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Bacteria that form long-term intracellular associations with host cells lose many genes, a process that often results in tiny, gene-dense, and stable genomes. Paradoxically, the some of the same evolutionary processes that drive genome reduction and simplification may also cause genome expansion and complexification. A bacterial endosymbiont of cicadas, Hodgkinia cicadicola, exemplifies this paradox. In many cicada species, a single Hodgkinia lineage with a tiny, gene-dense genome has split into several interdependent cell and genome lineages. Each new Hodgkinia lineage encodes a unique subset of the ancestral unsplit genome in a complementary way, such that the collective gene contents of all lineages match the total found in the ancestral single genome. This splitting creates genetically distinct Hodgkinia cells that must function together to carry out basic cellular processes. It also creates a gene dosage problem where some genes are encoded by only a small fraction of cells while others are much more abundant. Here, by sequencing DNA and RNA of Hodgkinia from different cicada species with different amounts of splitting-along with its structurally stable, unsplit partner endosymbiont Sulcia muelleri-we show that Hodgkinia does not transcriptionally compensate to rescue the wildly unbalanced gene and genome ratios that result from lineage splitting. We also find that Hodgkinia has a reduced capacity for basic transcriptional control independent of the splitting process. Our findings reveal another layer of degeneration further pushing the limits of canonical molecular and cell biology in Hodgkinia and may partially explain its propensity to go extinct through symbiont replacement., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Poly(ethylene terephthalate) (PET) is a synthetic polymer widely used globally. The high PET resistance to biotic degradation and its improper destination result in the accumulation of this plastic in the environment, largely affecting terrestrial and aquatic animals. This work investigated post-consumer PET (PC-PET) degradation using five commercial hydrolase enzymes (Novozym 51032, CalB, Palatase, Eversa, Lipozyme TL). Humicola insolens cutinase (HiC, Novozym 51032) was the most active among the enzymes studied. Several important reaction parameters (enzyme type, dual enzyme system, enzyme concentration, temperature, ultrasound treatment) were evaluated in PC-PET hydrolysis using HiC. The concentration and the proportion (molar ratio) of hydrolysis products, terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET), were significantly changed depending on the reaction temperature. The TPA released at 70 °C was 3.65-fold higher than at 50 °C. At higher temperatures, the conversion of MHET into TPA was favored. The enzymatic PET hydrolysis by HiC was very sensitive to the enzyme concentration, indicating that it strongly adsorbs on the polymer surface. The concentration of TPA, MHET, and BHET increased as the enzyme concentration increased, and a maximum was achieved using 40-50 vol % of HiC. The presented results add relevant data to optimizing enzyme-based PET recycling technologies., Competing Interests: Conflict of interestOn behalf of all authors, the corresponding author states that there is no conflict of interest., (© King Abdulaziz City for Science and Technology 2023, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Integration of multiple approaches is key to understand the evolutionary processes of local adaptation and speciation. Reptiles have successfully colonized desert environments, that is, extreme and arid conditions that constitute a strong selective pressure on organisms. Here, we studied genomic, physiological and morphological variations of the lizard Liolaemus fuscus to detect adaptations to the Atacama Desert. By comparing populations of L. fuscus inhabiting the Atacama Desert with populations from the Mediterranean forests from central Chile, we aimed at characterizing features related to desert adaptation. We combined ddRAD sequencing with physiological (evaporative water loss, metabolic rate and selected temperature) and morphological (linear and geometric morphometrics) measurements. We integrated the genomic and phenotypic data using redundancy analyses. Results showed strong genetic divergence, along with a high number of fixed loci between desert and forest populations. Analyses detected 110 fixed and 30 outlier loci located within genes, from which 43 were in coding regions, and 12 presented non-synonymous mutations. The candidate genes were associated with cellular membrane and development. Desert lizards presented lower evaporative water loss than those from the forest. Morphological data showed that desert lizards had smaller body size, different allometry, larger eyeballs and more dorsoventrally compressed heads. Our results suggest incipient speciation between desert and forest populations. The adaptive signal must be cautiously interpreted since genetic drift could also contribute to the divergence pattern. Nonetheless, we propose water and resource availability, and changes in habitat structure, as the most relevant challenges for desert reptiles. This study provides insights of the mechanisms that allow speciation as well as desert adaptation in reptiles at multiple levels, and highlights the benefit of integrating independent evidence., (© 2021 British Ecological Society.)

Documenting topical bioequivalence can be an extremely complex process, which is intrinsically dependent on the formulation technological features. According to EMA guideline, for simple formulations, BE may be demonstrated by documenting the qualitative (Q1), quantitative (Q2), microstructure (Q3) and performance (Q4) equivalence. Nevertheless, when addressing complex semisolids, equivalence regarding local availability should also be demonstrated. The purpose of this study is to pursue this strategy using two opposite scenarios: a simple dimetindene maleate 1 mg/g gel formulation and a diclofenac diethylammonium 23.2 mg/g emulgel, representing a complex formulation. For both formulations, Q1/Q2 test (TP) and reference products (RP) were used. Rheology, in vitro release (IVRT) and in vitro permeation methods (IVPT) were developed and validated for both products. For the dimetindene formulation, equivalence pertaining to Q4 was established. However, high variability was observed for some rheology endpoints, especially for the different RP batches. Therefore, equivalence could not be established for Q3 as per EMA requirements. Can some rheology endpoints be waived? Can we establish reasonable criteria that are overall feasible for generic manufacturers and at the same time safe for the patient? An attempt was made to propose a wider acceptance range based on the inter-batch variability of the RP. For that, the rationale presented in the EMA guideline on bioequivalence for highly variable products was used. For the diclofenac formulation, Q3 equivalence was likewise not established. Q4 equivalence was only found for some batch combinations and when applying a wider acceptance criterion (75-133%). Furthermore, IVPT equivalence also failed to be demonstrated. Nevertheless, since the TP displays an equivalent pharmacokinetic profile compared to the RP, the observed Q3, Q4 and local availability differences are not expected to be clinically significant. This study draws attention to an effective search to determine the most appropriate strategy for assessing topical bioequivalence on a case-by-case basis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The combination of two or more active pharmaceutical ingredients in the same dosage form - fixed-dose combination products - for topical administration represents a promising therapeutic approach for treating several pathologies, including pain. The pre-clinical development of fixed-dose combination products aims to characterize the interactions between the different APIs and ensure that the final medicinal product has the required safety characteristics. To this end, there are several regulatory accepted in vitro tests to assess the safety of medicinal products intended for cutaneous administration. In turn, the evaluation of anti-inflammatory activity should be based on models described in the scientific literature, as there are no models fully validated by competent entities. Therefore, this work presents the information regarding accepted in vitro tests to assess the safety of topical products and the most used methods to assess anti-inflammatory activity. Additionally, a new approach to select a fixed-dose combination product with the potential to enhance the therapeutic effects of the individual active pharmaceutical ingredients is rationalized by integrating the overall effects on several targets relevant for inflammation and pain management in one numeric index., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The combination in a fixed dose of two or more active pharmaceutical ingredients in the same pharmaceutical dosage form is an approach that has been used successfully in the treatment of several pathologies, including pain. In the preclinical development of a topical fixed-dose combination product with analgesic and anti-inflammatory activities for pain management, the main objective is to establish the nature of the interaction between the different active pharmaceutical ingredients while obtaining data on the medicinal product safety and efficacy. Despite the improvement of in vitro assays, animal models remain a fundamental strategy to characterise the interaction, efficacy and safety of active pharmaceutical ingredients at the physiological level, which cannot be reached by in vitro assays. Thus, the main goal of this review is to systematise the available animal models to evaluate the efficacy and safety of a new fixed-dose combination product for topical administration indicated for pain management. Particular emphasis is given to animal models that are accepted for regulatory purposes., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The genus Chilecicada Sanborn, 2014 is shown to be a complex of closely related species rather than a monospecific genus. Chilecicada citatatemporaria Sanborn Cole n. sp., C. culenesensis Sanborn Cole n. sp., C. curacaviensis Sanborn Cole n. sp., C. impartemporaria Sanborn Cole n. sp., C. magna Sanborn Cole n. sp., C. mapuchensis Sanborn n. sp., C. oraria Sanborn Cole n. sp., C. parrajaraorum Sanborn n. sp., C. partemporaria Sanborn Cole n. sp., C. pehuenchesensis Sanborn Cole n. sp., C. trifascia Sanborn n. sp., C. trifasciunca Sanborn Cole n. sp., and C. viridicitata Sanborn Cole n. sp. are described as new. Chilecicada occidentis Walker, 1850 is re-described to facilitate separation of the new species from the only previously known species. Song and cytochrome oxidase I analysis available for most species support the separation of the new taxa from the type species of the genus. Known species distributions and a key to the species of the genus are also provided. The new species increases the known cicada diversity 61.9% to 34 species, 91.2% of which are endemic to Chile.

Pain, a severe public health problem, can affect patient quality of life when inadequately controlled. Considering that pain pathophysiological mechanisms are complex, combining active pharmaceutical ingredients (APIs) with multiple and synergistic mechanisms of action represents a potentially more effective therapeutic approach than conventional monotherapy treatments. In turn, topical drug delivery has clear advantages over other routes of administration, such as high levels of efficacy, better safety profile and great patient compliance. In this context, the combination of two or more APIs in a single dosage form - fixed-dose combination product (FDC) - for topical administration may represent a promising therapeutic option in the field of pain management. Considering the above mentioned, the purpose of this manuscript is to address an overview of some general aspects regarding pain management and FDCs, as well as the regulatory environment that has to be taken into consideration during their development. Special emphasis will be given to fixed-dose combinations for topical administration with analgesic and/or anti-inflammatory activity. Market drivers of the topical FDC currently approved are ultimately pointed out, and new opportunities in pain management highlighted., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

Non-alcoholic steatohepatitis (NASH), one of the deleterious stages of non-alcoholic fatty liver disease, remains a significant cause of liver-related morbidity and mortality worldwide. In the current work, we used an exploratory data analysis to investigate time-dependent cellular and mitochondrial effects of different supra-physiological fatty acids (FA) overload strategies, in the presence or absence of fructose (F), on human hepatoma-derived HepG2 cells. We measured intracellular neutral lipid content and reactive oxygen species (ROS) levels, mitochondrial respiration and morphology, and caspases activity and cell death. FA-treatments induced a time-dependent increase in neutral lipid content, which was paralleled by an increase in ROS. Fructose, by itself, did not increase intracellular lipid content nor aggravated the effects of palmitic acid (PA) or free fatty acids mixture (FFA), although it led to an up-expression of hepatic fructokinase. Instead, F decreased mitochondrial phospholipid content, as well as OXPHOS subunits levels. Increased lipid accumulation and ROS in FA-treatments preceded mitochondrial dysfunction, comprising altered mitochondrial membrane potential (ΔΨm) and morphology, and decreased oxygen consumption rates, especially with PA. Consequently, supra-physiological PA alone or combined with F prompted the activation of caspase pathways leading to a time-dependent decrease in cell viability. Exploratory data analysis methods support this conclusion by clearly identifying the effects of FA treatments. In fact, unsupervised learning algorithms created homogeneous and cohesive clusters, with a clear separation between PA and FFA treated samples to identify a minimal subset of critical mitochondrial markers in order to attain a feasible model to predict cell death in NAFLD or for high throughput screening of possible therapeutic agents, with particular focus in measuring mitochondrial function.

Enzymes adapted to cold temperatures are commonly characterized for having higher Michaelis-Menten constants (K M ) values and lower optimum and denaturation temperature, when compared to other meso or thermophilic enzymes. Phenoloxidase (PO) enzymes are ubiquitous in nature, however, they have not been reported in spiders. It is the oxygen carrier protein hemocyanin (Hc), found at high concentrations in their hemolymph, which displays an inducible PO activity. Hence, we hypothesize that Hc-derived PO activity could show features of cold adaptation in alpine species. We analyzed the Hc from two species of Theraphosidae from different thermal environments: Euathlus condorito (2400 m a.s.l.) and Grammostola rosea (500 m a.s.l.). Hc was purified from the hemolymph of both spiders and was characterized by identifying subunit composition and measuring the sodium dodecyl sulfate (SDS)-induced PO activity. The high-altitude spider Hc showed higher PO activity under all conditions and higher apparent Michaelis-Menten constant. Moreover, the optimum temperature for PO activity was lower for E. condorito Hc. These findings suggest a potential adaptation at the level of Hc-derived PO activity in Euathlus condorito, giving insights on possible mechanisms used by this mygalomorph spider to occupy extremes and variable thermal environments., (Copyright © 2020. Published by Elsevier Inc.)

The fungal genus Massospora (Zoopagomycota: Entomophthorales) includes more than a dozen obligate, sexually transmissible pathogenic species that infect cicadas (Hemiptera) worldwide. At least two species are known to produce psychoactive compounds during infection, which has garnered considerable interest for this enigmatic genus. As with many Entomophthorales, the evolutionary relationships and host associations of Massospora spp. are not well understood. The acquisition of M. diceroproctae from Arizona, M. tettigatis from Chile, and M. platypediae from California and Colorado provided an opportunity to conduct molecular phylogenetic analyses and morphological studies to investigate whether these fungi represent a monophyletic group and delimit species boundaries. In a three-locus phylogenetic analysis including the D1-D2 domains of the nuclear 28S rRNA gene (28S), elongation factor 1 alpha-like ( EFL ), and beta-tubulin ( BTUB ), Massospora was resolved in a strongly supported monophyletic group containing four well-supported genealogically exclusive lineages, based on two of three methods of phylogenetic inference. There was incongruence among the single-gene trees: two methods of phylogenetic inference recovered trees with either the same topology as the three-gene concatenated tree ( EFL ) or a basal polytomy (28S, BTUB ). Massospora levispora and M. platypediae isolates formed a single lineage in all analyses and are synonymized here as M. levispora. Massospora diceroproctae was sister to M. cicadina in all three single-gene trees and on an extremely long branch relative to the other Massospora , and even the outgroup taxa, which may reflect an accelerated rate of molecular evolution and/or incomplete taxon sampling. The results of the morphological study presented here indicate that spore measurements may not be phylogenetically or diagnostically informative. Despite recent advances in understanding the ecology of Massospora , much about its host range and diversity remains unexplored. The emerging phylogenetic framework can provide a foundation for exploring coevolutionary relationships with cicada hosts and the evolution of behavior-altering compounds.

Preferential temperature as a physiological feature is crucial for spiders, since it determines the selection of key habitats for their survival and reproduction. In this work, we study the daily and geographical variation of the preferential temperature of the spider Sicarius thomisoides subjected to different degrees of daily thermal oscillation in their habitats. Preferred temperatures differ between coastal and inland populations, but in both cases, there is a marked bimodality in the daily pattern of temperature preference, with two peaks per day that would be given by the changes in the hours of activity. These nocturnal spiders select higher temperatures in the evening (active period) and select lower temperatures during late morning (resting period). In laboratory, spiders have preferred temperatures that differ from those found in their habitats, so they must tolerate or compensate non-preferred temperatures by active thermoregulation in natural conditions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Annona crassiflora Mart., popularly known as "Araticum", is a native tree of the Brazilian Cerrado used in folk medicine for treatment of pain and inflammatory diseases. We proposed to analyze analgesic and anti-inflammatory properties of the filtrate (F1) and the precipitate (F2) of the hydroalcoholic fraction from the leaves of Annona crassiflora Mart. in mice., Materials and Methods: Swiss mice were submitted to formalin-induced nociception test and tail-flick reflex test, to assess antinociceptive properties, and to the rota-rod test, for motor performance analyses. To evaluate anti-inflammatory properties, F1 and F2 were orally administered 1 h prior to the intrathoracic injection of carrageenan, zymosan, LPS, CXCL8, or vehicle in Balb/c mice and neutrophil infiltration was evaluated 4 h after injection., Results: F1 and F2 reduced the licking time in the second phase of formalin-induced nociception test, but only F2 showed a dose-dependent response. Neither F1 nor F2 reduced the latency time in the tail-flick reflex test. In addition, motor performance alteration was not observed in F1- or F2-treated mice. F2 treatment significantly inhibited the neutrophilia induced by carrageenan, LPS, or CXCL8, but not zymosan., Conclusions: The experimental data demonstrated that hydroalcoholic fractions of Annona crassiflora Mart. leaves have remarkable anti-inflammatory and antinociceptive activities.

Mitochondrial genomes can provide valuable information on the biology and evolutionary histories of their host organisms. Here, we present and characterize the complete coding regions of 107 mitochondrial genomes (mitogenomes) of cicadas (Insecta: Hemiptera: Auchenorrhyncha: Cicadoidea), representing 31 genera, 61 species, and 83 populations. We show that all cicada mitogenomes retain the organization and gene contents thought to be ancestral in insects, with some variability among cicada clades in the length of a region between the genes nad2 and cox1, which encodes 3 tRNAs. Phylogenetic analyses using these mitogenomes recapitulate a recent 5-gene classification of cicadas into families and subfamilies, but also identify a species that falls outside of the established taxonomic framework. While protein-coding genes are under strong purifying selection, tests of relative evolutionary rates reveal significant variation in evolutionary rates across taxa, highlighting the dynamic nature of mitochondrial genome evolution in cicadas. These data will serve as a useful reference for future research into the systematics, ecology, and evolution of the superfamily Cicadoidea., (© The American Genetic Association 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Xanthones represent a structurally diverse group of compounds with a broad range of biological and pharmacological activities, depending on the nature and position of various substituents in the dibenzo-γ-pyrone scaffold. Among the large number of natural and synthetic xanthone derivatives, carboxyxanthones are very interesting bioactive compounds as well as important chemical substrates for molecular modifications to obtain new derivatives. A remarkable example is 5,6-dimethylxanthone-4-acetic acid (DMXAA), a simple carboxyxanthone derivative, originally developed as an anti-tumor agent and the first of its class to enter phase III clinical trials. From DMXAA new bioactive analogues and derivatives were also described. In this review, a literature survey covering the report on carboxyxanthone derivatives is presented, emphasizing their biological activities as well as their application as suitable building blocks to obtain new bioactive derivatives. The data assembled in this review intends to highlight the therapeutic potential of carboxyxanthone derivatives and guide the design for new bioactive xanthone derivatives.