAT3. In Gmfb knockout hepatocytes, cytoskeleton-related gene expression was changed significantly, with an abnormal-appearing morphology of actin networks. In hepatocyte modeling, actin-filament turnover, STAT3 activation, and metabolite excretion show a strong reliance on the status of actin-filament organization. CONCLUSIONS: GMFB plays a significant role in liver regeneration by promoting acute inflammatory response in Kupffer cells and by intracellularly coordinating the responsive hepatocyte proliferation.

A meta-analysis of the clinical efficacy of rhBNP in treating patients with acute myocardial infarction and heart failure.

OBJECTIVE: To explore the clinical efficacy of rhBNP in patients with acute myocardial infarction (AMI) and heart failure (HF). METHODS: A systematic review and a meta-analysis were performed using the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. On May 30, 2020, we consulted the electronic databases PubMed, EBSCO, Elsevier, Springer, Wiley, and Cochrane using the keywords "acute coronary syndrome (ACS)", "brain natriuretic peptide (BNP)", and "acute myocardial infarction (AMI)". The quality of the data included in the study was assessed according to the Cochrane Handbook for Systematic Reviews of Interventions. The results of the clinical randomized controlled study reports were analyzed using Review Manager 5.1.0. RESULTS: A total of nine, clinical, randomized, controlled studies were included. The effective rate in the rhBNP group was significantly higher than it was in the control group (Z = 9.50, P < 0.00001). The patients in the rhBNP group showed remarkably shorter hospital stays (Z = 24.43, P < 0.00001) and markedly increased left ventricular ejection fractions (LVEF) (Z = 245.53, P < 0.00001). Compared with the LVEF in the control group, the LVEF in the rhBNP group was significantly increased (Z = 3.55, P = 0.0004), but the rate of cardiac hypotension (Z = 3.55, P = 0.0004) and the headache incidence rate in the rhBNP group (Z = 2.3, P = 0.04) were not elevated. The rhBNP group showed no increase in either the low heart rate (Z = 1.22, P = 0.22) or the rate of renal insufficiency (Z = 0.35, P = 0.73). CONCLUSION: The meta-analysis suggests that, compared with the conventional treatment of patients with AMI and HF, rhBNP can markedly improve the clinical efficacy and myocardial functions and shorten the hospital stays, without elevating the rate of adverse reactions, such as hypotension, headaches, low heart rate, and renal insufficiency.

Transition-Metal-Free Sulfuration/Annulation of Alkenes: Economical Access to Thiophenes Enabled by the Cleavage of Multiple C-H Bonds.

A novel, atom economical, and transition-metal-free strategy for the synthesis of thiophenes from substituted buta-1-enes with potassium sulfide has been presented. The reaction achieves double C-S bond formations via cleavage of multiple C-H bonds and provides an efficient approach to access various functionalized thiophenes. Moreover, the strategy can also be used for the synthesis of thiophenes from 1,4-diaryl-1,3-dienes. Mechanistically, DMSO plays a role of oxidant and S3•- in situ generated from K2S is involved.

Clinical observation of ulinastatin combined with CRRT in the treatment of early cardiopulmonary resuscitation.

The clinical efficacy of ulinastatin (UTI) combined with continuous renal replacement therapy (CRRT) in the treatment after early cardiopulmonary resuscitation (CPR) was evaluated. A total of 70 patients who were successfully treated with CPR in Ganzhou People's Hospital from October 2016 to March 2017 were selected as the subjects. The patients were randomly divided into control group (35 cases, conventional treatment) and UTI combined with CRRT group (35 cases, UTI + CRRT). The whole blood of patients was collected at 0, 3, 6 and 12 h after CPR. Reverse transcription-polymerase chain reaction assay was used to detect the changes of toll-like receptor 4 (TLR4) gene in mRNA levels between the two groups, i-STAT system 300 was used to analyze pH level, SO2, HCO3- and lactic acid (LAC) concentration; Abbott AXSYM system was used to detect the expression of cardiac troponin I (cTnI) in serum; the concentration of plasma malondialdehyde (MDA) was examined by a special kit; interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in patients was determined by enzyme-linked immunosorbent assay. The effect of UTI combined with CRRT in the early stage of CPR was analyzed. The levels of TLR4, cTnI, TNF-α, IL-6 and MDA in the plasma of patients in both groups were significantly increased (P<0.05), but the expression level in UTI + CRRT group was lower than that in control group (P<0.05). Compared with the control group, the HCO3- decreased significantly (P<0.05) in the UTI + CRRT group at 3 h, while the pH and SO2 did not change significantly. UTI + CRRT could significantly shorten the average recovery time of consciousness and the average recovery time of consciousness and spontaneous respiration in patients treated with CPR (P<0.05). Moreover, the score of APACHE II was significantly lower than that of control group (P<0.05). UTI combined with CRRT treatment can significantly improve the patient's condition after early CPR.

Copper-catalyzed three-component synthesis of benzothiazolethiones from o-iodoanilines, isocyanide, and potassium sulfide.

An efficient copper catalyzed strategy for the synthesis of a variety of benzothiazolethione derivatives has been developed. In the presence of CuCl, the three-component reaction of o-iodoanilines and K2S with p-toluenesulfonylmethyl isocyanide proceeded smoothly to obtain the corresponding benzothiazolethiones in good to excellent isolated yields. Notably, isocyanide functioned as a carbon source and K2S functioned as a sulfur source in this reaction.

Copper-catalyzed double C-S bonds formation via different paths: synthesis of benzothiazoles from N-benzyl-2-iodoaniline and potassium sulfide.

A new, highly efficient procedure for the synthesis of benzothiazoles from easily available N-benzyl-2-iodoaniline and potassium sulfide has been developed. The results show copper-catalyzed double C-S bond formation via a traditional cross-coupling reaction and an oxidative cross-coupling reaction.

Serine protease HTRA1 antagonizes transforming growth factor-ß signaling by cleaving its receptors and loss of HTRA1 in vivo enhances bone formation.

HTRA1 is a member of the High Temperature Requirement (HTRA1) family of serine proteases, which play a role in several biological and pathological processes. In part, HTRA1 regulation occurs by inhibiting the TGF-ß signaling pathway, however the mechanism of inhibition has not been fully defined. Previous studies have shown that HTRA1 is expressed in a variety of tissues, including sites of skeletal development. HTRA1 has also been implicated in the process of bone formation, although the precise manner of regulation is still unknown. This study investigated how HTRA1 regulates TGF-ß signaling and examined the in vivo effects of the loss of HTRA1. We demonstrated that recombinant HTRA1 was capable of cleaving both type II and type III TGF-ß receptors (TßRII and TßRIII) in vitro in a dose-dependent manner, but it did not affect the integrity of TßRI or TGF-ß. Overexpression of HTRA1 led to decreased levels of both TßRII and III on the cell surface but had no effect on TßRI. Silencing HTRA1 expression significantly increased TGF-ß binding to the cell surface and TGF-ß responsiveness within the cell. To examine the role of HTRA1 in vivo, we generated mice with a targeted gene deletion of HTRA1. Embryonic fibroblasts isolated from these mice displayed an increase in TGF-ß-induced expression of several genes known to promote bone formation. Importantly, the loss of HTRA1 in the knockout mice resulted in a marked increase in trabecular bone mass. This study has identified a novel regulatory mechanism by which HTRA1 antagonizes TGF-ß signaling, and has shown that HTRA1 plays a key role in the regulation of bone formation.

Glycosaminoglycan-binding properties and aggrecanase activities of truncated ADAMTSs: comparative analyses with ADAMTS-5, -9, -16 and -18.

Aggrecanases are ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) proteases capable of primary (patho)physiological cleavage at specific Glu-Xaa bonds within the core protein of the hyaluronan-binding proteoglycan aggrecan. Accumulating evidence suggests that regulation of the activity of one such aggrecanase, ADAMTS-4 (or Aggrecanase-1), involves post-translational C-terminal processing (truncation) which modulates both glycosaminoglycan (GAG)-binding affinity and enzymatic activity. In the present study, we compared the effects of C-terminal truncation on the GAG-binding properties and aggrecanase activity of ADAMTS-5 (Aggrecanase-2) relative to three other ADAMTS family members, ADAMTS-9, ADAMTS-16 and ADAMTS-18. Full-length recombinant human ADAMTS-5 (M(r) approximately 85 kDa; ADAMTS-5p85) underwent autolytic cleavage during expression by CHO/A2 cells, and co-purified with C-terminally truncated (tr) isoforms of M(r) approximately 60 kDa (ADAMTS-5p60 and M(r) approximately 45 kDa (ADAMTS-5p45). All three ADAMTS-5 isoforms bound to sulfated GAGs (heparin and chondroitin sulfate (CS)). An ADAMTS-5p45 structural mimetic, terminating at Phe628 and comprising the catalytic domain, disintegrin-like domain and thrombospondin type I repeat (TSR)-1 domain (designated trADAMTS-5F628), also bound to heparin, and exhibited potent aggrecanase activity toward cleavage sites both in the aggrecan CS-2-attachment region (at Glu1771-Ala1772) and in the interglobular domain (at Glu373-Ala374). Further truncation (deletion of the TSR-1 domain) of ADAMTS-5 significantly reduced aggrecanase activity, although appreciable GAG (heparin)-binding affinity was maintained. Other TSR-1 domain-bearing truncated ADAMTS constructs demonstrating either positive GAG-binding ability (trADAMTS-9F649) or negligible GAG-affinity (trADAMTS-16F647 and trADAMTS-18F650) displayed comparably low aggrecanase activities. Thus, the presence of TSR-1 on truncated ADAMTSs appears to be necessary, but not sufficient, for effective aggrecanase-mediated catalysis of target Glu-Xaa bonds. Similarly, GAG-binding ability, irrespective of the presence of a TSR-1 domain, does not necessarily empower truncated ADAMTSs with proficient aggrecanase activity.

Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS-4 (aggrecanase 1) or ADAMTS-5 (aggrecanase 2).

OBJECTIVE: To determine whether aggrecanase (ADAMTS) activities in articular cartilage can directly lead to the release of hyaluronan (HA) and hyaladherins (aggrecan G1 domain and link proteins), as may occur ex vivo during stimulation of cartilage explants with interleukin-1 (IL-1) or retinoic acid or in vivo in synovial joints during aging and joint pathology. METHODS: Bovine articular cartilage discs (live or freeze-killed) were cultured in the presence of IL-1 or were incubated in digestion buffer containing recombinant human ADAMTS-4 (rHuADAMTS-4; aggrecanase 1) or rHuADAMTS-5 (aggrecanase 2). Culture media, digestion supernatants, and tissue extracts were assayed for sulfated glycosaminoglycan (sGAG) content and analyzed by Western blotting to detect aggrecanase-generated G1 domain (using neoepitope monoclonal antibody AGG-C1/anti-NITEGE(373)) and link proteins (using monoclonal antibody 8-A-4), as well as by quantitative enzyme-linked immunosorbent assays to detect aggrecanase-generated G1 domain (G1-NITEGE(373)) and HA. RESULTS: IL-1 treatment of live cartilage explants induced a time-dependent release of sGAG, aggrecanase-generated G1 domain (G1-NITEGE(373)), and HA into the culture media. Exposure of live or freeze-killed articular cartilage discs to rHuADAMTS-4 or rHuADAMTS-5 resulted in a dose- and time-dependent release of sGAG and hyaluronan from the tissue, accompanied by a concomitant release of functionally intact hyaladherins (aggrecan G1-NITEGE(373) and link proteins). CONCLUSION: Coincident with aggrecanolysis, aggrecanase activities in articular cartilage may actuate the release of HA and associated hyaladherins, thereby further compromising the integrity of the cartilage matrix during degenerative joint diseases such as osteoarthritis.

ADAMTS-8 exhibits aggrecanase activity and is expressed in human articular cartilage.

Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family share common structural features including a disintegrin domain, a zinc metalloprotease domain, and at least one thrombospondin motif. Aberrant expression of several of these proteins has led to an understanding of their role in human disease; however, a link to function for many has not yet been made. One such uncharacterized family member, ADAMTS-8, shares significant protein sequence homology with a subgroup of ADAMTSs that includes ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-15. Each of these proteases has been shown to cleave 'aggrecanase-susceptible' site(s) within the extracellular matrix (ECM) proteoglycan aggrecan, and ADAMTS-4 and ADAMTS-5 have been postulated to play a role in the depletion of articular cartilage in osteoarthritic disease. Based on sequence relationships, in the present study we examined the ability of ADAMTS-8 to exhibit 'aggrecanase' activity. A neoepitope monoclonal antibody (MAb; AGG-C1; anti-NITEGE373) was developed and used to demonstrate the ability of ADAMTS-8 to cleave aggrecan at the aggrecanase-susceptible Glu373-Ala374 peptide bond. In addition, expression analyses demonstrated the presence of ADAMTS-8 mRNA transcripts in normal and osteoarthritic human cartilage.

Definition of two new epitopes on human immunodeficiency virus type 1 gag protein recognized by human CD8+ cytotoxic T lymphocyte clones.

We have established 3 CD8+ cytotoxic T lymphocyte (CTL) clones from the peripheral blood mononuclear cells of two HIV-1-seropositive asymptomatic donors. The epitopes recognized by these CTL clones were defined using synthetic peptides. The epitopes were located on HIV-1gag protein between amino acid (a.a.) 145 and 155 (QAISPRTLNAW), a.a.193 and 201 (GHQAAMQML), and a.a.260 and 267 (EIYKRWII), and were presented by HLA-A25, HLA-B38 and HLA-B8, respectively. The former 2 epitopes have not been previously defined. The HLA-A25-restricted epitope overlapped with HLA-B57-restricted and HLA-Cw3-restricted epitopes previously reported. In addition, this epitope overlapped with an HLA-DQ-restricted epitope recognized by CD4+ CTL. The HLA-B38-restricted epitope overlapped with HLA-A2-restricted and HLA-Bw52-restricted epitopes that were previously reported. The HLA-B38-restricted epitope between a.a.193 and 201 was highly conserved among HIV-1 strains. The results demonstrate that two new epitopes were defined in a region of gag protein that includes multiple epitopes presented by multiple HLA.

Autocatalytic cleavage of ADAMTS-4 (Aggrecanase-1) reveals multiple glycosaminoglycan-binding sites.

ADAMTS-4, also referred to as aggrecanase-1, is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans (GAGs) attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In the present study, we demonstrate that full-length ADAMTS-4 (M(r) approximately 68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms (M(r) approximately 53,000 and M(r) approximately 40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAG-binding thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the C-terminal cysteine (cys)-rich and/or spacer domains also effect binding of full-length ADAMTS-4 to sulfated GAGs. Binding-competition experiments conducted using native and deglycosylated aggrecan provided direct evidence for interaction of the ADAMTS-4 cysteine-rich/spacer domains with aggrecan GAGs. Furthermore, synthetic peptides mimicking putative (consensus) GAG-binding sequences located within the ADAMTS-4 cysteine-rich and spacer domains competitively blocked binding of sulfated GAGs to full-length ADAMTS-4, thereby identifying multiple GAG-binding sites, which may contribute to the regulation of ADAMTS-4 function.

Characterization of four murine homologs of the human ov-serpin monocyte neutrophil elastase inhibitor MNEI (SERPINB1).

The human ov-serpin monocyte neutrophil elastase inhibitor (MNEI) is encoded by a single gene SERPINB1. It is a highly efficient inhibitor of neutrophil granule proteases. Four murine genes with high sequence identity with MNEI were identified and fully sequenced, and these were named EIA, EIB, EIC, and EID. EIA, EIB and EIC showed the same seven-exon gene structure as SERPINB1. However, EIC included an additional, alternatively spliced, exon due to the insertion of an endogenous retrovirus-like sequence. EID lacked several exons and is a pseudogene. Reverse transcriptase-PCR showed that EIA, like MNEI, is expressed at high levels in many tissues. EIB is mainly expressed in brain, and EIC was only expressed as splicing variants unlikely to encode a functional serpin. Upon incubation with serine proteases, EIA formed inhibitory covalent complexes with pancreatic and neutrophil elastases, cathepsin G, proteinase-3, and chymotrypsin, as previously shown for MNEI, whereas EIB was only able to do so with cathepsin G. According to the new serpin nomenclature, the genes encoding EIA, EIB, EIC, and EID will be called Serpinb1, Serpinb1b, Serpinb1c, and Serpinb1-ps1. These data demonstrate that the four murine homologs of MNEI have met different evolutionary fates, and that EIA is the mouse ortholog of MNEI.