Prospects and Pitfalls: Next-Generation Tools to Control Mosquito-Transmitted Disease.

Mosquito-transmitted diseases, including malaria and dengue, are a major threat to human health around the globe, affecting millions each year. A diverse array of next-generation tools has been designed to eliminate mosquito populations or to replace them with mosquitoes that are less capable of transmitting key pathogens. Many of these new approaches have been built on recent advances in CRISPR/Cas9-based genome editing. These initiatives have driven the development of pathogen-resistant lines, new genetics-based sexing methods, and new methods of driving desirable genetic traits into mosquito populations. Many other emerging tools involve microorganisms, including two strategies involving Wolbachia that are achieving great success in the field. At the same time, other mosquito-associated bacteria, fungi, and even viruses represent untapped sources of new mosquitocidal or antipathogen compounds. Although there are still hurdles to be overcome, the prospect that such approaches will reduce the impact of these diseases is highly encouraging.

The impact of potassium peroxymonosulphate and chlorinated cyanurates on biofilms of Stenotrophomonas maltophilia: effects on biofilm control, regrowth, and mechanical properties.

The activity of two chlorinated isocyanurates (NaDCC and TCCA) and peroxymonosulphate (OXONE) was evaluated against biofilms of Stenotrophomonas maltophilia, an emerging pathogen isolated from drinking water (DW), and for the prevention of biofilm regrowth. After disinfection of pre-formed 48 h-old biofilms, the culturability was reduced up to 7 log, with OXONE, TCCA, and NaDCC showing more efficiency than free chlorine against biofilms formed on stainless steel. The regrowth of biofilms previously exposed to OXONE was reduced by 5 and 4 log CFU cm-2 in comparison to the unexposed biofilms and biofilms exposed to free chlorine, respectively. Rheometry analysis showed that biofilms presented properties of viscoelastic solid materials, regardless of the treatment. OXONE reduced the cohesiveness of the biofilm, given the significant decrease in the complex shear modulus (G*). AFM analysis revealed that biofilms had a fractured appearance and smaller bacterial aggregates dispersed throughout the surface after OXONE exposure than the control sample. In general, OXONE has been demonstrated to be a promising disinfectant to control DW biofilms, with a higher activity than chlorine. The results also show the impact of the biofilm mechanical properties on the efficacy of the disinfectants in biofilm control.

Phytochemicals Against Drug-Resistant Bacterial Biofilms and Use of Green Extraction Solvents to Increase Their Bioactivity.

Antimicrobial resistance has become one of the major global public health issues of the twenty-first century. One of the main factors in the limited action of antimicrobials is related to the ability of microorganisms, particularly bacteria, to form biofilms. These complex and well-organized communities allow the colonizing cells to acquire survival advantages over the same cells in suspension, including antibiotic resistance. A huge percentage of bacterial infections in humans are associated with biofilms, and many of them are chronic. Therefore, there is an urgent need to develop new products effective in controlling or eradicating biofilms. Plant secondary metabolites (phytochemicals) have demonstrated their potential as antibacterials against planktonic cells and sessile communities when used alone or in synergy with other molecules. This chapter covers recent advances in the activity of phytochemicals against biofilms, particularly those formed by drug-resistant bacteria. In addition, taking into account that the extraction step is crucial for the successful development of new bioactive compounds, the use of novel solvents that increase the phytochemical effect, such as natural deep eutectic solvents (NADES), as well as the recent applications of these solvents as antimicrobials are discussed.

Is the increment of diabetes mellitus in Brazil associated with the consumption of artificially sweetened beverages? A time trend analysis with 757,386 adults from 2006 to 2020.

OBJECTIVE: To describe trends in the prevalence of diabetes mellitus (DM) in Brazil and to analyze its association with the consumption of artificially sweetened beverages among individuals aged 18 years or older. STUDY DESIGN: This was a repeated cross-sectional study. METHODS: Annual data from VIGITEL surveys (2006-2020) were used, which included adults from all Brazilian state capitals. The outcome was the prevalence of DM (type 1 and type 2). The main exposure variable was consuming beverages like soft drinks and artificial juices, either in its 'diet, light, or zero' form. Covariates included sex, age, sociodemographic characteristics, smoking, alcohol consumption, physical activity, fruit consumption, and obesity. The temporal trend in the indicators and the etiological fraction (population attributable risk [PAR]) were calculated. Analyses were performed using Poisson regression. The association between DM and consumption of beverages was tested, excluding the year 2020 due to the pandemic; restricting the analysis to the final three years (2018-2020). RESULTS: Overall, 757,386 subjects were included. The prevalence of DM increased from 5.5% to 8.2%, with an annual growth of 0.17 percentage points (95% CI 0.11-0.24). Among those who consumed diet/light/zero beverages, the annual percentage change of DM was four times greater. The PAR corresponding to the consumption of diet/light/zero beverages on the occurrence of DM was 17%. CONCLUSIONS: An increasing prevalence of DM was observed, while diet/light/zero beverages consumption remains stable. A substantial reduction in the annual percentage change of DM could be observed if people stopped consuming diet/light soda/juice.

Rodent models of knee osteoarthritis for pain research.

Osteoarthritis (OA) is a chronic degenerative joint disease and a leading cause of disability worldwide. Pain is the main symptom, yet no current treatment can halt disease progression or effectively provide symptomatic relief. Numerous animal models have been described for studying OA and some for the associated OA pain. This review aims to update on current models used for studying OA pain, focusing on mice and rats. These models include surgical, chemical, mechanical, and spontaneous OA models. The impact of sex and age will also be addressed in the context of OA modelling. Although no single animal model has been shown ideal for studying OA pain, increased efforts to phenotype OA will likely impact the choice of models for pre-clinical and basic research studies.

Biofilm formation under high shear stress increases resilience to chemical and mechanical challenges.

The effect that the hydrodynamic conditions under which biofilms are formed has on their persistence is still unknown. This study assessed the behaviour of Pseudomonas fluorescens biofilms, formed on stainless steel under different shear stress (τw) conditions (1, 2 and 4 Pa), to chemical (benzalkonium chloride - BAC, glutaraldehyde - GLUT and sodium hypochlorite - SHC) and mechanical (20 Pa) treatments (alone and combined). The biofilms formed under different τw  showed different structural characteristics. Those formed under a higher τw were invariably more tolerant to chemical and mechanical stresses. SHC was the biocide which caused the highest biofilm killing and removal, followed by BAC. The sequential exposure to biocides and mechanical stress was found to be insufficient for effective biofilm control. A basal layer containing biofilm cells mostly in a viable state remained on the surface of the cylinders, particularly for the 2 and 4 Pa-generated biofilms.

Photodynamic therapy and combinatory treatments for the control of biofilm-associated infections.

The advent of antimicrobial resistance has added considerable impact to infectious diseases both in the number of infections and healthcare costs. Furthermore, the relentless emergence of multidrug-resistant bacteria, particularly in the biofilm state, has made mandatory the discovery of new alternative antimicrobial therapies that are capable to eradicate resistant bacteria and impair the development of new forms of resistance. Amongst the therapeutic strategies for treating biofilms, antimicrobial photodynamic therapy (aPDT) has shown great potential in inactivating several clinically relevant micro-organisms, including antibiotic-resistant 'priority bacteria' declared by the WHO as critical pathogens. Its antimicrobial effect is centred on the basis that harmless low-intensity light stimulates a non-toxic dye named photosensitizer, triggering the production of reactive oxygen species upon photostimulation. In addition, combination therapies of aPDT with other antimicrobial agents (e.g. antibiotics) have also drawn considerable attention, as it is a multi-target strategy. Therefore, the present review highlights the recent advances of aPDT against biofilms, also covering progress on combination therapy.

Isoflavones improve collagen I and glycosaminoglycans and prevent bone loss in type 1 diabetic rats.

Objective: The objective of this study was to evaluate the action of soy isoflavones (ISO) and 17ß-estradiol on collagen I (CollI) and sulfated glycosaminoglycans (GAGs) in the bone matrix of diabetic rats.Methods: Sixty adult female rats (Rattus norvegicus albinus) underwent ovariectomy, and then were randomized into six groups of 10 animals each: GI, sham control ovariectomized animals; GII, sham control diabetic (DM) ovariectomized animals; GIII, control ovariectomized animals receiving propylene glycol vehicle; GIV, control ovariectomized DM animals receiving propylene glycol vehicle; GV, ovariectomized DM animals treated with ISO (150 mg/kg by gavage); and GVI, ovariectomized DM animals treated with estrogen (17ß-estradiol, 10 mg/kg, subcutaneously). 17ß-Estradiol was used as a positive control when compared with ISO. To obtain significant depletion of the estrogen levels and subsequent bone loss, a postsurgical period of 90 days was observed. Treatments occurred during 30 consecutive days. After euthanasia, shafts of the animals' femurs were immersed in liquid nitrogen for molecular biology analysis, and the distal femurs were removed and processed for paraffin embedding.Results: ISO (GV) and 17ß-estradiol (GVI) improved bone formation, increasing GAGs and CollI formation when compared to the control group (GIV) (p < 0.05).Conclusions: ISO and 17ß-estradiol contribute to the decrease of bone loss in diabetic rats.

Influence of surface copper content on Stenotrophomonas maltophilia biofilm control using chlorine and mechanical stress.

This work aimed to evaluate the action of materials with different copper content (0, 57, 96 and 100%) on biofilm formation and control by chlorination and mechanical stress. Stenotrophomonas maltophilia isolated from drinking water was used as a model microorganism and biofilms were developed in a rotating cylinder reactor using realism-based shear stress conditions. Biofilms were characterized phenotypically and exposed to three control strategies: 10 mg l-1 of free chlorine for 10 min, an increased shear stress (a fluid velocity of 1.5 m s-1 for 30s), and a combination of both treatments. These shock treatments were not effective in biofilm control. The benefits from the use of copper surfaces was found essentially in reducing the numbers of non-damaged cells. Copper materials demonstrated better performance in biofilm prevention than chlorine. In general, copper alloys may have a positive public health impact by reducing the number of non-damaged cells in the water delivered after chlorine exposure.

Particle motion measurements near a rocky shore off Cabo Frio Island.

This paper describes the lessons learned from the experiment BIOCOM'19 carried out in January 2019, in a shallow water bay off the island of Cabo Frio (RJ, Brazil). A dual accelerometer vector sensor hydrophone was deployed for two days, near a rocky shore covered with a significant benthic fauna. The results show that the frequency band above approximately 1.5 kHz is mostly associated with invertebrate biological noise and that the acoustic and the particle motion fields have a similar behavior, following the usual dawn-dusk activity pattern, and a coherent directivity content. At low frequencies, below ∼300 Hz, the acoustic pressure and the particle acceleration fields have significantly different spectral content along time. Many of these differences are due to anthropogenic noise sources related with nearby boating activity, while during quiet periods, they may be attributed to the biological activity from the rocky shore.

Curvature of the elastic deformations in a nematic sample.

In this work we study the geometry of the elastic deformations of the uniaxial nematic liquid crystals at the bulk. We will show that, at this region of the sample, the elastic terms of the free energy can be separated as the sum of two kinds of elastic deformations, the first is proportional to the Gaussian curvature obtained from the director field of a three-dimensional nematic sample and the second is composed by those terms that cannot be expressed as resulting from this curvature. To achieve these results we will construct the metric of an unixial nematic sample using the fact that the director gives the direction of the anisotropy of the system. With this approach we will give analytical and geometrical arguments to show that the elastic terms determined by [Formula: see text], [Formula: see text] and [Formula: see text] are contained in a curvature term, while the terms fixed by the splay elastic term, [Formula: see text], and the bend elastic term, [Formula: see text], are not. The novelty here is that while [Formula: see text] and [Formula: see text] do not contribute the bulk elastic energy of a nematic sample, they have an important contribution to the curvature of the system.

Microcosm evaluation of the impact of oil contamination and chemical dispersant addition on bacterial communities and sediment remediation of an estuarine port environment.

AIM: To evaluate the interactive effects of oil contamination and chemical dispersant application on bacterial composition and sediment remediation of an estuarine port environment. METHODS AND RESULTS: A multifactorial controlled microcosm experiment was set up using sediment cores retrieved from an estuarine port area located at Ria de Aveiro lagoon (Aveiro, Portugal). An oil spill with and without chemical dispersant addition was simulated. Sediment oil hydrocarbon concentrations and benthic bacterial community structure were evaluated by GC-MS and 16S rRNA high-throughput sequencing respectively. Although initially (first 10 days) chemical dispersion of oil enhanced the concentrations of the heavier polycyclic aromatic hydrocarbons and of the C22 -C30 alkane group, with time (21 days), no significant differences in hydrocarbon concentrations were detected among treatments. Moreover, no significant changes were detected in the structure of sediment bacterial communities, which mainly consisted of operational taxonomic units related to hydrocarbon-contaminated marine environments. We hypothesize that the environmental background of the sampling site preconditioned the communities' response to additional contamination. CONCLUSION: This experimental microcosm study showed that the chemical dispersion of oil did not influence sediment remediation or bacterial community composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study showed that chemical dispersion of oil may not improve the remediation of port sediments. Further studies are needed to investigate the impact of chemical dispersants in combination with bioremediation strategies on the process of sediment remediation in port areas.

Soy isoflavones protect against oxidative stress and diminish apoptosis in ovary of middle-aged female rats.

Ovarian aging is characterized by declines in follicular reserve and oocyte quality due, in part, to increased oxidative stress and apoptosis. Soy isoflavones (ISOs) have been shown to improve ovarian lifespan by acting as antioxidant and antiapoptotic agents. We aimed at evaluating whether ISOs could modulate oxidative stress and reduce apoptosis and improve ovarian follicle survival in middle-aged female rats. Twelve ovary-intact female Wistar rats (12-month-old) were divided into groups: control (CTRL) and ISO, daily treated by gavage with vehicle or soy-ISO extract (150 mg/kg b.w), respectively. After 8 weeks, rats were euthanized and their ovaries removed for histomorphometric (% follicles) and apoptosis (cleaved-caspase-3/BCL2 immunostaining) evaluations, or subjected to biochemical assays to survey reactive oxygen species (ROS) and lipid peroxidation levels and total antioxidant capacity (TAC). The frequency of atretic follicles and number of cleaved-caspase-3-positive cells, as well as the ROS and lipid peroxidation levels, were significantly lower in ISO group compared to CTRL. A significantly higher number of BCL2-positive cells and TAC levels were also observed in ISO group. In conclusion, soy ISOs could decrease follicular atresia, apoptosis and oxidative stress, as well as increase the TAC in ovaries of female rats.

The use of selected phytochemicals with EDTA against Escherichia coli and Staphylococcus epidermidis single- and dual-species biofilms.

The aim of this study was the development of a novel and effective antibacterial formulation combining selected phytochemical compounds (quercetin, cuminaldehyde, indole-3-carbinol and vanillic acid) with ethylenediaminetetraacetic acid (EDTA), an aminopolycarboxylic acid. The antibacterial activity of the combinations was evaluated against Escherichia coli and Staphylococcus epidermidis in planktonic and sessile states as single and dual species. The compounds when applied individually demonstrated modest antibacterial activity. Nevertheless, synergy was observed when EDTA was combined with the selected phytochemicals, particularly with cuminaldehyde and indole-3-carbinol. These combinations were evaluated against single- and dual-species biofilms. An inactivation of 100% was obtained for almost all the biofilms, with E. coli biofilms showing the highest resistance. This study allowed the discovery of novel formulations of phytochemical compounds with antibacterial activity against E. coli and S. epidermidis single- and dual-species biofilms at concentrations close to the minimum bactericidal concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic combinations of EDTA and cuminaldehyde or indole-3-carbinol were effective against single- and dual-species E. coli and S. epidermidis planktonic cells and biofilms. The overall results highlight the role of phytochemical products as a green and sustainable source of antimicrobial potentiators to control bacteria in both planktonic and sessile states.

Factors influencing predator roadkills: The availability of prey in road verges.

Road mortality is the most noticeable effect of roads on wildlife. Road verges may provide important refuges for small mammals and rabbits, particularly when roads cross intensive agricultural or grazed areas. In these circumstances, the increasing use of verges by prey species may attract predators to road surroundings increasing the risk of roadkill. The aim of this study was to quantify the role of prey availability (small mammals and rabbits) on predator road casualties, taking into account road and surrounding landscape characteristics. We analyzed this effect on different predator species, such as, snakes, owls and mammal carnivores. The study took place in a 10 km stretch of a National Road (EN4) in southern Portugal. Relationships among predator mortality and explanatory variables (prey abundance, landscape characteristics, and road verge features) were evaluated using Multivariate Redundancy Analysis (RDA) followed by a variation partitioning. Our results show that, although landscape features explained most of the mortality variation, the prey availability was also very important. Roadkills of Montpellier snake and Egyptian mongoose are strongly associated with wild rabbit abundance on verges, while mortality of stone marten, barn owl and tawny owl is related with wood mouse abundance, Mediterranean forest (montado) density, and verge shrub density. Implications for verge management and implementation of mitigation actions are discussed. We suggest vegetation removal in verges to decrease shelter and food availability for prey, and/or the promotion of habitat for prey in areas distant from roads.

Combined effects of ovariectomy and streptozotocin-induced diabetes in the articular cartilage of rats.

AIM: To evaluate the combined effects of streptozotocin-induced diabetes (Di) and ovariectomy in the articular cartilage of rats. METHODS: Forty adult female Wistar rats were ovariectomized (OVX) or sham-operated. After recovery from surgery, the animals were assigned randomly into four groups: OVX control (OVX-C); OVX treated with 10 µg/kg/day of 17ß-estradiol (OVX-E); sham-operated subjected to Di (Sham-Di); and OVX subjected to Di (OVX-Di). After 60 days of treatment, the animals were euthanized and the distal femurs with articular cartilage were processed for paraffin-embedding. Sections were stained with hematoxylin and eosin for histomorphometry, Picro-Sirius Red for collagen, or Alcian Blue for glycosaminoglycan (GAG) content. To detect apoptosis, sections were stained with an antibody to cleaved caspase-3 (casp-3). RESULTS: Articular cartilage thickness and GAG content were significantly lower (p < 0.05) in the OVX-Di group, which also showed a higher number of casp-3-positive chondrocytes than the other groups. Interestingly, the higher percentage (p < 0.05) of mature collagen fibers was seen in the OVX-Di group, may be as a result of a reduced extracellular matrix remodeling of the articular cartilage. CONCLUSION: Our results indicate that the combination of ovariectomy and streptozotocin-induced diabetes produces more deleterious effects in articular cartilage of rats than either condition alone.

Prevention, removal and inactivation of Escherichia coli and Staphylococcus aureus biofilms using selected monoterpenes of essential oils.

AIMS: The aim of this study was to investigate the antibiofilm potential of five essential oil (EO) components with cyclic (sabinene-SAB, carveol-C1, carvone-C2) and acyclic (citronellol-C3 and citronellal-C4) structures against Escherichia coli and Staphylococcus aureus. METHODS AND RESULTS: The selected EO components prevented biofilm set-up, with C3 and C4 causing remarkable effects. When applied against pre-established biofilms, they promoted high biomass removal and inactivation of biofilm cells. Moreover, no viable E. coli biofilm cells were detected after exposure to SAB at 5 × MIC and 10 × MIC, and a significant viability decrease was observed for both bacteria with the other EO components. SAB, C3 and C4 caused the most prominent effects apparently due to their octanol-water partition coefficient (Po/w), the number of rotatable bonds (n-ROTB) and the free hydroxyl groups. CONCLUSIONS: The overall results demonstrated that the selected EO components, particularly SAB, C3 and C4 are of interest as new lead molecules to both prevent biofilm set-up and to control pre-established biofilms of E. coli and S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The tested EO components exhibited prominent antibiofilm properties against E. coli and S. aureus providing a novel and effective alternative/complementary approach to counteract chronic infections and the transmission of diseases in clinical settings.

Effects of soy isoflavones on the concentration of hyaluronic acid in the vagina of type 1 diabetic rats.

OBJECTIVE: To assess the effects of isoflavones and 17ß-estradiol on the vaginal epithelium extracellular matrix and hyaluronic acid (HA) in the diabetic rat model. METHODS: Sixty adult, virgin, female rats underwent ovariectomy, then randomization into six groups of ten animals each: GI, sham ovariectomized control animals; GII, sham ovariectomized control diabetic animals; GIII, control ovariectomized rats receiving propylene glycol vehicle; GIV, control ovariectomized diabetic animals receiving propylene glycol vehicle; GV, diabetic ovariectomized animals treated with soy isoflavones (150 mg/kg by gavage); GVI, ovariectomized diabetic rats treated with estrogen (17ß-estradiol, 10 mg/kg, subcutaneously). Treatment took place over 30 consecutive days. After euthanasia, a portion of the vagina was immersed in liquid nitrogen for RT-qPCR and Western blotting. Another portion was processed for paraffin embedding. Sections were stained with hematoxylin & eosin for histomorphometry and Picro Sirius Red for collagen quantification. RESULTS: Vaginal epithelium histomorphometry in GIII (15.3 ± 1.1 µm) and GIV (14.5 ± 1.8 µm) was thinner than in GV (41.3 ± 1.5 µm) and GVI (74.3 ± 1.6 µm). There was an increase in collagen content in GV (84.1 ± 1.2 µm) and GVI (88.2 ± 1.7 µm). HA quantification was higher in GV (0.38 ± 1.1 µg/mg) and GVI (0.49 ± 1.4 µg/mg) when compared with GIII (0.12 ± 1.1 µg/mg) and GIV (0.10 ± 1.2 µg/mg), p < 0.05. CONCLUSIONS: Soy isoflavones increase hyaluronic acid concentration in the vagina of diabetic ovariectomized rats. Such findings might help to attenuate the effects of vulvovaginal atrophy in women.

Antiparasitic Activity of Diterpenoids Against Trypanosoma cruzi.

Twenty-seven diterpenes, including abietanes, labdanes, abeoabietanes, halimanes, and pimaranes, have been evaluated against epimastigote and intracellular amastigote forms of Trypanosoma cruzi and also against LC5 and NCTC cell lines. Royleanones (3, 4, and 5) and a further abietane (12), obtained by purification of Plectranthus spp. extracts, were the most active compounds on epimastigotes, showing IC50 values similar (1.73 µg/mL, 12) or even lower (0.39, 0.99, and 1.20 µg/mL, 3, 4, and 5 respectively) than the positive control nifurtimox (2.3 µg/mL). On intracellular amastigotes, abietanes 3, 4, and 5 showed a significant activity with IC50 values of 0.83, < 0.31, and 0.62 µg/mL, respectively, but were less potent than the positive control nifurtimox (IC50 < 0.16 µg/mL). Compounds 3, 4, and 5 were not cytotoxic to LC5 and NCTC 929 cells at 1 µg/mL.

Effects of early and late treatment with soy isoflavones in the mammary gland of ovariectomized rats.

INTRODUCTION: Soy isoflavones have been shown to be an alternative to hormone therapy at menopause, without causing side-effects such as breast cancer. However, the effects of early and late treatment with isoflavones on the mammary gland remain controversial. OBJECTIVE: To investigate the effects of early and late treatment with soy isoflavones on the mammary gland of ovariectomized rats. METHODS: Thirty 3-month-old rats were ovariectomized and divided equally into groups: Control, treated with vehicle solution; or with 150 mg/kg/body weight of isoflavones by gavage; or subcutaneously treated with 10 µg/kg/body weight with 17ß-estradiol. Treatments started 3 days (early treatment) or 30 days (late treatment) after ovariectomy and lasted for 30 consecutive days. Thereafter, the animals were euthanized and the mammary glands were removed and processed for paraffin embedding. Sections were stained with hematoxylin and eosin for histomorphometry or subjected to immunohistochemical detection of Ki-67 and VEGF-A. RESULTS: The ductal, lobular and total epithelial fractions were similar between controls and the early/late isoflavone groups, but they were significantly higher in the groups treated with estradiol. In both epithelial and stromal regions, the immunoreactivity of VEGF-A and the percentage of Ki-67-positive cells were significantly higher in the groups treated with estradiol, while they were similar in the early/late isoflavone groups and control groups. CONCLUSION: Our results indicate that early and late treatment with soy isoflavones at the dose of 150 mg/kg/body weight does not show proliferative and angiogenic effects on the mammary gland of ovariectomized rats.