Cardiac resynchronization therapy using bi-ventricular pacing is proven effective in the management of heart failure (HF) with a wide QRS-complex. In the absence of QRS prolongation, however, device-based resynchronization is reported unsuitable. As an alternative, the present study tests a regenerative cell-based approach in the setting of narrow QRS-complex HF.

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1(H/H)) live shorter and show signs of accelerated aging. As wild-type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age-related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1-7) are a family of NAD(+)-dependent deacetylases that can delay age-related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD(+) and the ability of SIRT2 to maintain lysine-668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD(+) precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1(H/H) animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD(+) to delay diseases of aging in mammals is warranted.

Whole-genome gene expression analysis has been successfully utilized to diagnose, prognosticate, and identify potential therapeutic targets for high-risk cardiovascular diseases. However, the feasibility of this approach to identify outcome-related genes and dysregulated pathways following first-time myocardial infarction (AMI) remains unknown and may offer a novel strategy to detect affected expressome networks that predict long-term outcome.

Human papillomavirus (HPV) 6b L1 capsid protein was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system in silkworm larvae. Two constructs, full-length L1 (500 a.a) and C-terminal-deleted short L1 (479 a.a), and three PCR-manipulated antigenic loops at amino acids 55-56, 174-175, and 348-349 regions were incorporated with whole enhanced green fluorescent protein (EGFP). Expressed in full, short L1 proteins and variants were purified in heparin affinity column chromatography and confirmed by SDS-PAGE and western blot. The presence of self-assembled virus-like particles (VLPs) and EGFP incorporation on the surface of VLPs were confirmed by the observation of transmission electron and immunoelectron microscopies, respectively. HPV 6b L1 major capsid protein was successfully expressed in silkworm, and effective manipulation on the antigenic regions showed the path to versatile vaccine development based on HPV L1-VLPs.

Contractile discordance exacerbates cardiac dysfunction, aggravating heart failure outcome. Dissecting the genesis of mechanical dyssynchrony would enable an early diagnosis before advanced disease.

Cellular senescence, a stress-induced irreversible growth arrest often characterized by expression of p16(Ink4a) (encoded by the Ink4a/Arf locus, also known as Cdkn2a) and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time, and have been speculated to have a role in ageing. To explore the physiological relevance and consequences of naturally occurring senescent cells, here we use a previously established transgene, INK-ATTAC, to induce apoptosis in p16(Ink4a)-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. We show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. The clearance of p16(Ink4a)-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels and adipocytes, respectively. Thus, p16(Ink4a)-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in several organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

Na(V)1.5 is a mechanosensitive voltage-gated Na(+) channel encoded by the gene SCN5A, expressed in cardiac myocytes and required for phase 0 of the cardiac action potential (AP). In the cardiomyocyte, ranolazine inhibits depolarizing Na(+) current and delayed rectifier (I(Kr)) currents. Recently, ranolazine was also shown to be an inhibitor of Na(V)1.5 mechanosensitivity. Stretch also accelerates the firing frequency of the SA node, and fluid shear stress increases the beating rate of cultured cardiomyocytes in vitro. However, no cultured cell platform exists currently for examination of spontaneous electrical activity in response to mechanical stimulation. In the present study, flow of solution over atrial myocyte-derived HL-1 cultured cells was used to study shear stress mechanosensitivity of Na(+) current and spontaneous, endogenous rhythmic action potentials. In voltage-clamped HL-1 cells, bath flow increased peak Na(+) current by 14 ± 5%. In current-clamped cells, bath flow increased the frequency and decay rate of AP by 27 ± 12% and 18 ± 4%, respectively. Ranolazine blocked both responses to shear stress. This study suggests that cultured HL-1 cells are a viable in vitro model for detailed study of the effects of mechanical stimulation on spontaneous cardiac action potentials. Inhibition of the frequency and decay rate of action potentials in HL-1 cells are potential mechanisms behind the antiarrhythmic effect of ranolazine.

Embryonic stem cells possess a pluripotent transcriptional background with the developmental capacity for distinct cell fates. Simultaneous expression of genetic elements for multiple outcomes obscures cascades relevant to specific cell phenotypes. To map molecular patterns critical to cardiogenesis, we interrogated gene expression in stem cells undergoing guided differentiation, and defined a genomic paradigm responsible for confinement of pluripotency.

Allosteric regulation of heteromultimeric ATP-sensitive potassium (K(ATP)) channels is unique among protein systems as it implies transmission of ligand-induced structural adaptation at the regulatory SUR subunit, a member of ATP-binding cassette ABCC family, to the distinct pore-forming K+ (Kir6.x) channel module. Cooperative interaction between nucleotide binding domains (NBDs) of SUR is a prerequisite for K(ATP) channel gating, yet pathways of allosteric intersubunit communication remain uncertain. Here, we analyzed the role of the ED domain, a stretch of 15 negatively charged aspartate/glutamate amino acid residues (948-962) of the SUR2A isoform, in the regulation of cardiac K(ATP) channels. Disruption of the ED domain impeded cooperative NBDs interaction and interrupted the regulation of K(ATP) channel complexes by MgADP, potassium channel openers, and sulfonylurea drugs. Thus, the ED domain is a structural component of the allosteric pathway within the K(ATP) channel complex integrating transduction of diverse nucleotide-dependent states in the regulatory SUR subunit to the open/closed states of the K+-conducting channel pore.

Nanomaterials without chemical linkers or physical interactions that reside on a two-dimensional surface are attractive because of their electronic, optical and catalytic properties. An in situ method has been developed to fabricate gold nanoparticle (Au NP) films on different substrates, regardless of whether they are hydrophilic or hydrophobic surfaces, including glass, 96-well polystyrene plates, and polydimethylsiloxane (PDMS). A mixture of sodium formate (HCOONa) and chloroauric acid (HAuCl4) solution was used to prepare Au NP films at room temperature. An experimental study of the mechanism revealed that film formation is dependent on surface wettability and inter particle attraction. The as-fabricated Au NP films were further applied to the colorimetric detection of influenza virus. The response to the commercial target, New Caledonia/H1N1/1999 influenza virus, was linear in the range from 10 pg/ml to 10 μg/ml and limit of detection was 50.5 pg/ml. In the presence of clinically isolated influenza A virus (H3N2), the optical density of developed color was dependent on the virus concentration (10-50,000 PFU/ml). The limit of detection of this study was 24.3 PFU/ml, a limit 116 times lower than that of conventional ELISA (2824.3 PFU/ml). The sensitivity was also 500 times greater than that of commercial immunochromatography kits.

Hepatitis E virus (HEV) is one of the leading causes of acute viral hepatitis worldwide. In this work, a pulse-triggered ultrasensitive electrochemical sensor was fabricated using graphene quantum dots and gold-embedded polyaniline nanowires, prepared via an interfacial polymerization and then self-assembly approach. Introducing an external electrical pulse during the virus accumulation step increases the sensitivity towards HEV due to the expanded surface of the virus particle as well as the antibody-conjugated polyaniline chain length, compared to other conventional electrochemical sensors. The sensor was applied to various HEV genotypes, including G1, G3, G7 and ferret HEV obtained from cell culture supernatant and in a series of fecal specimen samples collected from G7 HEV-infected monkey. The sensitivity is similar to that detected by real-time quantitative reverse transcription-polymerase chain (RT-qPCR). These results suggests that the proposed sensor can pave the way for the development of robust, high-performance sensing methodologies for HEV detection.

Response to stem cell therapy in heart failure is heterogeneous, warranting a better understanding of outcome predictors. This study assessed left ventricular volume, a surrogate of disease severity, on cell therapy benefit. Small to large infarctions were induced in murine hearts to model moderate, advanced, and end-stage ischemic cardiomyopathy. At 1 month postinfarction, cardiomyopathic cohorts with comparable left ventricular enlargement and dysfunction were randomized 1:1 to those that either received sham treatment or epicardial delivery of cardiopoietic stem cells (CP). Progressive dilation and pump failure consistently developed in sham. In comparison, CP treatment produced significant benefit at 1 month post-therapy, albeit with an efficacy impacted by cardiomyopathic stage. Advanced ischemic cardiomyopathy was the most responsive to CP-mediated salvage, exhibiting both structural and functional restitution, with proteome deconvolution substantiating that cell therapy reversed infarction-induced remodeling of functional pathways. Moderate cardiomyopathy was less responsive to CP therapy, improving contractility but without reversing preexistent heart enlargement. In end-stage disease, CP therapy showed the least benefit. This proof-of-concept study thus demonstrates an optimal window, or "Goldilocks principle," of left ventricular enlargement for maximized stem cell-based cardiac repair. Disease severity grading, prior to cell therapy, should be considered to inform regenerative medicine interventions.

Insulin-like peptide 3 (INSL3) is a member of the relaxin/insulin superfamily and is expressed in testicular Leydig cells. Essential for fetal testis descent, INSL3 has been implicated in testicular and sperm function in adult males via interaction with relaxin/insulin-like family peptide receptor 2 (RXFP2). The INSL3 is typically prepared using chemical synthesis or overexpression in Escherichia coli followed by oxidative refolding and proteolysis. Here, we expressed and purified full-length porcine INSL3 (pINSL3) using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system. Biophysical measurements and proteomic analysis revealed that this recombinant pINSL3 exhibited the correct conformation, with the three critical disulfide bonds observed in native pINSL3, although partial cleavage occurred. In cAMP stimulation assays using RXFP2-expressing HEK293 cells, the recombinant pINSL3 possessed full biological activity. This is the first report concerning the production of fully active pINSL3 without post-expression treatments and provides an efficient production platform for expressing relaxin/insulin superfamily peptides.

Growth factors are signaling molecules which orchestrate cell growth, proliferation and differentiation. The majority are secreted proteins, exported through the classical endoplasmic reticulum (ER)/Golgi-dependent pathway, but a few are released by unconventional ER/Golgi-independent means. Human fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1), are canonical prototypes secreted by the unconventional and conventional pathway, respectively. We herein examined whether expression of these two growth factors in the Bombyx mori nucleopolyhedrovirus (BmNPV)-based silkworm expression system with its innate signal peptide, bombyxin, secures structural homogeneity at the signal peptide cleavage site regardless of the native secretory route. Proteomic analysis mapped structural microheterogeneity of signal peptide cleavage at the amino terminus of FGF2, whereas IGF1 displayed homogeneous amino-terminal cleavage with complete removal of the bombyxin signal peptide. A cell proliferation assay revealed potent functional activity of both FGF2 and IGF1, suggesting that FGF2 amino-terminal microheterogeneity does not alter mitogenic activity. These findings demonstrate that the occurrence of amino-terminal structural homogeneity may be associated with the original secretion mechanism of a particular growth factor. Furthermore, our results highlight the bombyxin signal peptide as a reliable secretion sequence applicable to mass production of functionally active secretory proteins in a silkworm-based expression platform.

Cardiopoietic stem cells have reached advanced clinical testing for ischemic heart failure. To profile their molecular influence on recipient hearts, systems proteomics was here applied in a chronic model of infarction randomized with and without human cardiopoietic stem cell treatment. Multidimensional label-free tandem mass spectrometry resolved and quantified 3987 proteins constituting the cardiac proteome. Infarction altered 450 proteins, reduced to 283 by stem cell treatment. Notably, cell therapy non-stochastically reversed a majority of infarction-provoked changes, remediating 85% of disease-affected protein clusters. Pathway and network analysis decoded functional reorganization, distinguished by prioritization of vasculogenesis, cardiac development, organ regeneration, and differentiation. Subproteome restoration nullified adverse ischemic effects, validated by echo-/electro-cardiographic documentation of improved cardiac chamber size, reduced QT prolongation and augmented ejection fraction post-cell therapy. Collectively, cardiopoietic stem cell intervention transitioned infarcted hearts from a cardiomyopathic trajectory towards pre-disease. Systems proteomics thus offers utility to delineate and interpret complex molecular regenerative outcomes.

Neosporosis, which is caused by Neospora caninum, is a well-known disease in the veterinary field. Infections in pregnant cattle lead to abortion via transplacental (congenitally from mother to fetus) transmission. In this study, a N. caninum profilin (NcPROF), was expressed in silkworm larvae by recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid and was purified from the hemolymph. Three NcPROF constructs were investigated, native NcPROF fused with an N-terminal PA tag (PA-NcPROF), PA-NcPROF fused with the signal sequence of bombyxin from B. mori (bx-PA-NcPROF), and bx-PA-NcPROF with additional C-terminal transmembrane and cytoplasmic domains of GP64 from BmNPV (bx-PA-NcPROF-GP64TM). All recombinant proteins were observed extra- and intracellularly in cultured Bm5 cells and silkworm larvae. The bx-PA-NcPROF-GP64TM was partly abnormally secreted, even though it has the transmembrane domain, and only it was pelleted by ultracentrifugation, but PA-NcPROF and bx-PA-NcPROF were not. Additionally, bx-PA-NcPROF-GP64TM was successfully purified from silkworm hemolymph by anti-PA agarose beads while PA-NcPROF and bx-PA-NcPROF were not. The purified bx-PA-NcPROF-GP64TM protein bound to its receptor, mouse Toll-like receptor 11 (TLR-11), and formed unique nanoparticles. These results suggest that profilin fused with GP64TM was secreted as a nanoparticle with binding affinity to its receptor and this nanoparticle formation is advantageous for the development of vaccines to N. caninum.

Stem cell paracrine activity is implicated in cardiac repair. Linkage between secretome functionality and therapeutic outcome was here interrogated by systems analytics of biobanked human cardiopoietic cells, a regenerative biologic in advanced clinical trials. Protein chip array identified 155 proteins differentially secreted by cardiopoietic cells with clinical benefit, expanded into a 520 node network, collectively revealing inherent vasculogenic properties along with cardiac and smooth muscle differentiation and development. Next generation RNA sequencing, refined by pathway analysis, pinpointed miR-146 dependent regulation upstream of the decoded secretome. Intracellular and extracellular integration unmasked commonality across cardio-vasculogenic processes. Mirroring the secretome pattern, infarcted hearts benefiting from cardiopoietic cell therapy restored the disease proteome engaging cardiovascular system functions. The cardiopoietic cell secretome thus confers a therapeutic molecular imprint on recipient hearts, with response informed by predictive systems profiling.

White spot syndrome virus (WSSV) is one of the most devastating pathogens in penaeid shrimp and can cause massive damage in shrimp aquaculture industries. Previously, the WSSV structural protein VP15 was identified as an antigenic reagent against WSSV infections. In this study, we truncated this protein into VP15(1-25), VP15(26-57), VP15(58-80), and VP15(1-25,58-80). The purified proteins from the E. coli expression system were assayed as potential protective agents in Kuruma shrimp (Marsupenaeus japonicus) using the prime-and-boost strategy. Among the four truncated constructs, VP15(26-57) provided a significant improvement in the shrimp survival rate after 20 days of viral infection. Subsequently, four peptides (KR11, SR11, SK10, and KK13) from VP15(26-57) were synthesized and applied in an in vivo assay. Our results showed that SR11 could significantly enhance the shrimp survival rate, as determined from the accumulated survival rate. Moreover, a multiligand binding protein with a role in the host immune response and a possible VP15-binding partner, MjgC1qR, from the host M. japonicus were employed to test its binding with the VP15 protein. GST pull-down assays revealed that MjgC1qR binds with VP15, VP15(26-57), and SR11. Taken together, we conclude that SR11 is a determinant antigenic peptide of VP15 conferring antiviral activity against WSSV.

In situ cell recruitment is a promising regenerative medicine strategy with the purpose of tissue regeneration without stem cell transplantation. This chemotaxis-based strategy is aimed at ensuring a restorative environment through the release of chemokines that promote site-specific migration of healing cell populations. Stromal cell-derived factor-1α (SDF-1α) is a critical chemokine that can regulate the migration of mesenchymal stem cells (MSCs). Accordingly, here, SDF-1α-loaded microporous oligo[poly(ethylene glycol) fumarate]/bis[2-(methacryloyloxy)ethyl] phosphate composites (SDF-1α/OPF/BP) were engineered and probed. SDF-1α/OPF/BP composites were loaded with escalating SDF-1α concentrations, namely, 0 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ml, and were cocultured with MSC. Scratching assay, Transwell assay, and three-dimensional migration model were utilized to assess the migration response of MSCs. Immunofluorescence staining of Runx2 and osteopontin (OPN), ELISA assay of osteocalcin (OCN) and alkaline phosphatase (ALP), and Alizarin Red S staining were conducted to assess the osteogenesis of MSCs. All SDF-1α/OPF/BP composites engendered a release of SDF-1α (>80%) during the first four days. SDF-1α released from the composites significantly promoted migration and osteogenic differentiation of MSCs documented by upregulated expression of osteogenic-related proteins, ALP, Runx2, OCN, and OPN. SDF-1α at 100 ng/ml was optimal for enhanced migration and osteogenic proficiency. Thus, designed SDF-1α/OPF/BP composites were competent in promoting the homing and osteogenesis of MSCs and thus offer a promising bioactive scaffold candidate for on-demand bone tissue regeneration.

Rous sarcoma virus-like particles (RSV-LPs) displaying hemagglutinins of H1N1 (A/New Caledonia/20/99) (H1) and H5N1 (A/Vietnam/1194/2004) (H5) of the influenza A virus were produced. The H1 has its transmembrane domain, but the H5 was fused with the transmembrane domain of glycoprotein 64 (BmGP64) from Bombyx mori nucleopolyhedrovirus (BmNPV). H1 and RSV Gag protein were coexpressed in the hemolymph of silkworm larvae, copurified, and confirmed RSV-LP displaying H1 (VLP/H1). Similarly, the RSV-LP displaying H5 (VLP/H5) production was also achieved. Using fetuin agarose column chromatography, RSV Gag protein-coexpressed H1 and H5 in silkworms were copurified from the hemolymph. By immuno-TEM, H1 and H5 were observed on the surface of an RSV-LP, indicating the formation of bivalent RSV-LP displaying two HAs (VLP/BivHA) in the hemolymph of silkworm larvae. VLP/H1 induced the hemagglutination of red blood cells (RBCs) of chicken and rabbit but not sheep, while VLP/H5 induced the hemagglutination of RBCs of chicken and sheep but not rabbit. Additionally, VLP/BivHA allowed the hemagglutination of RBCs of all three animals. Silkworm larvae can produce RSV-LPs displaying two HAs and is a promising tool to produce the bivalent enveloped VLPs for the vaccine platform.