Detection of Variants With Reduced Baloxavir Marboxil and Oseltamivir Susceptibility in Children With Influenza A During the 2019-2020 Influenza Season.

BACKGROUND: We aimed to detect influenza variants with reduced susceptibility to baloxavir marboxil (baloxavir) and oseltamivir and identify differences in the clinical course between children with and without these variants after antiviral treatment. METHODS: During the 2019-2020 influenza season, we enrolled children with confirmed influenza A (20 treated with baloxavir and 16 with oseltamivir). We analyzed patients' sequential viral RNA loads and infectious virus titers, the drug susceptibilities of clinical isolates, and amino acid substitutions in the viral polymerase acidic protein subunits or neuraminidase. We assessed patients' clinical information using questionnaires. RESULTS: All viral RNA loads and virus titers were significantly decreased after treatment, but we detected baloxavir-resistant and oseltamivir-resistant variants in 5 of 20 and 3 of 16 patients, respectively. The duration of fever was similar between patients with and without the variants, but infectious viral shedding lasted 3 days longer in patients with baloxavir-resistant variants. In addition, the duration to improvement of clinical symptoms was longer in these patients (75.0 vs 29.5 hours; P = .106). CONCLUSIONS: After antiviral treatment, the emergence of baloxavir-resistant variants may affect the patients' clinical course, but oseltamivir-resistant variants had no clinical impact.

Selective Inhibition of N-Methyl-d-aspartate Receptors with GluN2B Subunit Protects ß Cells against Stress-Induced Apoptotic Cell Death.

Participation of N-methyl-d-aspartate (NMDA) receptors (NMDARs) in the failure of pancreatic ß cells during development of type 2 diabetes mellitus is discussed. Our study investigates whether ß cell mass and function can be preserved by selectively addressing the GluN2B subunit of the NMDAR. NMDAR activation by NMDA and its coagonist glycine moderately influenced electrical activity and Ca2+ handling in islet cells at a threshold glucose concentration (4-5 mM) without affecting glucose-mediated insulin secretion. Exposure of islet cells to NMDA/glycine or a glucolipotoxic milieu increased apoptosis by 5% and 8%, respectively. The GluN2B-specific NMDAR antagonist WMS-1410 (0.1 and 1 µM) partly protected against this. In addition, WMS-1410 completely prevented the decrease in insulin secretion of about 32% provoked by a 24-hour-treatment with NMDA/glycine. WMS-1410 eliminated NMDA-induced changes in the oxidation status of the islet cells and elevated the sensitivity of intracellular calcium to 15 mM glucose. By contrast, WMS-1410 did not prevent the decline in glucose-stimulated insulin secretion occurring after glucolipotoxic culture. This lack of effect was due to a decrease in insulin content to 18% that obviously could not be compensated by the preservation of cell mass or the higher percentage of insulin release in relation to insulin content. In conclusion, the negative effects of permanent NMDAR activation were effectively counteracted by WMS-1410 as well as the apoptotic cell death induced by high glucose and lipid concentrations. Modulation of NMDARs containing the GluN2B subunit is suggested to preserve ß cell mass during development of type 2 diabetes mellitus. SIGNIFICANCE STATEMENT: Addressing NMDA receptors containing the GluN2B subunit in pancreatic islet cells has the potential to protect the ß cell mass that progressively declines during the development of type 2 diabetes. Furthermore, this study shows that harmful effects of permanent NMDAR activation can be effectively counteracted by the compound WMS-1410, a selective modulator for NMDARs containing the GluN2B subunit.

Extraction and Purification of R-Phycoerythrin Alpha Subunit from the Marine Red Algae Pyropia Yezoensis and Its Biological Activities.

Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria that is widely used as a fluorescent probe or as a colorant in the food and cosmetic industries. In this study, phycoerythrin was extracted from the red algae Pyropia yezoensis and purified by ammonium sulfate precipitation and various chromatography methods. The purified phycoerythrin was analyzed by UV-visible and fluorescence spectroscopy. The isolated pigment had the typical spectrum of R-phycoerythrin, with a trimmer state with absorbance maxima at 497, 536, and 565 nm. It was further purified and identified by LC-MS/MS and Mascot search. It showed a 100% sequence similarity with the R-phycoerythrin alpha subunit of Pyropia yezoensis. The molecular mass was 17.97 kDa. The antioxidant activity of the purified R-phycoerythrin alpha subunit was analyzed. It showed significant antioxidant activity in ABTS and FRAP assays and had significant cytotoxicity against HepG2 cells.

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human blood-brain barrier.

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system include neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-CoV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. The spike protein, which plays a key role in receptor recognition, is formed by the S1 subunit containing a receptor binding domain (RBD) and the S2 subunit. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is ubiquitously expressed throughout various vessel calibers in the frontal cortex. Moreover, ACE2 expression was upregulated in cases of hypertension and dementia. ACE2 was also detectable in primary hBMVECs maintained under cell culture conditions. Analysis of cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48 h exposure window. Introduction of spike proteins to invitro models of the blood-brain barrier (BBB) showed significant changes to barrier properties. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluidic model of the human BBB, a platform that more closely resembles the physiological conditions at this CNS interface. Evidence provided suggests that the SARS-CoV-2 spike proteins trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

Enolase and dipeptidyl peptidase IV protein sub-unit vaccines are not protective against a lethal Streptococcus suis serotype 2 challenge in a mouse model of infection.

BACKGROUND: Streptococcus suis is a major swine pathogen causing arthritis, meningitis and sudden death in post-weaning piglets and is also a zoonotic agent. S. suis comprises 35 different serotypes of which the serotype 2 is the most prevalent in both pigs and humans. In the absence of commercial vaccines, bacterins (mostly autogenous), are used in the field, with controversial results. In the past years, the focus has turned towards the development of sub-unit vaccine candidates. However, published results are sometimes contradictory regarding the protective effect of a same candidate. Moreover, the adjuvant used may significantly influence the protective capacity of a given antigen. This study focused on two protective candidates, the dipeptidyl peptidase IV (DPPIV) and the enolase (SsEno). Both proteins are involved in S. suis pathogenesis, and while contradictory protection results have been obtained with SsEno in the past, no data on the protective capacity of DPPIV was available. RESULTS: Results showed that among all the field strains tested, 86 and 88% were positive for the expression of the SsEno and DPPIV proteins, respectively, suggesting that they are widely expressed by strains of different serotypes. However, no protection was obtained after two vaccine doses in a CD-1 mouse model of infection, regardless of the use of four different adjuvants. Even though no protection was obtained, significant amounts of antibodies were produced against both antigens, and this regardless of the adjuvant used. CONCLUSIONS: Taken together, these results demonstrate that S. suis DPPIV and SsEno are probably not good vaccine candidates, at least not in the conditions evaluated in this study. Further studies in the natural host (pig) should still be carried out. Moreover, this work highlights the importance of confirming results obtained by different research groups.

Heterodimeric BMP-2/7 exhibits different osteoinductive effects in human and murine cells.

As robust osteoinductive cytokines, bone morphogenetic proteins (BMPs) play a significant role in bone tissue engineering. Constituted of two different polypeptides, heterodimeric BMPs are more effective than the homodimers in bone formation. While most studies focused on the murine cell lines, such as murine preosteoblasts MC3T3-E1, the role of heterodimeric BMPs in the osteogenic differentiation of human cells remains uncertain, which hinders their application to practical treatment. In this study, we compared the osteoinductive effects of BMP-2/7 heterodimer in human adipose-derived stem cells (hASCs) with their homodimers BMP-2 and BMP-7, in which MC3T3-E1 cells were utilized as a positive control. The results indicated that BMP-2/7 was not a stronger inducer during the osteogenic differentiation of hASCs as that for MC3T3-E1, and extracellular-signal-regulated kinase signaling played a role in the different effects of BMP-2/7 between hASCs and MC3T3-E1. Our study demonstrates the osteoinductive effects of heterodimeric BMP-2/7 present in a cell-specific pattern and cautions should be taken when applying heterodimeric BMP-2/7 to clinical practice.

Disulphide bond restrains the C-terminal region of thermostable direct hemolysin during folding to promote oligomerization.

Pore-forming toxins (PFTs) are typically produced as water-soluble monomers, which upon interacting with target cells assemble into transmembrane oligomeric pores. Vibrio parahaemolyticus thermostable direct hemolysin (TDH) is an atypical PFT that exists as a tetramer in solution, prior to membrane binding. The TDH structure highlights a core ß-sandwich domain similar to those found in the eukaryotic actinoporin family of PFTs. However, the TDH structure harbors an extended C-terminal region (CTR) that is not documented in the actinoporins. This CTR remains tethered to the ß-sandwich domain through an intra-molecular disulphide bond. Part of the CTR is positioned at the inter-protomer interface in the TDH tetramer. Here we show that the truncation, as well as mutation, of the CTR compromise tetrameric assembly, and the membrane-damaging activity of TDH. Our study also reveals that intra-protomer disulphide bond formation during the folding/assembly process of TDH restrains the CTR to mediate its participation in the formation of inter-protomer contact, thus facilitating TDH oligomerization. However, once tetramerization is achieved, disruption of the disulphide bond does not affect oligomeric assembly. Our study provides critical insights regarding the regulation of the oligomerization mechanism of TDH, which has not been previously documented in the PFT family.

ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols.

ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3ß3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (ß and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions.

Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata.

We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC50: 0.044 µmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477-17,638) and ß-subunit (Mw: 17,455-18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and ß-subunits. In addition, the LRY sequence was found in the ß-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and ß-subunits of PE (rPEα and rPEß, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEß: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins.

The B subunit of Escherichia coli heat-labile toxin alters the development and antigen-presenting capacity of dendritic cells.

Escherichia coli's heat-labile enterotoxin (Etx) and its non-toxic B subunit (EtxB) have been characterized as adjuvants capable of enhancing T cell responses to co-administered antigen. Here, we investigate the direct effect of intravenously administered EtxB on the size of the dendritic and myeloid cell populations in spleen. EtxB treatment appears to enhance the development and turnover of dendritic and myeloid cells from precursors within the spleen. EtxB treatment also gives a dendritic cell (DC) population with higher viability and lower activation status based on the reduced expression of MHC-II, CD80 and CD86. In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide. In in vitro bone marrow cultures, EtxB treatment was also found to enhance the development of DC from precursors dependent on Flt3L. In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional depletion of CD11c(+) DC. In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen-presenting cells on encounter with T cells.

Purification, characterization and partial sequence of a pro-inflammatory lectin from seeds of Canavalia oxyphylla Standl. & L. O. Williams.

Recent studies have shown that lectins are promising tools for use in various biotechnological processes, as well as studies of various pathological mechanisms, isolation, and characterization of glycoconjugates and understanding the mechanisms underlying pathological mechanisms conditions, including the inflammatory response. This study aimed to purify, characterize physicochemically, and predict the biological activity of Canavalia oxyphylla lectin (CoxyL) in vitro and in vivo. CoxyL was purified by a single-step affinity chromatography in Sephadex® G-50 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the pure lectin consists of a major band of 30 kDa (α-chain) and two minor components (ß-chain and γ-chain) of 16 and 13 kDa, respectively. These data were further confirmed by electrospray ionization mass spectrometry, suggesting that CoxyL is a typical ConA-like lectin. In comparison with the average molecular mass of α-chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. CoxyL presented hemagglutinating activity that was specifically inhibited by monosaccharides (D-glucose, D-mannose, and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). Moreover, CoxyL was shown to be thermostable, exhibiting full hemagglutinating activity up to 60°C, and it was pH-sensitive for 1 h, exhibiting maximal activity at pH 7.0. CoxyL caused toxicity to Artemia nauplii and induced paw edema in rats. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses.

The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1-dependent manner.

Shiga toxin (Stx) causes diarrhea-associated hemolytic uremic syndrome by damaging renal microvascular endothelium. The pentameric B subunits of Stx types 1 and 2 (Stx1B and Stx2B) are sufficient to stimulate acute VWF secretion from endothelial cells, but Stx1B and Stx2B exert distinct effects on Ca(2+) and cAMP pathways. Therefore, we investigated other signaling components in StxB-induced VWF exocytosis. Incubation of HUVECs with StxB transiently increased phospholipase D (PLD) activity. Inhibition of PLD activity or shRNA-mediated PLD1 knockdown abolished StxB-induced VWF secretion. In addition, treatment with StxB triggered actin polymerization, enhanced endothelial monolayer permeability, and activated RhoA. PLD activation and VWF secretion induced by Stx1B were abolished on protein kinase Cα (PKCα) inhibition or gene silencing but were only moderately reduced by Rho or Rho kinase inhibitors. Conversely, PLD activation and VWF exocytosis induced by Stx2B were reduced by Rho/Rho kinase inhibitors and dominant-negative RhoA, whereas attenuation of PKCα did not affect either process. Another PLD1 activator, ADP-ribosylation factor 6, was involved in VWF secretion induced by Stx1B or Stx2B, but not histamine. These data indicate that Stx1B and Stx2B induce acute VWF secretion in a PLD1-dependent manner but do so by differentially modulating PKCα, RhoA, and ADP-ribosylation factor 6.

Estimation of the subunit stoichiometry of the membrane-associated daptomycin oligomer by FRET.

Daptomycin is a lipopeptide antibiotic that kills Gram-positive bacteria by depolarizing their cell membranes. This antibacterial action of daptomycin is correlated with the formation of membrane-associated oligomers. We here examine the number of subunits contained in one oligomer using fluorescence resonance energy transfer (FRET). The results suggest that the oligomer contains approximately 6 to 7 subunits, or possibly twice this number if it spans both membrane monolayers.

Immunomodulatory activity of recombinant Ricin toxin binding Subunit B (RTB).

Ricin toxin binding subunit B (RTB) is one of the subunits of the ricin protein. RTB has been used as adjuvant, but little is known about its mechanism. In this study, we found that RTB increased not only nitric oxide (NO) release, but also tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in mouse macrophage cell line RAW264.7 cells. They subsequently exhibited enhanced ConA-induced T-cell and LPS-induced B-cell proliferative responses. We also examined the cytokines that were produced from splenocytes following in vitro RTB administration. Increased levels of IL-2, interferon (IFN)-γ and TNF-α were observed, while IL-4 and IL-5 were unaffected. These results demonstrate that recombinant RTB can act on the immune system and activate T-cells by introducing a Th1 immune response. Th1 cells might be the primary cellular target affected by RTB. Our results suggest that the recombinant RTB can promote the activation of macrophages and has a beneficial effect on immunomodulatory activity.

Melatonin protects porcine oocyte from copper exposure potentially by reducing oxidative stress potentially through the Nrf2 pathway.

Copper is widely used as a feeding additive to promote livestock growth. However, excessive copper can be excreted with feces, causing heavy metal pollution and aggravating environmental problems. At the same time, studies have found that excess copper can cause damage to reproductive function and reduce gamete quality. Here, we explored the effects of adding different concentrations of copper to the culture medium on porcine oocytes. First polar body extrusion rate, embryo development, and intracellular levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) ΔΨm, adenosine triphosphate(ATP) content, and acetylation of lysine 9 on histone H3 protein subunit (H3K9ac) were assessed. Results demonstrated that Cu exposure causes abnormalities in mitochondrial function and epigenetic modification, resulting in increased oxidative stress and levels of ROS, ultimately leading to a decreased porcine oocyte quality. In addition, we found melatonin can protect porcine oocytes from those damages. Notably, Nrf2 protein expression was significantly increased by copper exposure, meanwhile, Nrf2 signaling pathway inhibitor ML385 significantly attenuated the protective role of melatonin on oxidative stress induced by copper exposure. In summary, our study demonstrates that copper activates the Nrf2 pathway and impairs oocyte maturation by inducing oxidative stress, leading to poor quality of porcine oocytes, and the changes can be reversed by melatonin.

Combined delivery of Nogo-A antibody, neurotrophin-3 and the NMDA-NR2d subunit establishes a functional 'detour' in the hemisected spinal cord.

To encourage re-establishment of functional innervation of ipsilateral lumbar motoneurons by descending fibers after an intervening lateral thoracic (T10) hemisection (Hx), we treated adult rats with the following agents: (i) anti-Nogo-A antibodies to neutralize the growth-inhibitor Nogo-A; (ii) neurotrophin-3 (NT-3) via engineered fibroblasts to promote neuron survival and plasticity; and (iii) the NMDA-receptor 2d (NR2d) subunit via an HSV-1 amplicon vector to elevate NMDA receptor function by reversing the Mg(2+) block, thereby enhancing synaptic plasticity and promoting the effects of NT-3. Synaptic responses evoked by stimulation of the ventrolateral funiculus ipsilateral and rostral to the Hx were recorded intracellularly from ipsilateral lumbar motoneurons. In uninjured adult rats short-latency (1.7-ms) monosynaptic responses were observed. After Hx these monosynaptic responses were abolished. In the Nogo-Ab + NT-3 + NR2d group, long-latency (approximately 10 ms), probably polysynaptic, responses were recorded and these were not abolished by re-transection of the spinal cord through the Hx area. This suggests that these novel responses resulted from new connections established around the Hx. Anterograde anatomical tracing from the cervical grey matter ipsilateral to the Hx revealed increased numbers of axons re-crossing the midline below the lesion in the Nogo-Ab + NT-3 + NR2d group. The combined treatment resulted in slightly better motor function in the absence of adverse effects (e.g. pain). Together, these results suggest that the combination treatment with Nogo-Ab + NT-3 + NR2d can produce a functional 'detour' around the lesion in a laterally hemisected spinal cord. This novel combination treatment may help to improve function of the damaged spinal cord.

Heterologous expression and purification of the soybean 7S globulin α' subunit extension region: in vitro evidence of its involvement in cell cholesterol homeostasis.

In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin α' subunit, known to control cholesterol and triglyceride homeostasis, was described. In this work, a shorter version of the polypeptide chain, spanning 142 amino acid residues from the N-terminus and thus exclusively including the so-called extension region, was cloned and overexpressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed α'E, was 8-fold greater than the previous truncated version. The α'E polypeptide was purified by simple conventional biochemical techniques to make it available for biological assays. Human hepatoma cell lines (Hep G2) were used to monitor the uptake and degradation of labeled low-density lipoproteins (LDL), according to an established procedure. The LDL uptake (+86%) and degradation (+94%) by cells tested at the highest α'E dose (2 µM) were similar to those found in cells incubated with 1 µM simvastatin, a potent inhibitor of cholesterol biosynthesis. Additionally, the cell response to α'E was found to be dose-dependent. The present findings strongly suggest that this recombinant polypeptide, or a fragment thereof, is the molecular determinant for cholesterol homeostasis and open new prospects for understanding the mechanism involved in this biological response, as a gateway to its utilization in lipid-lowering therapies.

The native form of follicle-stimulating hormone is essential for the growth of mouse preantral follicles in vitro.

To assess whether the follicle-stimulating hormone (FSH) subunits observed in patients with gonadotroph adenomas (GA) can cause infertility, the effects of subunits and heterodimeric FSH on the in vitro follicle development were evaluated in mice. The partial forms of FSH in follicle culture did not induce development into pseudoantral follicles, whereas follicles cultured with native FSH developed into pseudoantral follicles and produced mature metaphase II oocyte. Therefore, intact FSH is needed for folliculogenesis, implying that production of FSH with a partial structure in GA may result in infertility.

Alpha-single chains of collagen type VI inhibit the fibrogenic effects of triple helical collagen VI in hepatic stellate cells.

The interaction of extracellular matrix (ECM) components with hepatic stellate cells (HSCs) is thought to perpetuate fibrosis by stimulating signaling pathways that drive HSC activation, survival and proliferation. Consequently, disrupting the interaction between ECM and HSCs is considered a therapeutical avenue although respective targets and underlying mechanisms remain to be established. Here we have interrogated the interaction between type VI collagen (CVI) and HSCs based on the observation that CVI is 10-fold upregulated during fibrosis, closely associates with HSCs in vivo and promotes cell proliferation and cell survival in cancer cell lines. We exposed primary rat HSCs and a rat hepatic stellate cell line (CFSC) to soluble CVI and determined the rate of proliferation, apoptosis and fibrogenesis in the absence of any additional growth factors. We find that CVI in nanomolar concentrations prevents serum starvation-induced apoptosis. This potent anti-apoptotic effect is accompanied by induction of proliferation and acquisition of a pronounced pro-fibrogenic phenotype characterized by increased α-smooth muscle actin, TGF-ß, collagen type I and TIMP-1 expression and diminished proteolytic MMP-13 expression. The CVI-HSC interaction can be disrupted with the monomeric α2(VI) and α3(VI) chains and abrogates the activating CVI effects. Further, functional relevant α3(VI)-derived 30 amino acid peptides lead to near-complete inhibition of the CVI effect. In conclusion, CVI serves as a potent mitogen and activating factor for HSCs. The antagonistic effects of the CVI monomeric chains and peptides point to linear peptide sequences that prevent activation of CVI receptors which may allow a targeted antifibrotic therapy.

Voltage-dependent calcium channel ß subunit-derived peptides reduce excitatory neurotransmission and arterial blood pressure.

AIMS: Voltage-dependent calcium channels (VDCCs) play an important role in various physiological functions in the nervous system and the cardiovascular system. In L-, N-, P/Q-, and R-type VDCCs, ß subunit assists the channels for membrane targeting and modulates channel properties. In this study, we investigated whether an inhibition of the ß subunit binding to α subunit, the pore-forming main subunit of VDCCs, have any effect on channel activation and physiological functions. MAIN METHODS: Peptides derived from the specific regions of ß subunit that bind to the α-interaction domain in I-II linker of α subunit were manufactured, presuming that the peptides interrupt α-ß subunit interaction in the channel complex. Then, they were tested on voltage-activated Ca2+ currents recorded in acutely isolated trigeminal ganglion (TG) neurons, excitatory postsynaptic currents (EPSCs) in the spinal dorsal horn neurons, and arterial blood pressure (BP) recorded from the rat femoral artery. KEY FINDINGS: When applied internally through patch pipettes, the peptides decreased the peak amplitudes of the voltage-activated Ca2+ currents. After fusing with HIV transactivator of transcription (TAT) sequence to penetrate cell membrane, the peptides significantly decreased the peak amplitudes of Ca2+ currents and the peak amplitudes of EPSCs upon the external application through bath solution. Furthermore, the TAT-fused peptides dose dependently reduced the rat BP when administered intravenously. SIGNIFICANCE: These data suggest that an interruption of α-ß subunit association in VDCC complex inhibits channel activation, thereby reducing VDCC-mediated physiological functions such as excitatory neurotransmission and arterial BP.