The Enhanced Expression of Cruzipain-Like Molecules in the Phytoflagellate Phytomonas serpens Recovered From the Invertebrate and Plant Hosts

Phytomonas serpens is a protozoan parasite that alternates its life cycle between two hosts: an invertebrate vector and the tomato fruit. This phytoflagellate is able to synthesize proteins displaying similarity to the cysteine peptidase named cruzipain, an important virulence factor from Trypanosoma cruzi, the etiologic agent of Chagas disease. Herein, the growth of P. serpens in complex medium (BHI) supplemented with natural tomato extract (NTE) resulted in the increased expression of cysteine peptidases, as verified by the hydrolysis of the fluorogenic substrate Z-Phe-Arg-AMC and by gelatin-SDS-PAGE. Phytoflagellates showed no changes in morphology, morphometry and viability, but the proliferation was slightly reduced when cultivated in the presence of NTE. The enhanced proteolytic activity was accompanied by a significant increase in the expression of cruzipain-like molecules, as verified by flow cytometry using anti-cruzipain antibodies. In parallel, parasites incubated under chemically defined conditions (PBS supplemented with glucose) and added of different concentration of NTE revealed an augmentation in the production of cruzipain-like molecules in a typically dose-dependent way. Similarly, P. serpens recovered from the infection of mature tomatoes showed an increase in the expression of molecules homologous to cruzipain; however, cells showed a smaller size compared to parasites grown in BHI medium. Furthermore, phytoflagellates incubated with dissected salivary glands from Oncopeltus fasciatus or recovered from the hemolymph of infected insects also showed a strong enhance in the expression of cruzipain-like molecules that is more relevant in the hemolymph. Collectively, our results showed that cysteine peptidases displaying similarities to cruzipain are more expressed during the life cycle of the phytoflagellate P. serpens both in the invertebrate and plant hosts.

NuA4 and H2A.Z control environmental responses and autotrophic growth in Arabidopsis

Nucleosomal acetyltransferase of H4 (NuA4) is an essential transcriptional coactivator in eukaryotes, but remains poorly characterized in plants. Here, we describe Arabidopsis homologs of the NuA4 scaffold proteins Enhancer of Polycomb-Like 1 (AtEPL1) and Esa1-Associated Factor 1 (AtEAF1). Loss of AtEAF1 results in inhibition of growth and chloroplast development. These effects are stronger in the Atepl1 mutant and are further enhanced by loss of Golden2-Like (GLK) transcription factors, suggesting that NuA4 activates nuclear plastid genes alongside GLK. We demonstrate that AtEPL1 is necessary for nucleosomal acetylation of histones H4 and H2A.Z by NuA4 in vitro. These chromatin marks are diminished genome-wide in Atepl1, while another active chromatin mark, H3K9 acetylation (H3K9ac), is locally enhanced. Expression of many chloroplast-related genes depends on NuA4, as they are downregulated with loss of H4ac and H2A.Zac. Finally, we demonstrate that NuA4 promotes H2A.Z deposition and by doing so prevents spurious activation of stress response genes.

ACSL4 Directs Intramuscular Adipogenesis and Fatty Acid Composition in Pigs

The intramuscular fat is a major quality trait of meat, affecting sensory attributes such as flavor and texture. Several previous GWAS studies identified Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) gene as the candidate gene to regulate intramuscular fat content in different pig populations, but the underlying molecular function of ACSL4 in adipogenesis within pig skeletal muscle is not fully investigated. In this study, we isolated porcine endogenous intramuscular adipocyte progenitors and performed ACSL4 loss- and gain-of-function experiments during adipogenic differentiation. Our data showed that ACSL4 is a positive regulator of adipogenesis in intramuscular fat cells isolated from pigs. More interestingly, the enhanced expression of ACSL4 in pig intramuscular adipocytes could increase the cellular content of monounsaturated and polyunsaturated fatty acids, such as gamma-L eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA). The above results not only confirmed the function of ACSL4 in pig intramuscular adipogenesis and meat quality attributes, but also provided new clues for the improvement of the nutritional value of pork for human health.

Targeting Oxidative Phosphorylation-Proteasome Activity in Extracellular Detached Cells Promotes Anoikis and Inhibits Metastasis

Metastasis arises owing to tumor cells’ capacity to evade pro-apoptotic signals. Anoikis—the apoptosis of detached cells (from the extracellular matrix (ECM)) is often circumvented by metastatic cells as a result of biochemical and molecular transformations. These facilitate cells’ ability to survive, invade and reattach to secondary sites. Here, we identified deregulated glucose metabolism, oxidative phosphorylation, and proteasome in anchorage-independent cells compared to adherent cells. Metformin an anti-diabetic drug that reduces blood glucose (also known to inhibit mitochondrial Complex I), and proteasome inhibitors were employed to target these changes. Metformin or proteasome inhibitors alone increased misfolded protein accumulation, sensitized tumor cells to anoikis, and impaired pulmonary metastasis in the B16F10 melanoma model. Mechanistically, metformin reduced cellular ATP production, activated AMPK to foster pro-apoptotic unfolded protein response (UPR) through enhanced expression of CHOP in ECM detached cells. Furthermore, AMPK inhibition reduced misfolded protein accumulation, thus highlight relevance of AMPK activation in facilitating metformin-induced stress and UPR cell death. Our findings provide insights into the molecular biology of anoikis resistance and identified metformin and proteasome inhibitors as potential therapeutic options for tumor metastasis.

Graphene Oxide reinforced Agarose-Hydroxyapatite Bioprinted 3-D Scaffolds for Bone Regeneration

Three-dimensional (3D) bioprinting is an emerging technology for fabricating cells, biomaterials and extracellular matrix (ECM) into customized shapes and patterns. Here, we report additive manufacturing to create a customized 3D bioactive constructs for regenerative medicine. We have attempted to emphasize the use of agarose and graphene oxide as a promising material for the conceptualization of bioink unpaid to its unique physicochemical properties. The 3D printed structure is able to regenerating bone tissues and regulates the cellular differentiation without any significant morphological changes. The presence of graphene oxide enhances the osteoinductive behavior of the developed scaffolds, which is further supplemented by encapsulating human mesenchymal stem cells (hMSCs) on the 3D printed scaffolds. A significant enhanced expression of early osteogenic markers like morphogenetic protein (BMP), Runx-2, collagen-1, osteopontin, osteocalcin as well as mineralized ECM are observed on agarose-hydroxyapatite and graphene oxide 3D printed scaffolds compared to agarose-hydroxyapatite 3D printed scaffolds. Thus, the outcomes of the developed 3D bioprinted scaffolds provide a promising strategy for development of personalized bone grafts for tissue regeneration.

Proteasome α6 Subunit Negatively Regulates the JAK/STAT Pathway and Blood Cell Activation in Drosophila melanogaster

JAK/STAT signaling regulates central biological functions such as development, cell differentiation and immune responses. In Drosophila, misregulated JAK/STAT signaling in blood cells (hemocytes) induces their aberrant activation. Using mass spectrometry to analyze proteins associated with a negative regulator of the JAK/STAT pathway, and by performing a genome-wide RNAi screen, we identified several components of the proteasome complex as negative regulators of JAK/STAT signaling in Drosophila. A selected proteasome component, Prosα6, was studied further. In S2 cells, Prosα6 silencing decreased the amount of the known negative regulator of the pathway, ET, leading to enhanced expression of a JAK/STAT pathway reporter gene. Silencing of Prosα6 in vivo resulted in activation of the JAK/STAT pathway, leading to the formation of lamellocytes, a specific hemocyte type indicative of hemocyte activation. This hemocyte phenotype could be partially rescued by simultaneous knockdown of either the Drosophila STAT transcription factor, or MAPKK in the JNK-pathway. Our results suggest a role for the proteasome complex components in the JAK/STAT pathway in Drosophila blood cells both in vitro and in vivo.

A tandem duplication in Drosophila melanogaster shows enhanced expression beyond the gene copy number

Tandem duplicated genes are common features of genomes, but the phenotypic consequences of their origins are not well understood. It is not known whether a simple doubling of gene expression should be expected, or else some other expression outcome. This study describes an experimental framework using engineered deletions to assess any contribution of locally-acting cis- and globally-acting trans-regulatory factors to expression interactions of particular tandem duplicated genes. Acsx1L (CG6300) and Acsx1R (CG11659) are tandem duplicates of a putative acyl-CoA synthetase gene found in D. melanogaster. Experimental deletions of the duplicated segments were used to investigate whether the presence of one tandem duplicated block influences the expression of its neighbor. Acsx1L, the gene in the left block, shows much higher expression than either its duplicate Acsx1R or the single Acsx1 in D. simulans. Acsx1L expression decreases drastically upon deleting the right-hand duplicated block. Crosses among wildtype and deletion strains show that high tandem expression is primarily due to cis-acting interactions between the duplicated blocks. No effect of these genes on cuticular hydrocarbons was detected. Sequence and phylogenetic analysis suggest that the duplication rose to fixation in D. melanogaster and has been subject to extensive gene conversion. Some strains actually carry three tandem copies, yet strains with three Acsx1s do not have higher expression levels than strains with two. Surveys of tandem duplicate expression have typically not found the expected twofold increase in expression. This study suggests that cis-regulatory interactions between duplicated blocks could be responsible for this trend.

MiR-126-HMGB1-HIF-1 Axis Regulates Endothelial Cell Inflammation during Exposure to Hypoxia-Acidosis

Crosstalk between molecular regulators miR-126, hypoxia-inducible factor 1-alpha (HIF-1-α), and high-mobility group box-1 (HMGB1) contributes to the regulation of inflammation and angiogenesis in multiple physiological and pathophysiological settings. Here, we present evidence of an overriding role for miR-126 in the regulation of HMGB1 and its downstream proinflammatory effectors in endothelial cells subjected to hypoxia with concurrent acidosis (H/A). Methods. Primary mouse endothelial cells (PMEC) were exposed to hypoxia or H/A to simulate short or chronic low-flow ischemia, respectively. RT-qPCR quantified mRNA transcripts, and proteins were measured by western blot. ROS were quantified by fluorogenic ELISA and luciferase reporter assays employed to confirm an active miR-126 target in the HMGB1 3 ′ UTR. Results. Enhanced expression of miR-126 in PMECs cultured under neutral hypoxia was suppressed under H/A, whereas the HMGB1 expression increased sequentially under both conditions. Enhanced expression of HMGB1 and downstream inflammation markers was blocked by the premiR-126 overexpression and optimized by antagomiR. Compared with neutral hypoxia, H/A suppressed the HIF-1α expression independently of miR-126. The results show that HMGB1 and downstream effectors are optimally induced by H/A relative to neutral hypoxia via crosstalk between hypoxia signaling, miR-126, and HIF-1α, whereas B-cell lymphoma 2(Bcl2), a HIF-1α, and miR-126 regulated gene expressed optimally under neutral hypoxia. Conclusion. Inflammatory responses of ECs to H/A are dynamically regulated by the combined actions of hypoxia, miR-126, and HIF-1α on the master regulator HMGB1. The findings may be relevant to vascular diseases including atherosclerotic occlusion and interiors of plaque where coexisting hypoxia and acidosis promote inflammation as a defining etiology.

The Distinct Roles of Sialyltransferases in Cancer Biology and Onco-Immunology

Aberrant glycosylation is a key feature of malignant transformation. Hypersialylation, the enhanced expression of sialic acid-terminated glycoconjugates on the cell surface, has been linked to immune evasion and metastatic spread, eventually by interaction with sialoglycan-binding lectins, including Siglecs and selectins. The biosynthesis of tumor-associated sialoglycans involves sialyltransferases, which are differentially expressed in cancer cells. In this review article, we provide an overview of the twenty human sialyltransferases and their roles in cancer biology and immunity. A better understanding of the individual contribution of select sialyltransferases to the tumor sialome may lead to more personalized strategies for the treatment of cancer.