Improved yield, stability, and cleavage reaction of a novel tobacco etch virus protease mutant.

The protease catalytic subunit of the nuclear inclusion protein A from tobacco etch virus (TEVp) is widely used to remove tags and fusion proteins from recombinant proteins. Some intrinsic drawbacks to its recombinant production have been studied for many years, such as low solubility, auto-proteolysis, and instability. Some point mutations have been incorporated in the amino acid protease sequence to improve its production. Here, a comprehensive review of each mutation reported so far has been made to incorporate them into a mutant called TEVp7M with a total of seven changes. This mutant with a His7tag at N-terminus was produced with remarkable purification yields (55 mg/L of culture) from the soluble fraction in a single step affinity purification. The stability of His7-TEVp7M was analyzed and compared with the single mutant TEVp S219V, making evident that His7-TEVp7M shows very constant thermal stability against pH variation, whereas TEVp S219V is highly sensitive to this change. The cleavage reaction was optimized by determining the amount of protease that could cleave a 100-fold excess substrate in the shortest possible time at 30 °C. Under these conditions, His7-TEVp7M was able to cleave His-tag in the buffers commonly used for affinity purification. Finally, a structural analysis of the mutations showed that four of them increased the polarity of the residues involved and, consequently, showed increased solubility of TEVp and fewer hydrophobic regions exposed to the solvent. Taken together, the seven changes studied in this work improved stability, solubility, and activity of TEVp producing enough protease to digest large amounts of tags or fusion proteins. KEY POINTS: • Production of excellent yields of a TEVp (TEVp7M) by incorporation of seven changes. • His-tag removal in an excess substrate in the common buffers used for purification. • Incorporated mutations improve polarity, stability, and activity of TEVp7M.

Vaccines against components of the renin-angiotensin system.

Even though effective drugs for treating hypertension are available, a great percentage of patients have inadequate control of their blood pressure. Unwanted side effects and inappropriate oral drug adherence are important factors that contribute to the global problem of uncontrolled hypertension. Vaccination could provide a revolutionary therapy with long-lasting effects, increasing patient compliance and therefore better control of high blood pressure. Nowadays, current immunization approaches against hypertension target renin, angiotensin I, angiotensin II, and angiotensin II type 1 receptor, key elements of the renin-angiotensin system. This article reviews the different vaccination attempts with proteins and peptides against the different molecules of the renin-angiotensin system in the last two decades, safety issues, and other novel prospects biomarkers in hypertension, and summarizes the potential of this immunomodulatory approach in clinical practice.

Peptidic vaccines: The new cure for heart diseases?

Cardiovascular disease continues to be the most common cause of death worldwide. The global burden is so high that numerous organizations are providing counseling recommendations and annual revisions of current pharmacological and non-pharmacological treatments as well as risk prediction for disease prevention and further progression. Although primary preventive interventions targeting risk factors such as obesity, hypertension, smoking, and sedentarism have led to a global decline in hospitalization rates, the aging population has overwhelmed these efforts on a global scale. This review focuses on peptidic vaccines, with the known and not well-known autoantigens in atheroma formation or acquired cardiac diseases, as novel potential immunotherapy approaches to counteract harmful heart disease continuance. We summarize how cancer immunomodulatory strategies started novel approaches to modulate the innate and adaptive immune responses, and how they can be targeted for therapeutic purposes in the cardiovascular system. Brief descriptions focused on the processes that start as either immunologic or non-immunologic, and the ultimate loss of cardiac muscle cell contractility as the outcome, are discussed. We conclude debating how novel strategies with nanoparticles and nanovaccines open a promising therapeutic option to reduce or prevent cardiovascular diseases.

Isotretinoin and Thalidomide Down-Regulate c-MYC Gene Expression and Modify Proteins Associated with Cancer in Hepatic Cells.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer. The number of cases is increasing and the trend for the next few years is not encouraging. HCC is usually detected in the advanced stages of the disease, and pharmacological therapies are not entirely effective. For this reason, it is necessary to search for new therapeutic options. The objective of this work was to evaluate the effect of the drugs isotretinoin and thalidomide on c-MYC expression and cancer-related proteins in an HCC cellular model. The expression of c-MYC was measured using RT-qPCR and western blot assays. In addition, luciferase activity assays were performed for the c-MYC promoters P1 and P2 using recombinant plasmids. Dose-response-time analyses were performed for isotretinoin or thalidomide in cells transfected with the c-MYC promoters. Finally, a proteome profile analysis of cells exposed to these two drugs was performed and the results were validated by western blot. We demonstrated that in HepG2 cells, isotretinoin and thalidomide reduced c-MYC mRNA expression levels, but this decrease in expression was linked to the regulation of P1 and P1-P2 c-MYC promoter activity in isotretinoin only. Thalidomide did not exert any effect on c-MYC promoters. Also, isotretinoin and thalidomide were capable of inducing and repressing proteins associated with cancer. In conclusion, isotretinoin and thalidomide down-regulate c-MYC mRNA expression and this is partially due to P1 or P2 promoter activity, suggesting that these drugs could be promising options for modulating the expression of oncogenes and tumor suppressor genes in HCC.

The algorithm used for the interpretation of doravirine transmitted drug resistance strongly influences clinical practice and guideline recommendations.

OBJECTIVES: We report the results of the reverse transcriptase (RT)/protease (PR) transmitted drug resistance (TDR) prevalence study in 2018, focusing on doravirine resistance-associated mutations and the differences observed when Stanford or French National Agency for AIDS Research (ANRS)/Spanish Network of AIDS Research (RIS)/IAS-USA resistance interpretation algorithms are used to describe clinically relevant resistance. METHODS: We used the WHO 2009 list to investigate the prevalence of NNRTI, NRTI and PI TDR, in treatment-naive HIV-1-infected patients, adding mutations E138A/G/K/Q/R, V106I, V108I, V179L, G190Q, H221Y, F227C/L/V, M230IDR, L234I, P236L and Y318F in RT. The prevalence of doravirine resistance-associated mutations, as described by Soulie et al. in 2019, was evaluated. Clinically relevant TDR was investigated using the latest versions of ANRS, RIS, IAS-USA and Stanford algorithms. RESULTS: NNRTI mutations were detected in 82 of 606 (13.5%) patients. We found 18 patients (3.0%) with NRTI mutations and 5 patients (0.8%) with PI mutations. We detected 11 patients harbouring doravirine resistance-associated mutations (prevalence of 1.8%). Furthermore, we observed important differences in clinically relevant resistance to doravirine when ANRS/RIS (0.7%), IAS-USA (0.5%) or Stanford algorithms (5.0%) were used. V106I, which was detected in 3.8% of the patients, was the main mutation driving these differences. V106I detection was not associated with any of the clinical, demographic or virological characteristics of the patients. CONCLUSIONS: The prevalence of NRTI and PI TDR remains constant in Spain. Doravirine TDR is very infrequent by RIS/ANRS/IAS-USA algorithms, in contrast with results using the Stanford algorithm. Further genotype-phenotype studies are necessary to elucidate the role of V106I in doravirine resistance.

Surveillance of transmitted drug resistance to integrase inhibitors in Spain: implications for clinical practice.

BACKGROUND: Integrase strand-transfer inhibitors (INSTIs) constitute at present one of the pillars of first-line ART. OBJECTIVES: To study the prevalence of and the trend in transmitted drug resistance (TDR) to INSTIs in ART-naive patients in Spain. METHODS: During the period 2012-17, 1109 patients from CoRIS were analysed. The Stanford algorithm v8.7 was used to evaluate TDR and transmission of clinically relevant resistance. To describe individual mutations/polymorphisms, the most recent IAS list (for INSTIs) and the 2009 WHO list update (for the backbone NRTIs used in combination with INSTIs in first-line treatment) were used. RESULTS: Clinically relevant resistance to the INSTI class was 0.2%: T66I, 0.1%, resistance to elvitegravir and intermediate resistance to raltegravir; and G163K, 0.1%, intermediate resistance to raltegravir and elvitegravir. No clinical resistance to dolutegravir or bictegravir was observed. The prevalence of INSTI TDR following the IAS-USA INSTI mutation list was 2.6%, with no trend towards changes in the prevalence throughout the study period. The overall prevalence of NRTI WHO mutations was 4.3%, whereas clinically relevant resistance to tenofovir, abacavir and emtricitabine/lamivudine was 1.7%, 1.9% and 0.7%, respectively. CONCLUSIONS: Given the low prevalence of clinically relevant resistance to INSTIs and first-line NRTIs in Spain, it is very unlikely that a newly diagnosed patient will present with clinical resistance to a first-line INSTI-based regimen. These patients may not benefit from INSTI and NRTI baseline resistance testing.

Anionic Carbosilane Dendrimers Destabilize the GP120-CD4 Complex Blocking HIV-1 Entry and Cell to Cell Fusion.

Cell-to-cell transmission is the most effective pathway for the spread of human immunodeficiency virus (HIV-1). Infected cells expose virus-encoded fusion proteins on their surface as a consequence of HIV-1 replicative cycle that interacts with noninfected cells through CD4 receptor and CXCR4 coreceptor leading to the formation of giant multinucleated cells known as syncytia. Our group previously described the potent activity of dendrimers against CCR5-tropic viruses. Nevertheless, the study of G1-S4, G2-S16, and G3-S16 dendrimers in the context of X4-HIV-1 tropic cell-cell fusion referred to syncytium formation remains still unknown. These dendrimers showed a suitable biocompatibility in all cell lines studied and our results demonstrated that anionic carbosilane dendrimers G1-S4, G2-S16, and G3-S16 significantly inhibit the X4-HIV-1 infection, as well as syncytia formation, in a dose dependent manner. We also demonstrated that G2-S16 and G1-S4 significantly reduced syncytia formation in HIV-1 Env-mediated cell-to-cell fusion model. Molecular modeling and in silico models showed that G2-S16 dendrimer interfered with gp120-CD4 complex and demonstrated its potential use for a treatment.

Silica nanoparticles induce cardiotoxicity interfering with energetic status and Ca2+ handling in adult rat cardiomyocytes.

Recent evidence has shown that nanoparticles that have been used to improve or create new functional properties for common products may pose potential risks to human health. Silicon dioxide (SiO2) has emerged as a promising therapy vector for the heart. However, its potential toxicity and mechanisms of damage remain poorly understood. This study provides the first exploration of SiO2-induced toxicity in cultured cardiomyocytes exposed to 7- or 670-nm SiO2 particles. We evaluated the mechanism of cell death in isolated adult cardiomyocytes exposed to 24-h incubation. The SiO2 cell membrane association and internalization were analyzed. SiO2 showed a dose-dependent cytotoxic effect with a half-maximal inhibitory concentration for the 7 nm (99.5 ± 12.4 µg/ml) and 670 nm (>1,500 µg/ml) particles, which indicates size-dependent toxicity. We evaluated cardiomyocyte shortening and intracellular Ca2+ handling, which showed impaired contractility and intracellular Ca2+ transient amplitude during ß-adrenergic stimulation in SiO2 treatment. The time to 50% Ca2+ decay increased 39%, and the Ca2+ spark frequency and amplitude decreased by 35 and 21%, respectively, which suggest a reduction in sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity. Moreover, SiO2 treatment depolarized the mitochondrial membrane potential and decreased ATP production by 55%. Notable glutathione depletion and H2O2 generation were also observed. These data indicate that SiO2 increases oxidative stress, which leads to mitochondrial dysfunction and low energy status; these underlie reduced SERCA activity, shortened Ca2+ release, and reduced cell shortening. This mechanism of SiO2 cardiotoxicity potentially plays an important role in the pathophysiology mechanism of heart failure, arrhythmias, and sudden death.NEW & NOTEWORTHY Silica particles are used as novel nanotechnology-based vehicles for diagnostics and therapeutics for the heart. However, their potential hazardous effects remain unknown. Here, the cardiotoxicity of silica nanoparticles in rat myocytes has been described for the first time, showing an impairment of mitochondrial function that interfered directly with Ca2+ handling.

Antineoplastic copper coordinated complexes (Casiopeinas) uncouple oxidative phosphorylation and induce mitochondrial permeability transition in cardiac mitochondria and cardiomyocytes.

Copper-based drugs, Casiopeinas (Cas), exhibit antiproliferative and antineoplastic activities in vitro and in vivo, respectively. Unfortunately, the clinical use of these novel chemotherapeutics could be limited by the development of dose-dependent cardiotoxicity. In addition, the molecular mechanisms underlying Cas cardiotoxicity and anticancer activity are not completely understood. Here, we explore the potential impact of Cas on the cardiac mitochondria energetics as the molecular mechanisms underlying Cas-induced cardiotoxicity. To explore the properties on mitochondrial metabolism, we determined Cas effects on respiration, membrane potential, membrane permeability, and redox state in isolated cardiac mitochondria. The effect of Cas on the mitochondrial membrane potential (Δψm) was also evaluated in isolated cardiomyocytes by confocal microscopy and flow cytometry. Cas IIIEa, IIgly, and IIIia predominately inhibited maximal NADH- and succinate-linked mitochondrial respiration, increased the state-4 respiration rate and reduced membrane potential, suggesting that Cas also act as mitochondrial uncouplers. Interestingly, cyclosporine A inhibited Cas-induced mitochondrial depolarization, suggesting the involvement of mitochondrial permeability transition pore (mPTP). Similarly to isolated mitochondria, in isolated cardiomyocytes, Cas treatment decreased the Δψm and cyclosporine A treatment prevented mitochondrial depolarization. The production of H2O2 increased in Cas-treated mitochondria, which might also increase the oxidation of mitochondrial proteins such as adenine nucleotide translocase. In accordance, an antioxidant scavenger (Tiron) significantly diminished Cas IIIia mitochondrial depolarization. Cas induces a prominent loss of membrane potential, associated with alterations in redox state, which increases mPTP opening, potentially due to thiol-dependent modifications of the pore, suggesting that direct or indirect inhibition of mPTP opening might reduce Cas-induced cardiotoxicity.

Impaired oxidative metabolism and calcium mishandling underlie cardiac dysfunction in a rat model of post-acute isoproterenol-induced cardiomyopathy.

Stress-induced cardiomyopathy, triggered by acute catecholamine discharge, is a syndrome characterized by transient, apical ballooning linked to acute heart failure and ventricular arrhythmias. Rats receiving an acute isoproterenol (ISO) overdose (OV) suffer cardiac apex ischemia-reperfusion damage and arrhythmia, and then undergo cardiac remodeling and dysfunction. Nevertheless, the subcellular mechanisms underlying cardiac dysfunction after acute damage subsides are not thoroughly understood. To address this question, Wistar rats received a single ISO injection (67 mg/kg). We found in vivo moderate systolic and diastolic dysfunction at 2 wk post-ISO-OV; however, systolic dysfunction recovered after 4 wk, while diastolic dysfunction worsened. At 2 wk post-ISO-OV, cardiac function was assessed ex vivo, while mitochondrial oxidative metabolism and stress were assessed in vitro, and Ca(2+) handling in ventricular myocytes. These were complemented with sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), and RyR2 expression studies. Ex vivo, basal mechanical performance index (MPI) and oxygen consumption rate (MVO2) were unchanged. Nevertheless, upon increase of metabolic demand, by ß-adrenergic stimulation (1-100 nM ISO), the MPI versus MVO2 relation decreased and shifted to the right, suggesting MPI and mitochondrial energy production uncoupling. Mitochondria showed decreased oxidative metabolism, membrane fragility, and enhanced oxidative stress. Myocytes presented systolic and diastolic Ca(2+) mishandling, and blunted response to ISO (100 nM), and all these without apparent changes in SERCA, PLB, or RyR2 expression. We suggest that post-ISO-OV mitochondrial dysfunction may underlie decreased cardiac contractility, mainly by depletion of ATP needed for myofilaments and Ca(2+) transport by SERCA, while exacerbated oxidative stress may enhance diastolic RyR2 activity.

Molecular Challenges and Opportunities in Climate Change-Induced Kidney Diseases.

As temperatures continue to modify due to weather changes, more regions are being exposed to extreme heat and cold. Physiological distress due to low and high temperatures can affect the heart, blood vessels, liver, and especially, the kidneys. Dehydration causes impaired cell function and heat itself triggers cellular stress. The decline in circulating plasma volume by sweat, which stresses the renal and cardiovascular systems, has been related to some molecules that are crucial players in preventing or provoking cellular damage. Hypovolemia and blood redistribution to cutaneous blood vessels reduce perfusion to the kidney triggering the activation of the renin-angiotensin-aldosterone system. In this review, we expose a deeper understanding of the modulation of molecules that interact with other proteins in humans to provide significant findings in the context of extreme heat and cold environments and renal damage reversal. We focus on the molecular changes exerted by temperature and dehydration in the renal system as both parameters are heavily implicated by weather change (e.g., vasopressin-induced fructose uptake, fructogenesis, and hypertension). We also discuss the compensatory mechanisms activated under extreme temperatures that can exert further kidney injury. To finalize, we place special emphasis on the renal mechanisms of protection against temperature extremes, focusing on two important protein groups: heat shock proteins and sirtuins.

HSP60-Derived Peptide as an LPS/TLR4 Modulator: An in silico Approach.

As a part of innate immunity mechanisms, the Toll-like receptor (TLR) signaling pathway serves as one of the mainstay lines of defense against pathogenic microorganisms and cell dysfunction. Nevertheless, TLR overactivation induces a systemic proinflammatory environment compromising organ function or causing the patient's death. TLRs modulators, specially those focused for TLR4, remain a promising approach for inflammatory diseases treatment, being peptide-based therapy a trendy approach. Heat shock protein 60 (HSP60) not only plays a pivotal role in the development of several maladies with strong inflammatory components but also HSP60 peptides possess anti-inflammatory properties in TLR4-mediated diseases, such as diabetes, arthritis, and atherosclerosis. The experimental treatment using HSP60 peptides has proven to be protective in preclinical models of the heart by hampering inflammation and modulating the activity of immune cells. Nonetheless, the effect that these peptides may exert directly on cells that express TLR and its role to inhibit overactivation remain elusive. The aim of this study is to evaluate by molecular docking, a 15 amino acid long-HSP60 peptide (Peptide-2) in the lipopolysaccharide (LPS) binding site of TLR4/MD2, finding most Peptide-2 resulting conformations posed into the hydrophobic pocket of MD2. This observation is supported by binding energy obtained for the control antagonist Eritoran, close to those of Peptide-2. This last does not undergo drastic structural changes, moving into a delimited space, and maintaining the same orientation during molecular dynamics simulation. Based on the two computational techniques applied, interaction patterns were defined for Peptide-2. With these results, it is plausible to propose a peptidic approach for TLR4 modulation as a new innovative therapy to the treatment of TLR4-related cardiovascular diseases.

Hydroxyapatite Nanoparticles in Drug Delivery: Physicochemistry and Applications.

Hydroxyapatite (HAP) has been the gold standard in the biomedical field due to its composition and similarity to human bone. Properties such as shape, size, morphology, and ionic substitution can be tailored through the use of different synthesis techniques and compounds. Regardless of the ability to determine its physicochemical properties, a conclusion for the correlation with the biological response it is yet to be found. Hence, a special focus on the most desirable properties for an appropriate biological response needs to be addressed. This review provides an overview of the fundamental properties of hydroxyapatite nanoparticles and the characterization of physicochemical properties involved in their biological response and role as a drug delivery system. A summary of the main chemical properties and applications of hydroxyapatite, the advantages of using nanoparticles, and the influence of shape, size, functional group, morphology, and crystalline phase in the biological response is presented. A special emphasis was placed on the analysis of chemical and physical interactions of the nanoparticles and the cargo, which was explained through the use of spectroscopic and physical techniques such as FTIR, Raman, XRD, SEM, DLS, and BET. We discuss the properties tailored for hydroxyapatite nanoparticles for a specific biomolecule based on the compilation of studies performed on proteins, peptides, drugs, and genetic material.

Reinforcement of Alginate-Gelatin Hydrogels with Bioceramics for Biomedical Applications: A Comparative Study.

This study states the preparation of novel ink with potential use for bone and cartilage tissue restoration. 3Dprint manufacturing allows customizing prostheses and complex morphologies of any traumatism. The quest for bioinks that increase the restoration rate based on printable polymers is a need. This study is focused on main steps, the synthesis of two bioceramic materials as WO3 and Na2Ti6O13, its integration into a biopolymeric-base matrix of Alginate and Gelatin to support the particles in a complete scaffold to trigger the potential nucleation of crystals of calcium phosphates, and its comparative study with independent systems of formulations with bioceramic particles as Al2O3, TiO2, and ZrO2. FT-IR and SEM studies result in hydroxyapatite's potential nucleation, which can generate bone or cartilage tissue regeneration systems with low or null cytotoxicity. These composites were tested by cell culture techniques to assess their biocompatibility. Moreover, the reinforcement was compared individually by mechanical tests with higher results on synthesized materials Na2Ti6O13 with 35 kPa and WO3 with 63 kPa. Finally, the integration of these composite materials formulated by Alginate/Gelatin and bioceramic has been characterized as functional for further manufacturing with the aid of novel biofabrication techniques such as 3D printing.

Cationic Dendrimer G2-S16 Inhibits Herpes Simplex Type 2 Infection and Protects Mice Vaginal Microbiome.

The G2-S16 polyanionic carbosilane dendrimer is a promising microbicide that inhibits HSV-2 infection in vitro and in vivo in mice models. This G2-S16 dendrimer inhibits HSV-2 infection even in the presence of semen. Murine models, such as BALB/c female mice, are generally used to characterize host-pathogen interactions within the vaginal tract. However, the composition of endogenous vaginal flora remains largely undefined with modern microbiome analyses. It is important to note that the G2-S16 dendrimer does not change healthy mouse vaginal microbiome where Pseudomonas (10.2-79.1%) and Janthinobacterium (0.7-13%) are the more abundant genera. The HSV-2 vaginally infected female mice showed a significant microbiome alteration because an increase of Staphylococcus (up to 98.8%) and Escherichia (30.76%) levels were observed becoming these bacteria the predominant genera. BALB/c female mice vaginally-treated with the G2-S16 dendrimer and infected with the HSV-2 maintained a healthy vaginal microbiome similar to uninfected female mice. Summarizing, the G2-S16 polyanionic carbosilane dendrimer inhibits the HSV-2 infection in the presence of semen and prevents the alteration of mice female vaginal microbiome.

Combination of G2-S16 dendrimer/dapivirine antiretroviral as a new HIV-1 microbicide.

Aim: To research the synergistic activity of G2-S16 dendrimer and dapivirine (DPV) antiretroviral as microbicide candidate to prevent HIV-1 infection. Materials & methods: We assess the toxicity of DPV on cell lines by MTT assay, the anti-HIV-1 activity of G2-S16 and DPV alone or combined at several fixed ratios. Finally, their ability to inhibit the bacterial growth in vitro was assayed. The analysis of combinatorial effects and the effective concentrations were performed with CalcuSyn software. Conclusion: Our results represent the first proof-of-concept study of G2-S16/DPV combination to develop a safe microbicide.

New anionic poly(alkylideneamine) dendrimers as microbicide agents against HIV-1 infection.

Acquired immune deficiency syndrome (AIDS) due to human immunodeficiency virus type-1 (HIV-1) represents one of the most important sexually transmitted infections (STI) worldwide. Great international efforts have been made to stop new infections but, to date, several compounds failed as microbicides at different stages of clinical trials. The quest to design new molecules that could prevent these infections is essential. In this work, we synthesized the first, second and third generations of anionic dendrimers having carboxylate and sulfonate terminal groups, respectively named G1C, G2C, G3C and G1S, G2S, and G3S, starting from a family of poly(alkylideneamine) dendrimers with nitrile termini. The anionic terminal groups of these dendrimers were expected to prompt them to act against HIV-1 infection. All dendrimers were fully characterized by 1H- and 13C-NMR, FTIR, MS and zeta potential techniques. Importantly, they were able to remain stable in the solid state and aqueous solutions at least for one and a half years. Screening of these six new dendrimers was then performed to shed light on their potential anti-HIV-1 activity and their mechanism of action. Results showed that the dendrimers were cytocompatible and that G1C and G1S dendrimers had important activity against R5-HIV-1NLAD8 and X4-HIV-1NL4.3 isolates by acting directly on viral particles and blocking their entry in host cells. Additionally, G1C and G1S dendrimers maintained their inhibitory effect at different pH values. Through a vaginal irritation assay carried out in BALB/c mice, the safety of these new dendrimers for topical application was also shown. Taken together, our results clearly show that G1C and G1S dendrimers are strong candidates for developing an effective microbicide to prevent HIV-1 new infections.

Poly(N-vinylcaprolactam) Nanogels with Antiviral Behavior against HIV-1 Infection.

Stimuli-responsive nanogels offer promising perspectives for the development of next generation formulations for biomedical applications. In this work, poly(N-vinylcaprolactam) nanogels were synthesized varying the concentration of monomer and crosslinking agent. Thus, the inhibitory effect of poly(N-vinylcaprolactam) nanogels against HIV-1 infection is presented for the first time. In particular, we have demonstrated that one of the synthesized poly(N-vinylcaprolactam) nanogels with initial concentration of 80 mg of vinylcaprolactam and 4% of crosslinking agent shows antiviral behavior against HIV-1 infection since this nanogel inhibits the viral replication in TZM.bl target cells.

Ex Vivo Cardiotoxicity of Antineoplastic Casiopeinas Is Mediated through Energetic Dysfunction and Triggered Mitochondrial-Dependent Apoptosis.

Casiopeinas are a group of copper-based antineoplastic molecules designed as a less toxic and more therapeutic alternative to cisplatin or Doxorubicin; however, there is scarce evidence about their toxic effects on the whole heart and cardiomyocytes. Given this, rat hearts were perfused with Casiopeinas or Doxorubicin and the effects on mechanical performance, energetics, and mitochondrial function were measured. As well, the effects of Casiopeinas-triggered cell death were explored in isolated cardiomyocytes. Casiopeinas III-Ea, II-gly, and III-ia induced a progressive and sustained inhibition of heart contractile function that was dose- and time-dependent with an IC50 of 1.3 ± 0.2, 5.5 ± 0.5, and 10 ± 0.7 µM, correspondingly. Myocardial oxygen consumption was not modified at their respective IC50, although ATP levels were significantly reduced, indicating energy impairment. Isolated mitochondria from Casiopeinas-treated hearts showed a significant loss of membrane potential and reduction of mitochondrial Ca2+ retention capacity. Interestingly, Cyclosporine A inhibited Casiopeinas-induced mitochondrial Ca2+ release, which suggests the involvement of the mitochondrial permeability transition pore opening. In addition, Casiopeinas reduced the viability of cardiomyocytes and stimulated the activation of caspases 3, 7, and 9, demonstrating a cell death mitochondrial-dependent mechanism. Finally, the early perfusion of Cyclosporine A in isolated hearts decreased Casiopeinas-induced dysfunction with reduction of their toxic effect. Our results suggest that heart cardiotoxicity of Casiopeinas is similar to that of Doxorubicin, involving heart mitochondrial dysfunction, loss of membrane potential, changes in energetic metabolites, and apoptosis triggered by mitochondrial permeability.

Differential cytotoxicity and internalization of graphene family nanomaterials in myocardial cells.

Given the well-known physical properties of graphene oxide (GO), numerous applications for this novel nanomaterial have been recently envisioned to improve the performance of biomedical devices. However, the toxicological assessment of GO, which strongly depends on the used material and the studied cell line, is a fundamental task that needs to be performed prior to its use in biomedical applications. Therefore, the toxicological characterization of GO is still ongoing. This study contributes to this, aiming to synthesize and characterize GO particles and thus investigate their toxic effects in myocardial cells. Herein, GO particles were produced from graphite using the Tour method and subsequent mild reduction was carried out to obtain low-reduced GO (LRGO) particles. A qualitative analysis of the viability, cellular uptake, and internalization of particles was carried out using GO (~54% content of oxygen) and LRGO (~37% content of oxygen) and graphite. GO and LRGO reduce the viability of cardiac cells at IC50 of 652.1±1.2 and 129.4±1.2µg/mL, respectively. This shows that LRGO particles produce a five-fold increase in cytotoxicity when compared to GO. The cell uptake pattern of GO and LRGO particles demonstrated that cardiac cells retain a similar complexity to control cells. Morphological alterations examined with electron microscopy showed that internalization by GO and LRGO-treated cells (100µg/mL) occurred affecting the cell structure. These results suggest that the viability of H9c2 cells can be associated with the surface chemistry of GO and LRGO, as defined by the amount of oxygen functionalities, the number of graphitic domains, and the size of particles. High angle annular dark-field scanning transmission electron microscopy, dynamic light-scattering, Fourier-transform infrared, Raman, and X-ray photoelectron spectroscopies were used to characterize the as-prepared materials.