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.

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.

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.

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.

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca2+ handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we analyzed the expression profile of α2A, α2B and α2C subtypes in mouse ventricle and conducted electrophysiological antagonist assay evaluating the contribution of these isoforms to the suppression of L-type Ca2+ current (ICaL). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of ICaL involves mainly the α2C, to a lesser extent the α2B, and not the α2A isoforms. RT-qPCR evaluation revealed the presence of adra2b and adra2c (α2B and α2C isoform genes, respectively), but was unable to identify the expression of adra2a (α2A isoform gene) in the mouse left ventricle. Immunoblotting confirmed the presence only of the α2B and the α2C proteins in this tissue. The identified α2-AR isoform-linked regulation of ICaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting.

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.

Biotin-dependent human acetyl-CoA carboxylases (ACCs) are integral in homeostatic lipid metabolism. By securing posttranslational biotinylation, ACCs perform coordinated catalytic functions allosterically regulated by phosphorylation/dephosphorylation and citrate. The production of authentic recombinant ACCs is heeded to provide a reliable tool for molecular studies and drug discovery. Here, we examined whether the human ACC2 (hACC2), an isoform of ACC produced using the silkworm BmNPV bacmid system, is equipped with proper posttranslational modifications to carry out catalytic functions as the silkworm harbors an inherent posttranslational modification machinery. Purified hACC2 possessed genuine biotinylation capacity probed by biotin-specific streptavidin and biotin antibodies. In addition, phosphorylated hACC2 displayed limited catalytic activity whereas dephosphorylated hACC2 revealed an enhanced enzymatic activity. Moreover, hACC2 polymerization, analyzed by native page gel analysis and atomic force microscopy imaging, was allosterically regulated by citrate and the phosphorylation/dephosphorylation modulated citrate-induced hACC2 polymerization process. Thus, the silkworm BmNPV bacmid system provides a reliable eukaryotic protein production platform for structural and functional analysis and therapeutic drug discovery applications implementing suitable posttranslational biotinylation and phosphorylation.

Metabolic processes that regulate muscle energy use are major determinants of bodily energy balance. Here, we find that sarcolemmal ATP-sensitive K(+) (K(ATP)) channels, which couple membrane excitability with cellular metabolic pathways, set muscle energy expenditure under physiological stimuli. Disruption of K(ATP) channel function provoked, under conditions of unaltered locomotor activity and blood substrate availability, an extra energy cost of cardiac and skeletal muscle performance. Inefficient fuel metabolism in K(ATP) channel-deficient striated muscles reduced glycogen and fat body depots, promoting a lean phenotype. The propensity to lesser body weight imposed by K(ATP) channel deficit persisted under a high-fat diet, yet obesity restriction was achieved at the cost of compromised physical endurance. Thus, sarcolemmal K(ATP) channels govern muscle energy economy, and their downregulation in a tissue-specific manner could present an antiobesity strategy by rendering muscle increasingly thermogenic at rest and less fuel efficient during exercise.

Conventional X-ray-based cystography uses radio-opaque materials, but this method uses harmful ionizing radiation and is not sensitive. In this study, we demonstrate nonionizing and noninvasive photoacoustic (PA) and fluorescence (FL) cystography using clinically relevant indocyanine green (ICG) in vivo. After transurethral injection of ICG into rats through a catheter, their bladders were photoacoustically and fluorescently visualized. A deeply positioned bladder below the skin surface (i.e., ~1.5-5 mm) was clearly visible in the PA and FL image using a laser pulse energy of less than 2 mJ/cm2 (1/15 of the safety limit). Then, the in vivo imaging results were validated through in situ studies. Our results suggest that dual modal cystography can provide a nonionizing and noninvasive imaging tool for bladder mapping.

Atrial fibrillation is a cardiac disease driven by numerous idiopathic etiologies. NUP155 is a nuclear pore complex protein that has been identified as a clinical driver of atrial fibrillation, yet the precise mechanism is unknown. The present study employs a systems biology algorithm to identify effects of NUP155 disruption on cardiogenicity in a model of stem cell-derived differentiation.

Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA). We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C-7°C-30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states.

Injectable polymers have attracted intensive attention in tissue engineering and drug delivery applications. Current injectable polymer systems often require free-radical or heavy-metal initiators and catalysts for the crosslinking process, which may be extremely toxic to the human body. Here, we report a novel polyhedral oligomeric silsesquioxane (POSS) based strain-promoted alkyne-azide cycloaddition (SPAAC) "click" organic-inorganic nanohybrids (click-ON) system that can be click-crosslinked without any toxic initiators or catalysts. The click-ON scaffolds supported excellent adhesion, proliferation, and osteogenesis of stem cells. In vivo evaluation using a rat cranial defect model showed outstanding bone formation with minimum cytotoxicity. Essential osteogenic alkaline phosphatase (ALP) and vascular CD31 marker expression were detected on the defect site, indicating excellent support of in vivo osteogenesis and vascularization. Using salt leaching techniques, an injectable porous click-ON cement was developed to create porous structures and support better in vivo bone regeneration. Beyond defect filling, the click-ON cement also showed promising application for spinal fusion using rabbits as a model. Compared to the current clinically used poly (methyl methacrylate) (PMMA) cement, this click-ON cement showed great advantages of low heat generation, better biocompatibility and biodegradability, and thus has great potential for bone and related tissue engineering applications.

The BubR1 gene encodes for a mitotic regulator that ensures accurate segregation of chromosomes through its role in the mitotic checkpoint and the establishment of proper microtubule-kinetochore attachments. Germline mutations that reduce BubR1 abundance cause aneuploidy, shorten lifespan and induce premature ageing phenotypes and cancer in both humans and mice. A reduced BubR1 expression level is also a feature of chronological ageing, but whether this age-related decline has biological consequences is unknown. Using a transgenic approach in mice, we show that sustained high-level expression of BubR1 preserves genomic integrity and reduces tumorigenesis, even in the presence of genetic alterations that strongly promote aneuplodization and cancer, such as oncogenic Ras. We find that BubR1 overabundance exerts its protective effect by correcting mitotic checkpoint impairment and microtubule-kinetochore attachment defects. Furthermore, sustained high-level expression of BubR1 extends lifespan and delays age-related deterioration and aneuploidy in several tissues. Collectively, these data uncover a generalized function for BubR1 in counteracting defects that cause whole-chromosome instability and suggest that modulating BubR1 provides a unique opportunity to extend healthy lifespan.

The ischemic stroke animal model evaluates the efficacy of reperfusion and neuroprotective strategies for ischemic injuries. Various conventional methods have been reported to induce the ischemic models; however, controlling specific neurological deficits, mortality rates, and the extent of the infarction is difficult as the size of the affected region is not precisely controlled. In this paper, we report a single laser-based localized target ischemic stroke model development method by simultaneous vessel monitoring and photothrombosis induction using photoacoustic microscopy (PAM), which has minimized the infarct size at precise location with high reproducibility. The proposed method has significantly reduced the infarcted region by illuminating the precise localization. The reproducibility and validity of suggested method have been demonstrated through repeated experiments and histological analyses. These results demonstrate that our method can provide the ischemic stroke model closest to the clinical pathology for brain ischemia research from inducement, occurrence mechanisms to the recovery process.

Acetyl-CoA carboxylase 2 (ACC2) is an isoform of ACC functioning as a negative regulator of fatty acid β-oxidation. Spot14, a thyroid hormone responsive protein, and Mig12, a Spot14 paralog, have recently been identified as regulators of fatty acid synthesis targeting ACC1, a distinctive subtype of ACC. Here, we examined whether Spot14/Mig12 modulates ACC2. Nanoscale protein topography mapped putative protein-protein interactions between purified human Spot14/Mig12 and ACC2, validated by functional assays. Human ACC2 displayed consistent enzymatic activity, and homogeneous particle distribution was probed by atomic force microscopy. Citrate-induced polymerization and enzymatic activity of ACC2 were restrained by the addition of the recombinant Spot14/Mig12 heterocomplex but only partially by the oligo-heterocomplex, demonstrating that the heterocomplex is a designated metabolic inhibitor of human ACC2. Moreover, Spot14/Mig12 demonstrated a sequestering role preventing an initial ACC2 nucleation step during filamentous polymer formation. Thus, the Spot14/Mig12 heterocomplex controls human ACC2 polymerization and catalytic function, emerging as a previously unrecognized molecular regulator in catalytic lipid metabolism.

Adenylate kinase2 (AK2) catalyzes trans-compartmental nucleotide exchange, but the functional implications of this mitochondrial intermembrane isoform is only partially understood. Here, transgenic AK2-/- null homozygosity was lethal early in embryo, indicating a mandatory role for intact AK2 in utero development. In the adult, conditional organ-specific ablation of AK2 precipitated abrupt heart failure with Krebs cycle and glycolytic metabolite buildup, suggesting a vital contribution to energy demanding cardiac performance. Depressed pump function recovered to pre-deletion levels overtime, suggestive of an adaptive response. Compensatory upregulation of phosphotransferase AK1, AK3, AK4 isozymes, creatine kinase isoforms, and hexokinase, along with remodeling of cell cycle/growth genes and mitochondrial ultrastructure supported organ rescue. Taken together, the requirement of AK2 in early embryonic stages, and the immediate collapse of heart performance in the AK2-deficient postnatal state underscore a primordial function of the AK2 isoform. Unsalvageable in embryo, loss of AK2 in the adult heart was recoverable, underscoring an AK2-integrated bioenergetics system with innate plasticity to maintain homeostasis on demand.

Plasmalemmal ATP sensitive potassium (KATP) channels are recognized metabolic sensors, yet their cellular reach is less well understood. Here, transgenic Kir6.2 null hearts devoid of the KATP channel pore underwent multiomics surveillance and systems interrogation versus wildtype counterparts. Despite maintained organ performance, the knockout proteome deviated beyond a discrete loss of constitutive KATP channel subunits. Multidimensional nano-flow liquid chromatography tandem mass spectrometry resolved 111 differentially expressed proteins and their expanded network neighborhood, dominated by metabolic process engagement. Independent multimodal chemometric gas and liquid chromatography mass spectrometry unveiled differential expression of over one quarter of measured metabolites discriminating the Kir6.2 deficient heart metabolome. Supervised class analogy ranking and unsupervised enrichment analysis prioritized nicotinamide adenine dinucleotide (NAD+), affirmed by extensive overrepresentation of NAD+ associated circuitry. The remodeled metabolome and proteome revealed functional convergence and an integrated signature of disease susceptibility. Deciphered cardiac patterns were traceable in the corresponding plasma metabolome, with tissue concordant plasma changes offering surrogate metabolite markers of myocardial latent vulnerability. Thus, Kir6.2 deficit precipitates multiome reorganization, mapping a comprehensive atlas of the KATP channel dependent landscape.

Urinary incontinence afflicts up to 40% of adult women in the United States. Stress urinary incontinence (SUI) accounts for approximately one-third of these cases, precipitating ~200,000 surgical procedures annually. Continence is maintained through the interplay of sub-urethral support and urethral sphincter coaptation, particularly during activities that increase intra-abdominal pressure. Currently, surgical correction of SUI focuses on the re-establishment of sub-urethral support. However, mesh-based repairs are associated with foreign body reactions and poor localized tissue healing, which leads to mesh exposure, prompting the pursuit of technologies that restore external urethral sphincter function and limit surgical risk. The present work utilizes a human platelet-derived CD41a and CD9 expressing extracellular vesicle product (PEP) enriched for NF-κB and PD-L1 and derived to ensure the preservation of lipid bilayer for enhanced stability and compatibility with hydrogel-based sustained delivery approaches. In vitro, the application of PEP to skeletal muscle satellite cells in vitro drove proliferation and differentiation in an NF-κB-dependent fashion, with full inhibition of impact on exposure to resveratrol. PEP biopotentiation of collagen-1 and fibrin glue hydrogel achieved sustained exosome release at 37 °C, creating an ultrastructural "bead on a string" pattern on scanning electron microscopy. Initial testing in a rodent model of latissimus dorsi injury documented activation of skeletal muscle proliferation of healing. In a porcine model of stress urinary incontinence, delivery of PEP-biopotentiated collagen-1 induced functional restoration of the external urethral sphincter. The histological evaluation found that sustained PEP release was associated with new skeletal muscle formation and polarization of local macrophages towards the regenerative M2 phenotype. The results provided herein serve as the first description of PEP-based biopotentiation of hydrogels implemented to restore skeletal muscle function and may serve as a promising approach for the nonsurgical management of SUI.

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the γ-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.

SUR2A is an ATP-binding protein that serves as a regulatory subunit of cardioprotective ATP-sensitive K(+) (K(ATP) ) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled K(ATP) channels, we have suggested that nicotinamide-rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide-rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild-type mice on nicotinamide-rich diet. The experiments focused on action membrane potential and intracellular Ca(2+) concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal K(ATP) channels and steady Ca(2+) levels in cardiomyocytes in response to β-adrenergic stimulation. In contrast, the same challenge in the wild-type was characterized by a lack of the channel activation and rise in intracellular Ca(2+) . Nicotinamide-rich diet was ineffective to increase physical endurance in mice lacking K(ATP) channels. This study has shown that nicotinamide-rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide-rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable.