Sex-specific effect of antenatal Zika virus infection on murine fetal growth, placental nutrient transporters, and nutrient sensor signaling pathways.

Maternal Zika virus (ZIKV) infection during pregnancy has been associated with severe intrauterine growth restriction (IUGR), placental damage, metabolism disturbances, and newborn neurological abnormalities. Here, we investigated the impact of maternal ZIKV infection on placental nutrient transporters and nutrient-sensitive pathways. Immunocompetent (C57BL/6) mice were injected with Low (103 PFU-ZIKVPE243) or High (5 × 107 PFU-ZIKVPE243) ZIKV titers at gestational day (GD) 12.5, and tissue was collected at GD18.5 (term). Fetal-placental growth was impaired in male fetuses, which exhibited higher placental expression of the ZIKV infective marker, eukaryotic translation initiation factor 2 (eIF2α), but lower levels of phospho-eIF2α. There were no differences in fetal-placental growth in female fetuses, which exhibited no significant alterations in placental ZIKV infective markers. Furthermore, ZIKV promoted increased expression of glucose transporter type 1 (Slc2a1/Glut1) and decreased levels of glucose-6-phosphate in female placentae, with no differences in amino acid transport potential. In contrast, ZIKV did not impact glucose transporters in male placentae but downregulated sodium-coupled neutral amino acid 2 (Snat2) transporter expression. We also observed sex-dependent differences in the hexosamine biosynthesis pathway (HBP) and O-GlcNAcylation in ZIKV-infected pregnancies, showing that ZIKV can disturb placental nutrient sensing. Our findings highlight molecular alterations in the placenta caused by maternal ZIKV infection, shedding light on nutrient transport, sensing, and availability. Our results also suggest that female and male placentae employ distinct coping mechanisms in response to ZIKV-induced metabolic changes, providing insights into therapeutic approaches for congenital Zika syndrome.

Interfacing manganese-based carbonaceous nanocomposites with plasma components: insights on protein interaction, structure and opsonization.

Metal encapsulation delivers a straightforward strategy to improve miscellaneous nanoparticle properties and qualifies the resulting nanocomposite for exceptional application, including bioimaging, drug release, and theranostic development. Besides crucial applications, investigations associated with the nanocomposite impact on the biological media are highly relevant from a pharmacological viewpoint. Such studies can be conducted by exploring nanocomposite attributes and all aspects of their interaction with proteins existing in biofluids. Based on these aspects, the present work examines manganese-encapsulated carbonaceous nanocomposite (MnCQD) and their interaction with plasma proteins. On one side, the obtained nanocomposite has almost spherical shapes (≈12 nm in size), an appropriate composition and interesting optical properties for bioimaging applications. On another side, MnCQD quenches the fluorescence of two plasma proteins (BSA and HTF) following a static mechanism, confirming the formation of the MnCQD-BSA and MnCQD-HTF complexes. Although hydrophobic forces guide the stability of both formed complexes, MnCQD binds preferentially to BSA compared to HTF, with affinity constants differing by almost an order of magnitude. Furthermore, HTF and BSA underwent modifications in their secondary structure provoked due to contact with the nanocomposite, which also presented neglectable opsonization levels when exposed to appropriate biological media. These results highlight the MnCQD outstanding potential to be employed in diverse bioapplications.Communicated by Ramaswamy H. Sarma.

Inhibition of O-GlcNAcylation Reduces Cell Viability and Autophagy and Increases Sensitivity to Chemotherapeutic Temozolomide in Glioblastoma.

Glioblastoma (GB) is the most aggressive primary malignant brain tumor and is associated with short survival. O-GlcNAcylation is an intracellular glycosylation that regulates protein function, enzymatic activity, protein stability, and subcellular localization. Aberrant O-GlcNAcylation is related to the tumorigenesis of different tumors, and mounting evidence supports O-GlcNAc transferase (OGT) as a potential therapeutic target. Here, we used two human GB cell lines alongside primary human astrocytes as a non-tumoral control to investigate the role of O-GlcNAcylation in cell proliferation, cell cycle, autophagy, and cell death. We observed that hyper O-GlcNAcylation promoted increased cellular proliferation, independent of alterations in the cell cycle, through the activation of autophagy. On the other hand, hypo O-GlcNAcylation inhibited autophagy, promoted cell death by apoptosis, and reduced cell proliferation. In addition, the decrease in O-GlcNAcylation sensitized GB cells to the chemotherapeutic temozolomide (TMZ) without affecting human astrocytes. Combined, these results indicated a role for O-GlcNAcylation in governing cell proliferation, autophagy, cell death, and TMZ response, thereby indicating possible therapeutic implications for treating GB. These findings pave the way for further research and the development of novel treatment approaches which may contribute to improved outcomes and increased survival rates for patients facing this challenging disease.

Development and clinical application of hydrogel formulations containing papain and urea for wound healing

Abstract Hydrogels are used for wound treatment, as they may contain one or more active components and protect the wound bed. Papain is one of the active substances that have been used with this purpose, alongside urea. In this paper, carboxypolymethylene hydrogels containing papain (2% and 10% concentrations) and urea (5% concentration) were produced. Physical-chemical stability was performed at 0, 7, 15 and 30 days at 2-8ºC, 25ºC and 40ºC, as well as the rheological aspects and proteolytic activity of papain by gel electrophoresis. Clinical efficacy of the formulations in patients with lower limb ulcers was also evaluated in a prospective, single-center, randomized, double-blind and comparative clinical trial. The results showed 7-day stability for the formulations under 25ºC, in addition to approximately 100% and 15% of protein activity for 10% and 2% papain hydrogel, respectively. The rheological profile was non-Newtonian for the 10% papain hydrogel tested. There were no significant differences regarding the mean time for healing of the lesions, although 10% papain presented a better approach to be used in all types of tissue present in the wound bed.

BDE-209 and TCDD enhance metastatic characteristics of melanoma cells after chronic exposure.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and BDE-209 (decabromodiphenyl ether) are persistent organic pollutants (POPs) produced by industrial activities and associated with several diseases. TCDD is a known human carcinogen, but few studies investigated about the effects of exposure to both compounds, i.e., whether BDE-209 and TCDD can render tumor cells more aggressive and metastatic. In the current study we investigated if the exposure of B16-F1 and B16-F10 melanoma murine cells to environmental relevant concentrations of TCDD and BDE-209 at 24 h and 15-day exposure modulates the expression of genes related to metastasis, making the cells more aggressive. Both pollutants did not affect cell viability but lead to increase of cell proliferation, including the upregulation of vimentin, MMP2, MMP9, MMP14 and PGK1 gene expression and downregulation of E-cadherin, TIMP2, TIMP3 and RECK, strongly suggesting changes in cell phenotypes defined as epithelial to mesenchymal transition (EMT) in BDE-209 and TCDD-exposed cells. Foremost, increased expression of metalloproteinases and decreased expression of their inhibitors made B16-F1 cells similar the more aggressive B16-F10 cell line. Also, the higher secretion of extracellular vesicles by cells after acute exposure to BDE-209 could be related with the phenotype changes. These results are a strong indication of the potential of BDE-209 and TCDD to modulate cell phenotype, leading to a more aggressive profile.

GM2/GM3 controls the organizational status of CD82/Met microdomains: further studies in GM2/GM3 complexation.

At cell surface gangliosides might associate with signal transducers proteins, grown factor receptors, integrins, small G-proteins and tetraspanins establishing microdomains, which play important role in cell adhesion, cell activation, motility, and growth. Previously, we reported that GM2 and GM3 form a heterodimer that interacts with the tetraspanin CD82, controlling epithelial cell mobility by inhibiting integrin-hepatocyte growth factor-induced cMet tyrosine kinase signaling. By using molecular dynamics simulations to study the molecular basis of GM2/GM3 interaction we demonstrate, here, that intracellular levels of Ca2+ mediate GM2/GM3 complexation via electrostatic interaction with their carboxyl groups, while hydrogen bonds between the ceramide groups likely aid stabilizing the complex. The presence of GM2/GM3 complex alters localization of CD82 on cell surface and therefore downstream signalization. These data contribute for the knowledge of how glycosylation may control signal transduction and phenotypic changes.

Enzymatic and structural properties of human glutamine:fructose-6-phosphate amidotransferase 2 (hGFAT2).

Glycoconjugates play a central role in several cellular processes, and alteration in their composition is associated with numerous human pathologies. Substrates for cellular glycosylation are synthesized in the hexosamine biosynthetic pathway, which is controlled by the glutamine:fructose-6-phosphate amidotransfera-se (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer; however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in Escherichia coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the expected ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much more slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerize fructose-6-phosphate into glucose-6-phosphate even in the presence of equimolar amounts of glutamine, which results in unproductive glutamine hydrolysis. Structural analysis of a three-dimensional model of rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in the glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations suggest that this loop is the most flexible portion of the protein and plays a key role on conformational states of hGFAT2. Thus, our study provides the first comprehensive set of data on the structure, kinetics, and mechanics of hGFAT2, which will certainly contribute to further studies on the (patho)physiology of hGFAT2.

The emerging role of neutrophil extracellular traps in severe acute respiratory syndrome coronavirus 2 (COVID-19).

The novel coronavirus SARS-CoV-2 causes COVID-19, a highly pathogenic viral infection threatening millions. The majority of the individuals infected are asymptomatic or mildly symptomatic showing typical clinical signs of common cold. However, approximately 20% of the patients can progress to acute respiratory distress syndrome (ARDS), evolving to death in about 5% of cases. Recently, angiotensin-converting enzyme 2 (ACE2) has been shown to be a functional receptor for virus entry into host target cells. The upregulation of ACE2 in patients with comorbidities may represent a propensity for increased viral load and spreading of infection to extrapulmonary tissues. This systemic infection is associated with higher neutrophil to lymphocyte ratio in infected tissues and high levels of pro-inflammatory cytokines leading to an extensive microthrombus formation with multiorgan failure. Herein we investigated whether SARS-CoV-2 can stimulate extracellular neutrophils traps (NETs) in a process called NETosis. We demonstrated for the first time that SARS-CoV-2 in fact is able to activate NETosis in human neutrophils. Our findings indicated that this process is associated with increased levels of intracellular Reactive Oxygen Species (ROS) in neutrophils. The ROS-NET pathway plays a role in thrombosis formation and our study suggest the importance of this target for therapy approaches against disease.

Hyperglycemia Enhances Cancer Immune Evasion by Inducing Alternative Macrophage Polarization through Increased O-GlcNAcylation.

Diabetes mellitus (DM) significantly increases the risk for cancer and cancer progression. Hyperglycemia is the defining characteristic of DM and tightly correlates with a poor prognosis in patients with cancer. The hexosamine biosynthetic pathway (HBP) is emerging as a pivotal cascade linking high glucose, tumor progression, and impaired immune function. Here we show that enhanced glucose flow through the HBP drives cancer progression and immune evasion by increasing O-GlcNAcylation in tumor-associated macrophages (TAM). Increased O-GlcNAc skewed macrophage polarization to a M2-like phenotype supporting tumor progression. Finally, we found an upregulation of M2 markers on TAMs in DM2 patients with colorectal cancer compared with nondiabetic normoglycemic patients. Our results provide evidence for a new and targetable mechanism of cancer immune evasion in patients with hyperglycemia, advocating for strict control of hyperglycemia in patients with cancer.

Biological evaluation and molecular modeling of peptidomimetic compounds as inhibitors for O-GlcNAc transferase (OGT).

The vital enzyme O-linked ß-N-acetylglucosamine transferase (OGT) catalyzes the O-GlcNAcylation of intracellular proteins coupling the metabolic status to cellular signaling and transcription pathways. Aberrant levels of O-GlcNAc and OGT have been linked to metabolic diseases as cancer and diabetes. Here, a new series of peptidomimetic OGT inhibitors was identified highlighting the compound LQMed 330, which presented better IC50 compared to the most potent inhibitors found in the literature. Molecular modeling study of selected inhibitors into the OGT binding site provided insight into the behavior by which these compounds interact with the enzyme. The results obtained in this study provided new perspectives on the design and synthesis of highly specific OGT inhibitors.

Trends in Nanomedicines for Cancer Treatment.

BACKGROUND: Cancer is characterized by abnormal cell growth and considered one of the leading causes of death around the world. Pharmaceutical Nanotechnology has been extensively studied for the optimization of cancer treatment. OBJECTIVE: Comprehend the panorama of Pharmaceutical Nanotechnology in cancer treatment, through a survey about nanomedicines applied in clinical studies, approved for use and patented. METHODS: Acknowledged products under clinical study and nanomedicines commercialized found in scientific articles through research on the following databases: Pubmed, Science Direct, Scielo and Lilacs. Derwent tool was used for patent research. RESULTS: Nanomedicines based on nanoparticles, polymer micelles, liposomes, dendrimers and nanoemulsions were studied, along with cancer therapies such as Photodynamic Therapy, Infrared Phototherapy Hyperthermia, Magnetic Hyperthermia, Radiotherapy, Gene Therapy and Nanoimmunotherapy. Great advancement has been observed over nanotechnology applied to cancer treatment, mainly for nanoparticles and liposomes. CONCLUSION: The combination of drugs in nanosystems helps to increase efficacy and decrease toxicity. Based on the results encountered, nanoparticles and liposomes were the most commonly used nanocarriers for drug encapsulation. In addition, although few nanomedicines are commercially available, this specific research field is continuously growing.

Trends in Nanotechnology for in vivo Cancer Diagnosis: Products and Patents.

BACKGROUND: Cancer is a set of diseases formed by abnormal growth of cells leading to the formation of the tumor. The diagnosis can be made through symptoms' evaluation or imaging tests, however, the techniques are limited and the tumor detection may be late. Thus, pharmaceutical nanotechnology has emerged to optimize the cancer diagnosis through nanostructured contrast agent's development. OBJECTIVE: This review aims to identify commercialized nanomedicines and patents for cancer diagnosis. METHODS: The databases used for scientific articles research were Pubmed, Science Direct, Scielo and Lilacs. Research on companies' websites and articles for the recognition of commercial nanomedicines was performed. The Derwent tool was applied for patent research. RESULTS: This article aimed to research on nanosystems based on nanoparticles, dendrimers, liposomes, composites and quantum dots, associated to imaging techniques. Commercialized products based on metal and composite nanoparticles, associated with magnetic resonance and computed tomography, have been observed. The research conducted through Derwent tool displayed a small number of patents using nanotechnology for cancer diagnosis. Among these patents, the most significant number was related to the use of systems based on metal nanoparticles, composites and quantum dots. CONCLUSION: Although few systems are found in the market and patented, nanotechnology appears as a promising field for the development of new nanosystems in order to optimize and accelerate the cancer diagnosis.

CD43 sialoglycoprotein modulates cardiac inflammation and murine susceptibility to Trypanosoma cruzi infection.

CD43 (leukosialin) is a large sialoglycoprotein abundantly expressed on the surface of most cells from the hematopoietic lineage. CD43 is directly involved in the contact between cells participating in a series of events such as signaling, adherence and host parasite interactions. In this study we examined the role of CD43 in the immune response against Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, a potential life-threatening illness endemic in 21 Latin American countries according to the WHO. The acute stage of infection is marked by intense parasitemia and cardiac tissue parasitism, resulting in the recruitment of inflammatory cells and acute damage to the heart tissue. We show here that CD43-/- mice were more resistant to infection due to increased cytotoxicity of antigen specific CD8+ T cells and reduced inflammatory infiltration in the cardiac tissue, both contributing to lower cardiomyocyte damage. In addition, we demonstrate that the induction of acute myocarditis involves the engagement of CD43 cytoplasmic tripeptide sequence KRR to ezrin-radixin-moiesin cytoskeletal proteins. Together, our results show the participation of CD43 in different events involved in the pathogenesis of T. cruzi infection, contributing to a better overall understanding of the mechanisms underlying the pathogenesis of acute chagasic cardiomyopathy.

Emerging role of glycosylation in the polarization of tumor-associated macrophages.

Tumors are formed by several cell types interacting in a complex environment of soluble and matrix molecules. The crosstalk between the cells and extracellular components control tumor fate. Macrophages are highly plastic and diverse immune cells that are known to be key regulators of this complex network, which is mostly because they can adjust their metabolism and reprogram their phenotype and effector function. Here, we review the studies that disclose the central role of metabolism and tumor microenvironment in shaping the phenotype and function of macrophages, highlighting the importance of the hexosamine biosynthetic pathway. We further discuss growing evidence of nutrient-sensitive protein modifications such as O-GlcNAcylation and extracellular glycosylation in the function and polarization of tumor-associated macrophages.

Targeting the Hexosamine Biosynthetic Pathway Prevents Plasmodium Developmental Cycle and Disease Pathology in Vertebrate Host.

Cerebral malaria (CM) is a clinical syndrome involving irreversible and lethal signs of brain injury associated to infection by parasites of the genus Plasmodium. The pathogenesis of CM derives from infection-induced proinflammatory cytokines associated with cytoadherence of parasitized red blood cells to brain microvasculature. Glycoconjugates are very abundant in the surface of Plasmodium spp., and are critical mediators of parasite virulence in host-pathogen interactions. Herein, we show that 6-Diazo-5-oxo-L-norleucine (DON) therapeutically used for blocking hexosamine biosynthetic pathway leads to recovery in experimental murine cerebral malaria. DON-induced protection was associated with decreased parasitism, which severely reduced Plasmodium transmission to mosquitoes. These findings point to a potential use of DON in combination therapies against malaria.

Hexosamine Biosynthetic Pathway and Glycosylation Regulate Cell Migration in Melanoma Cells.

The Hexosamine Biosynthetic Pathway (HBP) is a branch of glycolysis responsible for the production of a key substrate for protein glycosylation, UDP-GlcNAc. Cancer cells present altered glucose metabolism and aberrant glycosylation, pointing to alterations on HBP. Recently it was demonstrated that HBP influences many aspects of tumor biology, including the development of metastasis. In this work we characterize HBP in melanoma cells and analyze its importance to cellular processes related to the metastatic phenotype. We demonstrate that an increase in HBP flux, as well as increased O-GlcNAcylation, leads to decreased cell motility and migration in melanoma cells. In addition, inhibition of N- and O-glycosylation glycosylation reduces cell migration. High HBP flux and inhibition of N-glycosylation decrease the activity of metalloproteases 2 and 9. Our data demonstrates that modulation of HBP and different types of glycosylation impact cell migration.

Duffy binding-like 1α adhesin from Plasmodium falciparum recognizes ABH histo-blood group saccharide in a type specific manner.

The ability of erythrocytes, infected by Plasmodium falciparum, to adhere to endothelial cells (cytoadherence) and to capture uninfected erythrocyte (rosetting) is the leading cause of death by severe malaria. Evidences link the binding of the adhesin Duffy Binding Like1-α (DBL1α) domain to the ABH histo-blood antigens with formation of rosettes. Inspired by this very close relationship between the disease susceptibility and individual blood type, here we investigate the structural requirements involved in the interaction of DBL1α with A, B and H histo-blood determinants and their subtypes. Our results evidence the high preference of DBL1α to A epitopes, in comparison to B and H epitopes. DBL1α interacts with ABH epitopes in subtype specific manner, presenting a remarkable affinity for type 2 structures, Fucα1-2Galß1-4GlcNAcß1, particularly the A2 epitope. The contacts made by DBL1α binding pocket and the ABH histo-blood groups were mapped by theoretical methods and supported by NMR experiments.

N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs.

Disorders of N-linked glycosylation are increasingly reported in the literature. However, the targets that are responsible for the associated developmental and physiological defects are largely unknown. Bone morphogenetic proteins (BMPs) act as highly dynamic complexes to regulate several functions during development. The range and strength of BMP activity depend on interactions with glycosylated protein complexes in the extracellular milieu. Here, we investigate the role of glycosylation for the function of the conserved extracellular BMP antagonist Short gastrulation (Sog). We identify conserved N-glycosylated sites and describe the effect of mutating these residues on BMP pathway activity in Drosophila Functional analysis reveals that loss of individual Sog glycosylation sites enhances BMP antagonism and/or increases the spatial range of Sog effects in the tissue. Mechanistically, we provide evidence that N-terminal and stem glycosylation controls extracellular Sog levels and distribution. The identification of similar residues in vertebrate Chordin proteins suggests that N-glycosylation may be an evolutionarily conserved process that adds complexity to the regulation of BMP activity.

Prevalence of IgG Autoantibodies against GD3 Ganglioside in Acute Zika Virus Infection.

Zika virus (ZIKV) disease has become a global health emergency with devastating effects on public health. Recent evidences implicate the virus as an emergent neuropathological agent promoting serious pathologies of the human nervous system, that include destructive and malformation consequences such as development of ocular and fetal brain lesions, microcephaly in neonates, and Guillain-Barré syndrome (GBS) in adults. These neurological disorders of both central and peripheral nervous systems are thought to be associated to the neurotropic properties of the virus that has ability to infect neural stem cells as well as peripheral neurons, a hallmark of its pathogenicity. The presence of autoantibodies against gangliosides plays a pivotal role in the etiogenesis of GBS and a variety of neurological disorders. Gangliosides are a class of galactose-containing cerebrosides mainly expressed in nervous system tissues playing a critical role in the physiology of neural cells and neurogenesis. Herein, our findings indicate that patients at acute phase of ZIKV infection without any neurological signs show increased levels of IgG autoantibody against GD3 gangliosides, a class of glycolipid found to be highly expressed in neural stem cell acting in the maintenance of their self-renewal cellular capacity. It is possible that a pathological threshold of these antibodies is only acquired in secondary or subsequent infections. In the light of these evidences, we propose that the target of GD3 by autoimmune responses may possibly has an effect in the neuropathy and neurogenesis disorder seen during ZIKV infection.