Function and Characterization of an Alanine Dehydrogenase Homolog From Nocardia seriolae

Fish nocardiosis is a chronic, systemic, granulomatous disease in aquaculture. Nocardia seriolae has been reported to be one of the main pathogenic bacteria of fish nocardiosis. There are few studies on the associated virulence factors and pathogenesis of N. seriolae. Alanine dehydrogenase (ALD), which may be a secreted protein, was discovered by analysis using bioinformatics methods throughout the whole genomic sequence of N. seriolae. Nevertheless, the roles of ALD and its homologs in the pathogenesis of N. seriolae are not demonstrated. In this study, the function of N. seriolae ALD (NsALD) was preliminarily investigated by gene cloning, host cell subcellular localization, secreted protein identification, and cell apoptosis detection. Identification of the extracellular products of N. seriolae via mass spectrometry (MS) analysis revealed that NsALD is a secreted protein. In addition, subcellular localization of NsALD-GFP recombinant protein in fathead minnow (FHM) cells showed that the strong green fluorescence co-localized with the mitochondria. Moreover, apoptosis assays demonstrated that the overexpression of NsALD induces apoptosis in FHM cells. This study may lay the foundation for further exploration of the function of NsALD and facilitate further understanding of the pathogenic mechanism and the associated virulence factors of N. seriolae.

Insights into the Mechanisms of Action of MDA-7/IL-24: A Ubiquitous Cancer-Suppressing Protein

Melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24), a secreted protein of the IL-10 family, was first identified more than two decades ago as a novel gene differentially expressed in terminally differentiating human metastatic melanoma cells. MDA-7/IL-24 functions as a potent tumor suppressor exerting a diverse array of functions including the inhibition of tumor growth, invasion, angiogenesis, and metastasis, and induction of potent “bystander” antitumor activity and synergy with conventional cancer therapeutics. MDA-7/IL-24 induces cancer-specific cell death through apoptosis or toxic autophagy, which was initially established in vitro and in preclinical animal models in vivo and later in a Phase I clinical trial in patients with advanced cancers. This review summarizes the history and our current understanding of the molecular/biological mechanisms of MDA-7/IL-24 action rendering it a potent cancer suppressor.

A self-generated Toddler gradient guides mesodermal cell migration

The sculpting of germ layers during gastrulation relies on coordinated migration of progenitor cells, yet the cues controlling these long-range directed movements remain largely unknown. While directional migration often relies on a chemokine gradient generated from a localized source, we find that zebrafish ventrolateral mesoderm is guided by the uniformly expressed and secreted protein Toddler/ELABELA/Apela, acting as a self-generated gradient. We show that the Apelin receptor, which is specifically expressed in mesodermal cells, has a dual role during gastrulation, acting as a scavenger receptor to generate a Toddler gradient, and as a chemokine receptor to sense this guidance cue. Thus, we uncover a single receptor-based self-generated gradient as the enigmatic guidance cue that can robustly steer the directional migration of mesoderm through the complex and continuously changing environment of the gastrulating embryo.

S100A9 is a functional effector of infarct wall thinning after myocardial infarction

Neutrophils infiltrate into the left ventricle (LV) early after myocardial infarction (MI) and launch a pro-inflammatory response. Along with neutrophil infiltration, LV wall thinning due to cardiomyocyte necrosis also peaks at day 1 in the mouse model of MI. To understand the correlation, we examined a previously published dataset that included day 0 (n=10) and MI day 1 (n=10) neutrophil proteome and echocardiography assessments. Out of 123 proteins, 4 proteins positively correlated with the infarct wall thinning index (1/wall thickness): histone 1.2 (r=0.62, p=0.004), S100A9 (r=0.60, p=0.005), histone 3.1 (r=0.55, p=0.01), and fibrinogen (r=0.47, p=0.04). As S100A9 was the highest ranked secreted protein, we hypothesized that S100A9 is a functional effector of infarct wall thinning. We exogenously administered S100A8/A9 at the time of MI to mice (C57BL/6J, male, 3-6 months of age, n=7M (D1), and n=5M (D3)) and compared to saline vehicle control treated mice (n=6M (D1) and n=6M (D3)) at MI days 1 and 3. At MI day 3, the S100A8/A9 group showed a 22% increase in the wall thinning index compared to saline (p=0.02), along with higher dilation and lower ejection fraction. The decline in cardiac physiology occurred subsequent to increased neutrophil and macrophage infiltration at MI day 1 and increased macrophage infiltration at D3. Our results reveal that S100A9 is a functional effector of infarct wall thinning.

Ecology of Hantaviruses in Rodent Reservoirs and Their Early Innate Immune Responses in Human Model Systems

The spillover of zoonotic RNA viruses is responsible for a great deal of the disease outbreaks in human populations. These spillover events are set to continue due to anthropogenic and environmental changes that impact the distribution of these viruses. The viruses in the family Hantaviridae are classified as one of these emerging zoonotic RNA viruses. The spillover of the viruses in this family are responsible for two severe human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These viruses are distributed across the globe and are responsible for a large number of human disease cases with potentially high mortality rates each year. Unfortunately, there is a lack of surveillance efforts to identify hantaviruses in most countries making accurate diagnosis or recognition of hantavirus cases complicated. To address the potential public health impact of hantaviruses, we surveyed rodents in a rural region of Ukraine, and identified a high prevalence which underscores the potential for human disease in this country. As it is challenging to address how hantaviruses infect humans, I established approaches to evaluate the early innate immune response in primary lung microvascular endothelial cells (HLMVECs) with pathogenic and nonpathogenic hantaviruses. Surprisingly, my findings challenged some of the current dogma in that there were not dramatic difference between pathogenic and nonpathogenic viruses. This work highlights the critical need for advancement of cell culture models to probe the immune response. To understand the ecology of hantaviruses in their reservoirs their prevalence was studied in northwestern Ukraine. A field capture study was conducted at two sites which each had distinct habitats and contained nine capture lines. During this survey, we captured 424 small mammals, consisting of species across three orders. The most abundant species were Myodes glareolus, the bank vole (45%); Apodemus flavicollis, the yellow-necked mouse (29%); and Apodemus agrarius, the striped field mouse (14.6%). Out of the collection, it was determined that 79 animals were seropositive by immunofluorescent assay (IFA), from which 15.7% were M. glareolus, 20.5% A. flavicollis, and 33.9% A. agrarius. These finding were of interest as M. glareolus and Apodemus spp. harbor Puumala orthohantavirus and Dobrava-Belgrade orthohantavirus viruses, respectively, which are responsible for causing HFRS in humans. IFA reciprocal titer showed a wide distribution indicating new infections are occurring. No relationship was found between species diversity and the proportion of hantavirus seropositive animals captured at these sites. Population analysis on M. glareolus and Apodemus spp. revealed that neither sex nor age was associated with being seropositive. To define the early innate immune responses during human infection by hantaviruses, this research studies the responses in HLMVECs, the primary cells of infection in humans, infected by the pathogenic viruses, Andes orthohantavirus (ANDV) and Hantaan orthohantavirus (HTNV), and the nonpathogenic virus, Prospect Hill orthohantavirus (PHV). A curated list of 39 host genes were studied across multiple time points during the first 72 hours of infection of HLMVECs from a male donor by these three viruses. mRNA level analysis revealed the mRNA levels of only CCL5, CXCL10, CXCL11, IDO1, IFNB1, IRF7, and TLR3 we increased during infection of each viruses. The measurement of CCL5, CXCL10, CXCL11, IDO, and IFN-β secreted protein levels in the same HLMVEC donor during infection confirmed gene expression findings. The study of host immune responses to hantavirus infection was expanded to include HLMVECs from an additional male and two female donors. Measurement of secreted protein levels of CCL5, CXCL10, CXCL11, IDO, and IFN-β by each of the four donors revealed that levels of these proteins are upregulated during infection by each of the viruses. Pair wise analysis on these secreted protein levels by each of the donors during hantavirus infection suggests that donor characteristics and virus species together drive different outcomes. However, female donors had higher levels of CXCL10, IDO, and IFN-β and these increased protein levels were species specific. Lastly, the suppression of immune response involved in cell death were examined and it was found that ANDV is capable of inhibiting cell death in HLMVECs. In summary, the findings presented, show the critical need to understand and define the early innate immune responses to hantaviral infection in human models as well as the necessity of understanding the ecology of hantaviruses in their reservoir hosts.

scEMC10, a novel circulating inhibitor of adipocyte thermogenesis, is upregulated in human obesity and its neutralizing antibody prevents diet-induced obesity

Secreted isoform of endoplasmic reticulum membrane complex subunit 10 (scEMC10) is a poorly characterised secreted protein of largely unknown physiological function. Here we demonstrate that scEMC10 is upregulated in humans with obesity and is positively associated with insulin resistance. Consistent with a causal role for scEMC10 in obesity, Emc10-/- mice are resistant to diet-induced obesity due to an increase in energy expenditure. Furthermore, neutralization of circulating scEMC10 using a monoclonal antibody reduces body weight and enhances insulin sensitivity in obese mice. Mechanistically, we provide evidence that scEMC10 binds to the catalytic subunit of PKA and inhibits its stimulatory action on CREB while ablation of EMC10 promotes thermogenesis in adipocytes via activation of the PKA signalling pathway and its downstream targets. Taken together, our data identify scEMC10 as a novel circulating inhibitor of thermogenesis and a potential therapeutic target for obesity and its cardiometabolic complications.

Following the SPARC: tracking Secreted Protein Acidic and Rich in Cysteine associated with LX-2´s extracellular vesicles as a non-destructive modality to evaluate lipid-based antifibrotic treatments

Uncovering the complex cellular mechanisms underlying hepatic fibrogenesis, a highly dynamic and active process ultimately responsible for liver failure if left untreated, could expedite the development of effective treatments and noninvasive diagnostic modalities for this often silent pathology. The biochemical complexity of extracellular vesicles (EVs) and their role in intercellular communication make them an attractive tool to look for biomarkers that might become a viable alternative to invasive liver biopsies. We developed a solid set of methods to isolate and characterize EVs from differently treated human hepatic stellate cell (HSC) line LX-2 in vitro, and we investigated the biological effect they exert onto naïve LX-2, proving that EVs do play an active role in fibrogenesis. Electrical/asymmetric flow field-flow fractionation (EAF4) revealed EV subpopulations with different physicochemical behaviors. Proteomic data from our samples was mined for EV-associated proteins whose expression correlated with HSC treatment. Consequently, we chose the secreted protein acidic and cysteine rich (SPARC), a matricellular protein previously reported to be upregulated in activated HSCs, as a proof-of-concept protein to explore the feasibility of using fluorescence nanoparticle tracking analysis as a non-destructive tool for the determination of HSCs’ fibrogenic phenotype based on EVs. We could thus use EVs to directly evaluate the efficacy of treatment with S80, a lipid rich (>75 %) in polyenylphosphatidylcholines (PPC). We found that PPC-rich S80 reduces the relative presence of SPARC-positive EVs. For the first time, we could correlate the cellular response to lipid-based antifibrotic treatment to the relative presence of a candidate protein marker associated with the released EVs. In addition to providing novel insights into PPC treatments, our findings pave the way for more precise and less invasive diagnostic analyses of hepatic fibrogenesis.

Measuring Exercise-Induced Secreted Protein Acidic and Rich in Cysteine Expression as a Molecular Tool to Optimize Personalized Medicine

The numerous exercise benefits for health as well as applications for diseases has lead to exercise being prescribed in many pathological conditions. Secreted protein acidic and rich in cysteine (SPARC) gene expression is stimulated by exercise and SPARC has been suggested as a molecular mediator of exercise. Therefore, we suggest using this property for personalized medicine. This can be achieved by prescribing the exercise with a pattern (duration, intensity, etc.) that corresponds to the optimum SPARC/Sparc expression. We expect this approach to optimize the exercise therapy in both the preventive and curative contexts. In the research field, measuring exercise -dependent expression of Sparc would represent a molecular tool to further optimize the selection of exercise animal models as well.