Longitudinal study comparing IgG antibodies induced by heterologous prime-boost COVID-19 vaccine.

Vaccines are critical cost-effective tools to control the COVID-19 pandemic. The heterologous prime-boost vaccination has been used by many countries to overcome supply issues, so the effectiveness and safety of this strategy need to be better clarified. This study aims to verify the effect of heterologous prime-boost COVID-19 vaccination on healthcare professionals from Dante Pazzanese Hospital in Brazil. It was performed serological assays of vaccinated individuals after 2-dose of CoronaVac (Sinovac; n = 89) or ChAdOx1 nCoV-19 (Oxford-AstraZeneca; n = 166) followed by a BNT162b2 booster (Pfizer-BioNTech; n = 255). The serum antibodies anti-S (spike), anti-N (nucleocapsid), and anti-RBD (receptor binding domain) were assessed by enzyme-linked immunosorbent assay. The heterologous booster dose induced a 10-fold higher anti-Spike antibody regardless of the 2-dose of a prime vaccine. It was strikingly observed that BNT162b2 enhanced levels of anti-spike antibodies, even in those individuals who did not previously respond to the 2-dose of CoronaVac. In conclusion, the heterologous scheme of vaccination using mRNA as a booster vaccine efficiently enhanced the antibody response against SARS-CoV-2, especially benefiting those elderly who were seronegative with a virus-inactivated vaccine.

Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding.

The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

Inhibition of Epithelial-Mesenchymal Transition and Metastasis by Combined TGFbeta Knockdown and Metformin Treatment in a Canine Mammary Cancer Xenograft Model.

Epithelial mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties, generating metastases. Transforming growth factor beta (TGF-ß) is associated with this malignancy by having the ability to induce EMT. Metformin, has been shown to inhibit EMT in breast cancer cells. Based on this evidence we hypothesize that treatment with metformin and the silencing of TGF-ß, inhibits the EMT in cancer cells. Canine metastatic mammary tumor cell line CF41 was stably transduced with a shRNA-lentivirus, reducing expression level of TGF-ß1. This was combined with metformin treatment, to look at effects on cell migration and the expression of EMT markers. For in vivo study, unmodified or TGF-ß1sh cells were injected in the inguinal region of nude athymic female mice followed by metformin treatment. The mice's lungs were collected and metastatic nodules were subsequently assessed for EMT markers expression. The migration rate was lower in TGF-ß1sh cells and when combined with metformin treatment. Metformin treatment reduced N-cadherin and increased E-cadherin expression in both CF41 and TGF-ß1sh cells. Was demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-ß1sh tumors. This paralleled a decreased N-cadherin and vimentin expression, and increased E-cadherin and claudin-7 expression in lung metastases. This study confirms the benefits of TGF-ß1 silencing in addition to metformin as potential therapeutic agents for breast cancer patients, by blocking EMT process. To the best of our knowledge, we are the first to report metformin treatment in cells with TGF-ß1 silencing and their effect on EMT.

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor.

The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD and 15N-HSQC NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target.

Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos.

Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.

Extracellular vesicles produced by immunomodulatory cells harboring OX40 ligand and 4-1BB ligand enhance antitumor immunity.

Genetically modified tumor cells harboring immunomodulators may be used as therapeutic vaccines to stimulate antitumor immunity. The therapeutic benefit of these tumor vaccines is extensively investigated and mechanisms by which they boost antitumor response may be further explored. Tumor cells are large secretors of extracellular vesicles (EVs). These EVs are able to vehiculate RNA and proteins to target cells, and engineered EVs also vehiculate recombinant proteins. In this study, we explore immunomodulatory properties of EVs derived from antitumor vaccines expressing the TNFSF ligands 4-1BBL and OX40L, modulating immune response mediated by immune cells and eliminating tumors. Our results suggest that the EVs secreted by genetically modified tumor cells harboring TNFSF ligands can induce T cell proliferation, inhibit the transcription factor FoxP3, associated with the maintenance of Treg phenotype, and enhance antitumor activity mediated by immune cells. The immunomodulatory extracellular vesicles have potential to be further engineered for developing new approaches for cancer therapy.

GLS2 is protumorigenic in breast cancers.

Many types of cancers have a well-established dependence on glutamine metabolism to support survival and growth, a process linked to glutaminase 1 (GLS) isoforms. Conversely, GLS2 variants often have tumor-suppressing activity. Triple-negative (TN) breast cancer (testing negative for estrogen, progesterone, and Her2 receptors) has elevated GLS protein levels and reportedly depends on exogenous glutamine and GLS activity for survival. Despite having high GLS levels, we verified that several breast cancer cells (including TN cells) express endogenous GLS2, defying its role as a bona fide tumor suppressor. Moreover, ectopic GLS2 expression rescued cell proliferation, TCA anaplerosis, redox balance, and mitochondrial function after GLS inhibition by the small molecule currently in clinical trials CB-839 or GLS knockdown of GLS-dependent cell lines. In several cell lines, GLS2 knockdown decreased cell proliferation and glutamine-linked metabolic phenotypes. Strikingly, long-term treatment of TN cells with another GLS-exclusive inhibitor bis-2'-(5-phenylacetamide-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) selected for a drug-resistant population with increased endogenous GLS2 and restored proliferative capacity. GLS2 was linked to enhanced in vitro cell migration and invasion, mesenchymal markers (through the ERK-ZEB1-vimentin axis under certain conditions) and in vivo lung metastasis. Of concern, GLS2 amplification or overexpression is linked to an overall, disease-free and distant metastasis-free worse survival prognosis in breast cancer. Altogether, these data establish an unforeseen role of GLS2 in sustaining tumor proliferation and underlying metastasis in breast cancer and provide an initial framework for exploring GLS2 as a novel therapeutic target.

Therapeutic Potential of Melatonin in the Regulation of MiR-148a-3p and Angiogenic Factors in Breast Cancer.

BACKGROUND: The high mortality rate of breast cancer is related to the occurrence of metastasis, a process that is promoted by tumor angiogenesis. MicroRNAs are small molecules of noncoding mRNA that play a key role in gene regulation and are directly involved in the progression and angiogenesis of various tumor types, including breast cancer. Several miRNAs have been described as promoters or suppressors angiogenesis and may be associated with tumor growth and metastasis. Melatonin is an oncostatic agent with a capacity of modifying the expression of innumerable genes and miRNAs related to cancer. OBJECTIVE: The aim of this study was to evaluate the role of melatonin and the tumor suppressor miR- 148a-3p on angiogenesis of breast cancer. METHOD: MDA-MB-231 cells were treated with melatonin and modified with the overexpression of miR-148a-3p. The relative quantification in real-time of miR-148a-3p, IGF-IR and VEGF was performed by real-time PCR. The protein expression of these targets was performed by immunocytochemistry and immunohistochemistry. Survival, migration and invasion rates of tumor cells were evaluated. Finally, the xenograft model of breast cancer was performed to confirm the role of melatonin in the tumor. RESULTS: The melatonin was able to increase the gene level of miR-148a-3p and decreased the gene and protein expression of IGF-1R and VEGF, both in vitro and in vivo. In addition, it also had an inhibitory effect on the survival, migration and invasion of breast tumor cells. CONCLUSION: Our results confirm the role of melatonin in the regulation of miR-148a-3p and decrease of angiogenic factors.

Opposing action of NCoR1 and PGC-1α in mitochondrial redox homeostasis.

The ability to respond to fluctuations of reactive oxygen species (ROS) within the cell is a central aspect of mammalian physiology. This dynamic process depends on the coordinated action of transcriptional factors to promote the expression of genes encoding for antioxidant enzymes. Here, we demonstrate that the transcriptional coregulators, PGC-1α and NCoR1, are essential mediators of mitochondrial redox homeostasis in skeletal muscle cells. Our findings reveal an antagonistic role of these coregulators in modulating mitochondrial antioxidant induction through Sod2 transcriptional control. Importantly, the activation of this mechanism by either PGC-1α overexpression or NCoR1 knockdown attenuates mitochondrial ROS levels and prevents cell death caused by lipid overload in skeletal muscle cells. The opposing actions of coactivators and corepressors, therefore, exert a commanding role over cellular antioxidant capacity.

Screening for PPAR Non-Agonist Ligands Followed by Characterization of a Hit, AM-879, with Additional No-Adipogenic and cdk5-Mediated Phosphorylation Inhibition Properties.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor, playing key roles in maintenance of adipose tissue and in regulation of glucose and lipid homeostasis. This receptor is the target of thiazolidinediones, a class of antidiabetic drugs, which improve insulin sensitization and regulate glycemia in type 2 diabetes. Despite the beneficial effects of drugs, such as rosiglitazone and pioglitazone, their use is associated with several side effects, including weight gain, heart failure, and liver disease, since these drugs induce full activation of the receptor. By contrast, a promising activation-independent mechanism that involves the inhibition of cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation has been related to the insulin-sensitizing effects induced by these drugs. Thus, we aimed to identify novel PPARγ ligands that do not possess agonist properties by conducting a mini-trial with 80 compounds using the sequential steps of thermal shift assay, 8-anilino-1-naphthalenesulfonic acid fluorescence quenching, and a cell-based transactivation assay. We identified two non-agonist PPARγ ligands, AM-879 and P11, and one partial-agonist, R32. Using fluorescence anisotropy, we show that AM-879 does not dissociate the NCOR corepressor in vitro, and it has only a small effect on TRAP coactivator recruitment. In cells, AM-879 could not induce adipocyte differentiation or positively regulate the expression of genes associated with adipogenesis. In addition, AM-879 inhibited CDK5-mediated phosphorylation of PPARγ in vitro. Taken together, these findings supported an interaction between AM-879 and PPARγ; this interaction was identified by the analysis of the crystal structure of the PPARγ:AM-879 complex and evidenced by AM-879's mechanism of action as a putative PPARγ non-agonist with antidiabetic properties. Moreover, we present an optimized assay pipeline capable of detecting ligands that physically bind to PPARγ but do not cause its activation as a new strategy to identify ligands for this nuclear receptor.

Hydrocephalus and arthrogryposis in an immunocompetent mouse model of ZIKA teratogeny: A developmental study.

The teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5-9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes.

Viral Inhibition Mechanism Mediated by Surface-Modified Silica Nanoparticles.

Vaccines and therapies are not available for several diseases caused by viruses, thus viral infections result in morbidity and mortality of millions of people every year. Nanoparticles are considered to be potentially effective in inhibiting viral infections. However, critical issues related to their use include their toxicity and their mechanisms of antiviral action, which are not yet completely elucidated. To tackle these problems, we synthesized silica nanoparticles with distinct surface properties and evaluated their biocompatibility and antiviral efficacy. We show that nanoparticles exhibited no significant toxicity to mammalian cells, while declines up to 50% in the viral transduction ability of two distinct recombinant viruses were observed. We designed experiments to address the mechanism of antiviral action of our nanoparticles and found that their hydrophobic/hydrophilic characters play a crucial role. Our results reveal that the use of functionalized silica particles is a promising approach for controlling viral infection and offer promising strategies for viral control.

Astroglioma conditioned medium increases synaptic elimination and correlates with major histocompatibility complex of class I (MHC I) upregulation in PC12Cells.

Astrocytes are multifunctional glial cells that actively participate in synaptic plasticity in health and disease. Little is known about molecular interactions between neurons and glial cells that result in synaptic stability or elimination. In this sense, the main histocompatibility complex of class I (MHC I) has been shown to play a role in the synaptic plasticity process during development and after lesion of the CNS. MHC I levels in neurons appear to be influenced by astrocyte secreted molecules, which may generate endoplasmic reticulum stress. In vitro studies are of relevance since cell contact can be avoided by the use of astrocyte conditioned medium, allowing investigation of soluble factors isolated from cell direct interaction. Thus, we investigated synaptic preservation by synaptophysin and MHC I immunolabeling in PC12 neuron-like cells exposed to NG97 astroglioma conditioned medium (CM). For that, PC12 cells were cultured and differentiated into neuron-like profile with nerve growth factor. MHC I was induced with interferon beta treatment (IFN), and the effects were compared to PC12 exposure to NG97 CM. Overall, the results show that NG97 CM increases, more than IFN alone, the expression of MHC I, negatively influencing synaptic stability. This indicates that glial soluble factors influence synapse elimination, compatible to in vivo synaptic stripping process, in a cell contact independent fashion. In turn, our results indicate that deleterious effects of astroglioma are not only restricted to rapid growth ratio of the tumor, but also correlated with secretion of stress-related molecules that directly affect neuronal networks.

Adipose tissue-derived stem cells from humans and mice differ in proliferative capacity and genome stability in long-term cultures.

Adipose tissue-derived stem cells (ASCs) are among the more attractive adult stem cell options for potential therapeutic applications. Here, we studied and compared the basic biological characteristics of ASCs isolated from humans (hASCs) and mice (mASCs) and maintained in identical culture conditions, which must be examined prior to considering further potential clinical applications. hASCs and mASCs were compared for immunophenotype, differentiation potential, cell growth characteristics, senescence, nuclear morphology, and DNA content. Although both strains of ASCs displayed a similar immunophenotype, the percentage of CD73(+) cells was markedly lower and CD31(+) was higher in mASC than in hASC cultures. The mean population doubling time was 98.08 ± 6.15 h for hASCs and 52.58 ± 3.74 h for mASCs. The frequency of nuclear aberrations was noticeably lower in hASCs than in mASCs regardless of the passage number. Moreover, as the cells went through several in vitro passages, mASCs showed changes in DNA content and cell cycle kinetics (frequency of hypodiploid, G0/G1, G2/M, and hyperdiploid cells), whereas all of these parameters remained constant in hASCs. Collectively, these results suggest that mASCs display higher proliferative capacity and are more unstable than hASCs in long-term cultures. These results underscore the need to consider specificities among model systems that may influence outcomes when designing potential human applications.