Anthocyanin-Loaded Polymers as Promising Nature-Based, Responsive, and Bioactive Materials.

Anthocyanins are a specific group of molecules found in nature that have recently received increasing attention due to their interesting biological and colorimetric properties that have been successfully applied in several fields such as food preservation and biomedicine. Consequently, reviews devoted to a general overview of these flavonoids have proliferated in recent years. Meanwhile, the incorporation of anthocyanins into polymeric systems has become an interesting strategy to widen the applicability of these molecules and develop new smart and functional polymers in the above cited areas. However, anthocyanin-based polymers have been scarcely reviewed in the literature. Accordingly, this review aims to be a systematic summary of the most recent approaches for the incorporation of anthocyanins into macro-, micro-, or nanostructured polymers. Moreover, this work describes the fundamentals of the applicability of smart anthocyanin-based polymers and offers an updated review of their most interesting applications as sensors, biological regulators, and active materials.

Silk fibroin and sericin polymer blends for sustainable battery separators.

Natural polymers are a promising alternative for reducing the environmental impact of batteries. For this reason, it is still necessary to study their behavior and implement its use in these devices, especially in separator membranes. This work reports on new separator membranes based on silk fibroin (SF) and silk sericin (SS) prepared by salt leaching method. The effect of the different SS relative content on the physiochemical properties of the membranes and on the electrochemical performance of the corresponding batteries with lithium iron phosphate (LFP) as cathodes has been reported. It is observed that the increasing of SS content leads to a decrease of the overall crystallinity of the membranes. All SF/SS membranes presented a well-defined porosity above 75% with a uniform distribution of interconnected micropores. The electrolyte uptake and the ionic conductivity are dependent on the relative SS content. The addition of 10 wt% of SS into SF membranes, induce a high ionic conductivity of 4.09 mS.cm-1 and high lithium transference number (0.52), due to the improvement of the Li+ ions conduction paths within the blended structure. Charge/discharge tests performed in Lithium/C-LFP half-cells reveal a discharge capacity of 85 mAh.g-1 at 2C after 100 cycles for batteries with a SF/SS separator, containing a 10 wt% of SS, which suggests a stabilizing effect of Sericin on discharge capacity. Further, a 50% and 35% of capacity of retention and capacity fade, respectively, is observed. The presented SF/SS membrane show high electrochemical stability, being suitable for implementation in a next generation of sustainable battery systems. This could allow the SS valorization considering that 150,000 tons of SS are abandoned each year, reducing the contamination of environmental effluents.

Processing Strategies to Obtain Highly Porous Silk Fibroin Structures with Tailored Microstructure and Molecular Characteristics and Their Applicability in Water Remediation.

The present work reports on the control of silk fibroin (SF) porous structures performance through various processing methods. The study includes the analysis of two dissolving techniques (CaCl2/H2O/EtOH ternary and LiBr/H2O binary solutions), three regeneration methods (gelation, lyophilization and gas foaming) and one post-processing (EtOH). In all the cases, followed steps lead to SF structures with porosity values above 94% and large surface areas. Also, results about samples microstructure, secondary organization, crystallinity and water behavior, reveal a direct correlation between processing and SF properties. Thanks to the achieved progress, the SF varying porous structures were evaluated for metalloids (As5+ and As3+) and heavy metals (Cr6+ and Cr3+) adsorption, observing a direct relationship between samples processing and ionic species adsorption ability. Thus, it is shown that the control of the properties of SF based porous structures through processing, represents a suitable and ecofriendly approach for the development of bio-based materials for environmental applications.

Hydrogel-based magnetoelectric microenvironments for tissue stimulation.

The development of strategies to mimic the natural environment of tissues with engineered scaffolds remains one of the biggest challenges of tissue engineering. Hydrogels appear as suitable materials for this purpose due to their substantial water content, biocompatibility, and for being able to carry nanomaterials that introduce new functionalities to the hydrogel. The incorporation of magnetically responsive and, in particular, magnetoelectric materials into the hydrogel-based scaffolds are a promising approach for bone tissue engineering applications once it can promote not only tissue regeneration through magnetic to mechanic to electrical conversion/stimuli but also the external control of the scaffold by the application of magnetic fields. This work reports on a new CoFe2O4/ Methacrylated Gellan Gum (GGMA)/poly(vinylidene fluoride) (PVDF) hydrogel-based scaffold with 20 kPa Young's modulus and cell viability superior to 80%. The ≈ 1 µm thick PVDF/CoFe2O4 spheres added to GGMA gel (2 wt.%) exhibit 20 emu.g-1 magnetization saturation, 2.7 kOe magnetic coercivity and ß-phase contents ≈ 78%, leading to a piezoelectric response |d33| of ≈ 22 pC N-1 and a magnetoelectric response of Δ|d33| ≈ 6 pC N-1 at a DC magnetic field of 220 m T, as verified for the CoFe2O4/PVDF spheres with 20 wt.% filler content. Such characteristics allow novel tissue regeneration strategies approaches once CoFe2O4/GGMA/PVDF has a porous 3-D structure, biocompatibility, bioresorbability, and mechanical/electrical dynamic responses that can be triggered by an applied external magnetic field.

Poly(L-lactide)/branched ß-cyclodextrin blends: Thermal, morphological and mechanical properties.

In this work we develop poly(L-lactide)/branched ß-cyclodextrin (bßCD) blends in an attempt to obtain new biocompatible and biodegradable materials to be used in the emerging fields of pharmaceutical, biomedicine and food industry. Ionic branched ß-cyclodextrin (bßCD) was obtained by polycondensation of the ß-CD monomer and it was blended with a commercially available PLLA. Fourier transform infrared spectroscopy (FTIR) has been applied to study the occurring interactions between both partners. Thermal properties of blends have been analyzed by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), while the phase structure of the blends was analyzed by scanning electron microscopy (SEM). Finally, dynamic mechanical analysis (DMA) has been used to provide further insights into the features controlling miscibility between PLLA and bßCD. Results show the presence of a single phase irrespectively of the blend composition. Overall, this work opens new perspectives for the development of naturally available materials with tunable functional properties for applications in which cyclodextrins emerge as a new class of promising candidates.

Short communication: Biological and genetic characterization of HIV type 1 subtype B and nonsubtype B transmitted viruses: usefulness for vaccine candidate assessment.

Due to the extraordinary degree of genetic diversity of HIV-1 and the structural complexity of its envelope glycoproteins, designing an effective vaccine is difficult, requiring the development of viral reagents to assess vaccine-elicited neutralizing antibodies. The aim of this study was to improve on our previously developed panel of HIV-1 strains of different genetic forms, focusing on strains from acute and recently acquired infections as the most representative of the transmitted viruses. HIV-1 primary isolates were expanded in peripheral blood mononuclear cells. Viral stocks of 40 ml each were produced. Syncytium-inducing (SI) phenotype, coreceptor use, and TCID(50)/ml were determined. Near full-length HIV-1 genomes were amplified by RT-nested PCR in four overlapping segments. Phylogenetic analyses were performed with neighbor-joining trees and bootscanning. Forty-four HIV-1 strains were included in the panel. Twenty-four (54.1%) strains were from early infections (16 acute and 8 recent); of them, 21 (87%) were sexually transmitted. NSI/R5 phenotype was detected in 37 (84.1%) viruses and SI/R5,X4 in another 7 (15.9%). TCID(50)/ml ranged between 10(4) and 10(6.6). Twelve different genetic forms constituted this panel: subtypes A1, B, C, F1, and G; circulating recombinant forms CRF02_AG, CRF14_BG, and CRF24_BG; and unique recombinant forms CRF02_AG/A3, BF1, CRF12_BF/B, and DF1G. In conclusion, in this study, we report the development of a comprehensive and well-characterized panel of HIV-1 isolates for assessing neutralization in HIV vaccine research. This panel is available for distribution through the Programme EVA Centre for AIDS Reagents, National Institute for Biological Standard and Control (NIBSC).

Incidence of non-B subtypes of HIV-1 in Galicia, Spain: high frequency and diversity of HIV-1 among men who have sex with men.

An increase in HIV transmission among men who have sex with men (MSM) has been reported in eight regions of Spain from 2003 to 2007. In order to study the incidence of HIV-1 genetic forms in Galicia, northwest of Spain, in particular the spread of HIV-1 variants among MSM, 93 newly diagnosed HIV-1 patients, including those with acute and recently acquired infections, were studied for a year from August 2008 to August 2009. Thirty eight (41%) were MSM. Of them, nine (24%) were infected by non-B viruses, including seven different genetic forms. The analysis of transmission clusters showed that 23 (60%) MSM grouped in different clusters and mostly in large clusters. Resistance mutations were detected in six (16%) MSM.

Chronic periadolescent cannabinoid treatment enhances adult hippocampal PSA-NCAM expression in male Wistar rats but only has marginal effects on anxiety, learning and memory.

Pubertal and adolescent exposure to cannabinoids is associated with enduring alterations in anxiety and memory. However, periadolescence virtually remains unexplored. Here, we measured anxiety in the Elevated Plus Maze (EPM) in adult Wistar rats treated at periadolescence (P28-P38) with the cannabinoid agonist CP 55,940 (CP) (0.4 mg/kg; 2 ml/kg i.p., 1 daily injection), and we also defined their recognition memory in the novel object paradigm and spatial learning and memory in the water maze. Additionally, we measured the expression of hippocampal PSA-NCAM (Polysialic Acid-Neural Cell Adhesion Molecule) and long-term potentiation (LTP) as well as, given their role in mnemonic processing, the levels of plasma corticosterone and estradiol. We found that CP had no robust effects on anxiety or in recognition memory. In the water maze, only a slight decreased percentage of failed trials in the reference memory task and an improvement in an indirect index of attention were observed. However, we detected an up-regulation of hippocampal PSA-NCAM expression, only in CP-males, although this effect was not related to changes in LTP. No hormonal alterations were evident. Based on our data, minimal long-term effects on anxiety, learning and memory appear to result from cannabinoid exposure during the periadolescent period.

Development of a panel of well-characterized human immunodeficiency virus type 1 isolates from newly diagnosed patients including acute and recent infections.

The aim of this study was the development of a panel constituted by well-defined HIV-1 strains of different genetic forms, with a particular focus on isolates from acute and recent infections. Fourteen HIV-1 isolates, including four from acute and five from recent infections, were expanded in peripheral blood mononuclear cells. SI phenotype, coreceptors use, and TCID(50)/ml were determined. V3 net charge was calculated. Near full-length genomes were amplified by RT-nested PCR in four overlapping segments. Phylogenetic analyses were performed with neighbor-joining trees and bootscanning. Analysis of cysteine residues, lengths of variable regions, and potential N-linked glycosylation sites in gp120 and gp41 was performed. Viral stocks were produced. Thirteen strains were NSI/R5 and one SI/R5,X4. TCID(50)/ml ranged between 10(4.6) and 10(6). V3 net charge was <+5 in 12 sequences and +5 in two sequences. Near full-length HIV-1 genomes analysis identified viruses of the following genetic forms: eight subtype B, three subtype C, two CRF02_AG, and one subtype G. Cysteine residues that form the V1,V2,V3, and V4 loops were highly conserved. The number of potential N-linked glycosylation sites in gp120 and gp41 ranged between 24-29 and 4-6, respectively. Seven potential N-linked glycosylation sites in gp120 and three in gp41 were conserved. V1, V2, V4, and V5 variable regions exhibited substantial length variation. In addition, an analysis of transmitted and natural resistance to current antiretroviral drugs in these strains was performed. It is worth mentioning that the 13S mutation in the V3 sequence, associated with resistance to maraviroc, was observed in a subtype B strain that harbored resistance mutations to nucleoside reverse transcriptase inhibitors and to T20. The availability of a panel including strains from acute and recent infections should be a valuable resource for optimizing and standardizing vaccine candidate assessment. Near full-length genome characterization may be necessary for evaluating clade-specific reactivities.

Long-term monitoring of genotypic and phenotypic resistance to T20 in treated patients infected with HIV-1.

The aim of this study was to investigate the susceptibility to T20 and the dynamics of amino acid changes in HR1 and HR2 of gp41 of HIV-1 obtained from plasma, peripheral blood mononuclear cells (PBMC), and primary isolates (PI) in four highly antiretroviral-experienced patients. These patients received T20 plus an antiretroviral regimen and were followed-up over a period of 40-72 weeks. In one non-responder patient, N43D substitution was detected at 12 weeks of treatment, in association with a value of T20-IC50 of 10 microg/ml (10-fold increase). Double mutations N42T + N43D were observed in plasma RNA at 32 weeks and remained detectable up to 16 weeks after the withdrawal of the drug. The S138A substitution in HR2 was observed in plasma RNA at 32 weeks, and both in plasma RNA and in PI DNA at 40 weeks, associated with an increase of the T20-IC50 to 25 microg/ml (25-fold increase). Mutations V101G and E137K, not reported previously, were also observed in the HR2 region. Whether these new substitutions play a role in T20 resistance needs to be examined. In three temporary responders, coinciding with viral load rebound, G36D, and N42T substitutions were observed at 12, 24, and 40 weeks. G36D mutation was associated with a value of T20-IC50 of 5 microg/ml. The HR2 S138A mutation was detected after the detection of HR1 substitutions and was associated with an increase in the level of T20-IC50 to 125 microg/ml (125-fold increase) All these data reinforce the role of gp41 amino acids 36-45 and the potential influence of the HR2 S138A mutation in the genotypic/phenotypic resistance to T20.

Analysis of drug resistance-associated mutations in treatment-naïve individuals infected with different genetic forms of HIV-1 circulating in countries of the former Soviet Union.

There are few data on drug resistance-associated mutations in the former Soviet Union since, studies have usually been focused on the env or gag genes for subtype information. This study examines the prevalence and patterns of resistance-associated mutations to reverse transcriptase and protease inhibitors (RTI, PRI) in 278 HIV-1-infected treatment-naïve subjects from countries of Eastern Europe, and defines characteristic polymorphisms of RT and PR sequences in HIV-1 subtype A viruses. Blood samples were collected between 1997 and 2004. Plasma RNA was used for PR-RT amplification by reverse transcription coupled with nested PCR and sequencing. Phylogenetic analysis was done with neighbor-joining trees and bootscanning. Analysis of drug resistance mutations, with Stanford University HIV Drug Resistance Database's algorithm, resulted in an overall prevalence of 12.9% resistance to RTI and 3.9% to PRI. The most frequent substitutions in the RT region were at positions 62 and 236. V77I substitution in PR was found in 47.8% of samples. Polymorphisms in subtype A sequences were identified. This is the first study reporting the prevalence and patterns of both PRI and RTI resistance-associated mutations in naïve HIV-1 infected patients from the former Soviet Union. These data underline the importance of genotypic resistance testing of chronically HIV-1-infected patients before initiating treatment, in order to select the most suitable drug regimen.

Natural resistance-associated mutations to Enfuvirtide (T20) and polymorphisms in the gp41 region of different HIV-1 genetic forms from T20 naive patients.

BACKGROUND: The natural occurrence of primary resistance mutations in reverse transcriptase (RT) and protease (PR) genes of HIV-1 isolates from untreated patients has been reported and it may have important implications for the response to drug treatment. It is predictable that the same occurs in the HR1 region of gp41 sequence from patients who have never received T20 therapy, and in this regard it would be important to know not only the mutation frequencies at HR1 region but also the natural polymorphisms at resistance-associated positions present in the absence of this drug. OBJECTIVES: The objectives of this study are to investigate the existence of natural resistance-associated mutations to T20 in HR1 gp41 region corresponding to different HIV-1 genetic forms from T20 naive patients and to determine their prevalence. STUDY DESIGN: Two hundred HIV-1 gp41 sequences were included: subtype B: 164 (81.3%); subtype A: 15 (8.2%); subtype G: 10 (4.6%); subtype F: 6 (3.5%); subtype C: 3 (1.8%); subtype K: 1 (0.6%); and subtype D: 1 (0.6%). We analyzed the resistance-associated mutations previously described: Q32H/R, G36D/S, I37V, V38A/M, Q39R/H, Q40H, N42T/D/Q/H, N43D/S/K/Q, L44M, L45M, R46M and V69I. RESULTS: Natural resistance mutations to T20 were found at a high frequency: 10.5%, corresponding to 9.1% in subtype B and 16.7% in non-B subtype samples. Polymorphisms were more frequent in non-B and recombinant forms than in subtype B (p<0.001). Different substitutions were related to subtypes: N42S in subtypes A, B, G and C, but not in F, Q56R in subtype A from CRF02_AG, and L54M in subtype B from CRF14_BG. CONCLUSIONS: To our knowledge this is the first study describing natural-resistance to T20 among different HIV-1 subtypes, warranting a study of the biological significance of this mutations and their clinical relevance. The detection of differences between subtypes may have an influence on the rate and patterns of resistance in patients undergoing T20 treatment.

Involvement of 5-HT1A receptors in behavioural effects of the cannabinoid receptor agonist CP 55,940 in male rats.

We have studied the possible interaction between the cannabinoid receptor agonist CP 55,940 (1 and 50 microg/kg) and the 5-HT1A receptor antagonist WAY 100635 (1 mg/kg) in the modulation of plus-maze and holeboard activity in Wistar adult male rats. In the plus-maze, the higher dose of CP 55,940 induced an anxiogenic-like effect, whereas the lower dose induced anxiolytic-like responses. The 5-HT1A antagonist, which was silent in this test, attenuated the anxiogenic, but not the anxiolytic, effect of CP 55,940. In the holeboard, the higher dose of CP 55,940 significantly decreased head-dipping duration, and WAY 100635, which did not affect exploratory head-dipping when administered alone, antagonized this effect. The administration of WAY 100635 significantly increased grooming behaviour, and this effect was inhibited by the two doses of CP 55,940, which did not exert any effect, per se, on this parameter. We provide the first evidence implicating 5-HT1A receptors in anxiety-related behavioural responses to a cannabinoid agonist.

Genotypic resistance mutations to antiretroviral drugs in HIV-1 B and non-B subtypes from Cuba.

OBJECTIVES: To determine the prevalence of drug resistance and to analyze the subtyping in HIV-1 samples from Cuba. METHODS: From an estimated total number of 1,950 HIV-1-infected persons in Cuba, a sample of 103 patients were studied, 76 of whom had received drug treatment for HIV and 27 who had not. The RNA plasma viral load was measured, and automated sequencing was used to assess resistance mutations to reverse transcriptase inhibitors (RTIs) and to protease inhibitors (PIs). Subtyping in the V3 region was performed using heteroduplex mobility assay (HMA). In order to corroborate the HMA results, sequencing of env (C2-V3-C3) was done with one-third of the samples in each of the subtype groups detected by HMA. RESULTS: Out of the 103 samples, 81 of them (78.6%) were classified as subtype B, 19 (18.5%) as subtype A, and 3 (2.9%) as subtype C. The prevalence of resistance mutations was 26.2% to RTIs, none to PIs alone, and 3.9% to both categories of drugs. The prevalence of resistance to nucleoside RTIs (NRTIs) was 27.6% in treated patients and 7.4% in the untreated patients, and for nonnucleoside RTIs (NNRTIs) it was 5.3% and 0%, respectively. Among treated patients a low frequency (2.6%) of dual resistance to zidovudine (ZDV) plus lamivudine (3TC) and abacavir (ABC) was detected, and multidrug resistance to NRTIs was not found. In relation to PIs together with RTIs, the prevalence of resistance was 5.3% for treated patients and 0% for untreated patients. CONCLUSIONS: Even though Cuba is generally considered an area where subtype B is dominant, we detected a high proportion of non-B subtype viruses. The low prevalence of resistance mutations to RTIs and PIs reflects the delay in introducing these drugs to Cuba. Multidrug resistance to RTIs was not found, so, as of now, the use of these drugs continues to be an option for Cuban patients.

Analysis of HIV type 1 protease and reverse transcriptase sequences from Venezuela for drug resistance-associated mutations and subtype classification: a UNAIDS study.

We report the first study on prevalence of antiretroviral drug-associated resistance mutations in Venezuela. Protease and reverse transcriptase (RT) coding regions were analyzed in DNA samples obtained from 100 HIV-1-infected individuals. Primary resistance mutations to RT inhibitors were identified in 26% of patients treated with these drugs. Transmission of HIV-1-resistant strains was detected in a drug-naive patient (3%). Primary resistance mutations to protease inhibitors (PIs) were present in 9% of the 44 PI-treated patients and in 1 PI-naive individual. Phylogenetic analysis of these samples has resulted in the most extensive survey, to date, of HIV-1 genetic forms circulating in Venezuela. Ninety-nine samples clustered with subtype B, and 1 individual harbored the first B/F recombinant virus reported in Venezuela, with protease clustering with subtype F and RT with subtype B. In addition, this isolate had a new insertion (Glu-34 duplication) in the protease gene.

HIV-1 genetic diversity in Galicia Spain: BG intersubtype recombinant viruses circulating among injecting drug users.

BACKGROUND: The HIV-1 epidemics in Western Europe are dominated by B subtype viruses. Non-B subtype is largely restricted to individuals infected outside of Europe and to their direct contacts and is generally acquired by the heterosexual route. METHODS: Protease and a segment of reverse transcriptase were amplified and sequenced from plasma RNA in 451 individuals from seven cities of Galicia, north-western Spain. Subtype sequence homologies were determined using the BLAST algorithm. Non-B sequences were examined by phylogenetic analysis and intersubtype recombination by bootscanning. The env V3 region was analysed in all non-B and in 38 B subtype viruses. RESULTS: Ten different non-B genetic forms were identified in 20 (4.4%) individuals. Subtypes were concordant between pol and V3 in five viruses; 14 (70%) infections were with intersubtype recombinant viruses, and one individual had a dual B+G infection. Seven recombinant viruses were phylogenetically related to five reported recombinant forms. Three non-recombinant G and six recombinant BG viruses formed a monophyletic cluster for pol. All but three individuals with non-B infections were native Spanish. Only 6 of 16 individuals referred to sexual contacts with sub-Saharan Africans. Twelve (60%) non-B subtype infections, including all with G and BG viruses, were in injecting drug users (IDU). CONCLUSIONS: Non-B subtype viruses were identified in 4.4%, with a high diversity of genetic forms, including 70% infections with intersubtype recombinant viruses. The majority of individuals with non-B infections were IDU, most of them without known contacts with non-European sources, and among whom BG recombinant viruses are circulating.

Detection and quantification of multiple drug resistance mutations in HIV-1 reverse transcriptase by an oligonucleotide ligation assay.

OBJECTIVES: To develop an assay for the early detection and quantification of minor human immunodeficiency virus-1 populations bearing multiple drug resistance (MDR) mutations. STUDY DESIGN/METHODS: The oligonucleotide ligation assay (OLA) is based on ligation of probe and detector oligonucleotides annealed to a polymerase chain reaction amplicon strand with detection by an enzyme immunoassay. In OLA-MDR, oligonucleotides were designed to detect MDR mutations. The method was validated with wild-type and MDR mutant clones mixed at different proportions. RESULTS: K103N mutants were detected as minor populations (5%-30%) by OLA in 6 of 18 samples from patients treated with nonnucleoside reverse transcription inhibitors and classified as wild type by sequencing. In one patient, the kinetics of the increase of MDR mutants could be followed in sequential samples, with K103N being detected earlier by OLA than by sequencing. Q151M mutants were detected as minor populations (13%-24%) by OLA but not by sequencing in 4 samples. CONCLUSIONS: Oligonucleotide ligation assay MDR exhibits higher sensitivity than sequencing for detection of minor MDR mutant populations.