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Warming is increasingly recognized to magnify the toxicity of chemicals. Yet, limited understanding of the underlying physiological mechanisms constrains our ability to predict these interactions between warming and toxicants, and how these may evolve under global climate change scenarios. Here, a common-garden experiment was conducted with two resurrected subpopulations of Daphna magna, separated by ca. 40 years in time, of which the recent subpopulation evolved higher heat tolerance. Daphnia were exposed to low (150 µg L-1) and high (300 µg L-1) zinc levels across a large thermal gradient (12°C - 30°C). After 96-h exposure, bioenergetic traits, oxidative damage to lipids and hemoglobin levels were quantified. Both zinc levels caused stronger reductions in net energy budget at higher temperatures in both subpopulations. At the highest temperature, low zinc reduced energy consumption in the old subpopulation, whereas the recent subpopulation evolved an increased energy consumption, resulting in a greater decline in the net energy budget. Across treatment groups, survival correlated positively with the net energy budget and oxidative damage, and hemoglobin levels correlated positively with the acute heat tolerance. These findings suggest reductions in the net energy budget to be a key driver of the widespread phenomenon whereby toxicants become more lethal at higher temperatures, and reductions in hemoglobin to contribute to the toxicant-induced declines in heat tolerance. Overall, our study underscores the importance of incorporating physiological mechanisms to understand and predict the current and future sensitivity of organisms to contaminants under ongoing climate change.

Although allosteric regulation has been pointed out as one of the cornerstones of biological function, it has been a scarcely studied phenomenon in Archaea carbohydrate metabolism. Given its central role in metabolism, we experimentally investigated how allosteric regulation and its underlying kinetic mechanism evolved along evolutionary pathways within the archaeal ADP-dependent kinase family. Using ancestral sequence reconstruction, we resurrected key ancestors of this family and show that AMP regulation is an ancestral feature retained exclusively in lineages encoding bifunctional ADP-dependent phosphofructokinase (PFK)/glucokinase (GK) enzymes, which are restricted to methanogenic organisms, whereas it is lost in lineage-specific PFK enzymes. Notably, although AMP-dependent allosteric regulation is conserved among bifunctional ADP-PFK/GK enzymes, the kinetic mechanisms underlying activation are not. Instead, we observed a diversity of activation mechanisms (increased affinity for substrates, enhanced catalytic efficiency, or a combination of both), distributed along a 2-billion-year evolutionary trajectory, and that persists across different temperatures studied, both in extant and ancestral enzymes. These results highlight that the structural scaffold of this protein family is evolutionarily robust, preserving function while allowing substantial diversification of the underlying activation mechanisms under sequence variation. Based on these findings, we propose the concept of mechanistic drift, in which evolutionary pressures primarily act on adaptive functional traits that confer an adaptive advantage, rather than on the specific molecular mechanisms by which they are achieved. This framework has broad implications for macromolecular evolution, illustrating how long-term functional conservation can coexist with extensive physicochemical mechanistic diversity.

A new subgenus, Leonidasdeanea, of the genus Wyeomyia Theobald, 1901 is established based on morphological and molecular analyses. It comprises three sibling species: Wyeomyia chalcocephala Dyar & Knab, Wy. flui (Bonne-Wepster & Bonne) and Wy. luciae Senevet, Chabelard & Abonnenc, the last of which is resurrected from synonymy with Wy. chalcocephala. The larva, pupa and adult female and male, including the genitalia, of these three species are redescribed, along with a summary of the systematics, bionomics and distribution of the new subgenus. In addition, Wy.surinamensis Bruijning, 1959 is formally recognized as a junior synonym of Wy. flui.

The 100 kDa hexokinase (HK) enzyme family represents an attractive model to investigate the molecular origins of allosteric regulation in multidomain enzymes. Extant HK homologs are subject to various allosteric phenomena, including activation and inhibition by both homotropic and heterotropic ligands. Here, we report the results of a phylogenetic investigation of this enzyme family using the recently developed Topiary ancestral sequence reconstruction pipeline. The results agree with prior studies that used a smaller number of sequences from individual HK domains and suggest that modern HK3 isozymes diverged first from a 100 kDa ancestor, followed by gene duplication and divergence of the HK2 isozymes. A subsequent gene duplication event led to divergence of HK1 and the hexokinase domain-containing protein 1. To probe the ability of Topiary to yield functional, allosterically regulated ancestral enzymes, we resurrected and biochemically characterized two HKs from early vertebrate evolution, Anc1 and Anc2. Both enzymes were functionally similar to extant HK1 and possessed a low activity, regulatory N-terminal domain that governs allosteric regulation of the C-terminal active site by two heterotropic effectors, glucose 6-phosphate and inorganic phosphate. Neither ancestor was subject to homotropic regulation by substrate glucose, a characteristic observed in several extant HK3 family members. Our phylogenetic analysis provides a foundation for investigating the evolution of allostery in this enzyme family. It also demonstrates the need to sequence and biochemically characterize additional full-length HKs, especially those from jawless vertebrates, to enable more robust inferences of ancestral regulatory traits.

Four new species of the genus Callistethus Blanchard, 1851, including C. sinocontinentalis Zhao, sp. nov., C. abditus Zhao, sp. nov., C. huadongensis Zhao, sp. nov. and C. setimarginatus Zhao, sp. nov., are described mainly from the Chinese mainland. The identity of C. plagiicollis (Fairmaire, 1886) is clarified, and C. impictus (Bates, 1888) is resurrected as a valid species through examination of type material. Callistethus chrysochroma (Arrow, 1917) comb. nov. and C. xanthochroma (Arrow, 1917) comb. nov. are transferred from the genus Anomala Samouelle, 1819 to the genus Callistethus. Lectotypes are designated for C. plagiicollis, C. impictus, C. xanthochroma, and C. xanthonotus (Arrow, 1917). Callistethus isidai Miyake, 1987 stat. rev. and C. planicauda Fujioka & Kobayashi, 2012 stat. nov. are elevated from subspecies to species.

Species of the marine bivalve family Laternulidae are frequently misidentified with some having particularly tangled nomenclatural histories. To redress these problems all available type specimens or new images of them, totalling 52 of the 56 nominal species, were examined and assessed. Concurrently, new molecular analyses identified major clades and corroborated species boundaries based on shell characters. Two clades of laternulids were recognised as subfamilies Laternulinae and Exolaternulinae the latter introduced herein and characterised by presence of a lithodesma. A new classification of the family is presented. The depth of insertion of the pallial sinus is identified as a useful character separating species with similar shell shapes. Two new genera Cryolaternula and Parilaternula are proposed. Twenty-eight putative species are recognised with some resurrected from prior synonymy. Six new species are described: Laternula pristissinus Taylor & Glover, L. inopinata Taylor & Glover, L. omissa Taylor & Glover, Parilaternula acuta Taylor, Fukuda & Haga, P. certa Taylor & Glover and P. delicata Taylor, Glover & Hong. To stabilise the name Exolaternula a lectotype is selected for the type species, Anatina truncata Lamarck, 1818. Exolaternulinae species are distributed from the Arabian Gulf eastwards to Japan and eastern Russia and northern Australia at the southern limit. Laternulinae species occur around Australia, northern Indian Ocean, through the central Indo-W. Pacific to Japan, with Cryolaternula confined to the Southern Ocean within the Antarctic Convergence.

Theodore Cantor described Coronella violacea in 1839 for a colubrid snake collected from "Rungpore" (now Rangpur, Bangladesh). It was applied to several species in the genus Oligodon until Smith (1940) treated the name as a junior synonym of Oligodon cyclurus (Cantor, 1839). Evidence from the unpublished notes and watercolor drawings of Cantor indicate Coronella violacea is not a synonym of O. cyclurus, but is conspecific with Oligodon kheriensis Acharji & Ray, 1936. The collector of the holotype of Coronella violacea is identified as Honoratus Bonnevie, a Norwegian-born merchant and indigo plantation owner who lived in Rangpur during Cantor's residency in British India. Precedence cannot be reversed in favor of O. kheriensis, so the older name is resurrected as Oligodon violaceus stat. reinstat., with O. kheriensis relegated as a junior synonym. Since the holotype of Coronella violacea is considered lost, the holotype of Oligodon kheriensis is designated as the neotype, an act that objectively links the two nomina with one another.

Stipitate stereoid fungi are saprotrophic basidiomycetes characterized by a leathery basidiome, a central-to-lateral stipe and infundibuliform pilei. Although numerous species of stipitate stereoid fungi have been recorded worldwide, understanding of their phylogenetic relationships remains extremely limited, and research on this group of fungi in China is insufficient. In this study, specimens of the three stipitate stereoid genera, namely Podoscypha s. l., Cymatoderma s. l. and Stereopsis s. l., from southern China were investigated. Phylogenetic analyses of the internal transcribed spacer (ITS) regions and the large subunit of the nuclear ribosomal RNA gene (LSU) using maximum likelihood (ML) and Bayesian inference (BI) methods revealed that all three genera are polyphyletic. Consequently, Podoscypha s. s. and Cymatoderma s. s. were delimited, and Cladoderris-previously synonymized with Cymatoderma-was resurrected. Cladoderris is characterized by an imbricate basidiome, tomentose pilei and basidiospores typically shorter than 4 μm in length. Three new species, Podoscypha casiae, Stereopsis buccinata and Cladoderris perennis, were described and illustrated. The morphological distinctions and affinities between the new species and closely related taxa were discussed, the thresholds for the intraspecific and interspecific demarcation within the three genera in this study were provided, and identification keys for the species of each genus were presented.

Fifteen species belonging to the Longitarsus cyanipennis species group are revised. All species are described or redescribed, accompanied by images of external morphology and genitalia. A key to the species of the group is provided. Eleven new species are described: L. bryanti Liang & Konstantinov sp. nov., L. jinpingensis Liang & Konstantinov sp. nov., L. lijiangensis Liang & Konstantinov sp. nov., L. lodayi Liang & Konstantinov sp. nov., L. mauliki Liang & Konstantinov sp. nov., L. latipenis Liang & Konstantinov sp. nov., L. medogensis Liang & Konstantinov sp. nov., L. radialis Liang & Konstantinov sp. nov., L. viridis Liang & Konstantinov sp. nov., L. volkovitshi Liang & Konstantinov sp. nov., and L. zhejiangensis Liang & Konstantinov sp. nov. Longitarsus warchalowskii Scherer, 1969 is resurrected from synonymy with L. indigonaceus Lopatin, 1963, while L. indigonaceus Lopatin, 1963 syn. nov. is proposed as a junior synonym of L. cyanipennis Bryant, 1924. Amongst the species group, four species are recorded in Mêdog County, i.e., L. warchalowskii, L. bryantisp. nov., L. latipenis sp. nov., and L. medogensissp. nov.

Azomycin, as a representative example of valued nitro compounds, suffers from harsh reaction conditions and low yields in its traditional synthesis methods. Biocatalytic synthesis using N-oxygenase offers a green alternative but is constrained by the enzyme's limited efficiency and stability. To overcome these limitations, this study employed ancestral sequence reconstruction to identify a high-performance N-oxygenase. Seven ancestral node proteins, inferred from 83 homologous sequences, were resurrected and screened. The derived enzyme ASR-90 exhibited superior catalytic efficiency (kcat/Km) toward 2-aminoimidazole, exceeding its parent enzyme and a key mutant by 546-fold and 92-fold, respectively. Furthermore, ASR-90 exhibited improved acid tolerance (optimal pH 4.5) and a 4-fold longer half-life at 50 °C. Under optimal conditions, it achieved 50.9% substrate conversion in 20 min─2.7-fold higher than that of the best reported enzyme. The substrate scope analysis revealed that ASR-90 displayed catalytic ability on 16 non-natural substrates. Molecular dynamics simulations indicated that the enhanced catalytic efficiency stems from increased substrate binding stability and affinity. Its broad substrate scope and preference for aromatic amines are attributed to a balanced active-site pocket, electrostatic guidance, and a continuous aromatic track within the substrate channel. This study establishes ancestral sequence reconstruction as an effective strategy for developing robust N-oxygenases, facilitating the green biosynthesis of azomycin and its derivatives.

Protein regulation is essential for cellular function and mis-regulation commonly causes disease. Despite this fact, we know little about how new regulatory strategies first emerge and how they evolve to act in concert to control complex physiological processes. Glucokinase (GCK), the body's glucose sensor, lies at the heart of vertebrate glucose homeostasis and its activity is tightly controlled by multiple regulatory mechanisms. In the pancreas and liver, GCK is regulated by a unique form of monomeric allostery originating from the unliganded enzyme's conformational dynamics. In the liver, GCK and GKRP form an inhibitory protein-protein interaction that sequesters GCK within the hepatocyte nucleus. Using a vertical, evolutionary approach, we resurrected extinct GCKs and GKRPs along correlated evolutionary trajectories. Using enzyme kinetics, limited proteolysis, hydrogen-deuterium exchange, high resolution NMR, and X-ray crystallography we determined the historical and molecular origins of protein regulation. Prior to the emergence of jawed vertebrates, a non-regulated GCK ancestor underwent a conformational expansion leading to monomeric allostery. This novel conformation includes an intrinsically disordered substrate binding loop. Paradoxically, the emergence of disorder did not require sequence change in the loop. The new GCK conformation also exposed a hydrophobic cleft. In the jawed vertebrate GKRP ancestor, a de novo loop insertion enabled exaptation of the pre-existing hydrophobic patch in GCK. Our results demonstrate how multiple, distinct regulatory strategies can arise at a central homeostatic control point through evolutionary addition of novel conformations. Additionally, our results provide a general mechanism for the emergence of heteromeric protein-protein interactions. Glucose homeostasis was a key innovation in vertebrate evolution. Here, we uncover the evolutionary basis of regulation in two key homeostatic proteins, glucokinase (GCK) and glucokinase regulatory protein (GKRP). We find that the unique cooperativity of vertebrate GCK resulted from an expansion of this enzyme's conformational landscape. This expansion included sampling a new state and the emergence of intrinsic disorder, which did not require substitutions in the disordered region itself. We also discover that the GCK-GKRP interaction emerged when a pre-existing hydrophobic surface - a structural spandrel resulting from prior conformational expansion - was co-opted by loop insertion in GKRP, facilitating a new, inhibitory heteromeric interaction. Our results demonstrate how multiple, mechanistically distinct regulatory strategies arise from an ability to sample new protein conformations.

The genus Dryophylax currently comprises 15 widely distributed South American species of snakes. Despite recent efforts to clarify the systematic status of the genus, several species remain taxonomically poorly defined. One example is Dryophylaxnattereri (Mikan 1820), regarded first as a junior synonym of Thamnodynastes strigilis (Thunberg 1787), and later as a junior synonym of Thamnodynastes pallidus (Linnaeus 1758). Despite several historical taxonomic studies addressing other congeners, D. nattereri has been often neglected, and no author has attempted to test species boundaries. Recently, several authors referred to this taxon as Dryophylax cf. nattereri, after it was resurrected without a proper taxonomic justification. Here, we demonstrate that D.nattereri is a valid species, based on morphological data gathered from a large series of individuals distributed throughout its known distribution. We designated a neotype for D.nattereri and provide a detailed description of the species diagnostic characters, with additional data on external morphological variation, skull osteology and hemipenial morphology.

The resurrection plant Haberlea rhodopensis is a rare species endemic to Greece and Bulgaria, renowned for its exceptional desiccation tolerance and rich phytochemical composition. This study investigated the antioxidant, cytoprotective, and wound-healing-associated effects of H. rhodopensis ethanolic extract (HEE) in human keratinocytes (HaCaT cells) under oxidative and cytotoxic stress conditions. Antioxidant capacity was initially evaluated using a plasmid DNA protection assay, in which HEE attenuated oxidative DNA damage induced by a Fenton reaction system and preserved the native supercoiled structure of pUC19 plasmid DNA. Cytotoxicity screening using the sulforhodamine B (SRB) assay and real-time proliferation monitoring (HoloMonitor® M4) identified 20 μg/mL as a non-toxic pre-treatment concentration (EC10). Under hydrogen peroxide (H2O2)-induced oxidative stress, HEE pre-treatment maintained cell viability and significantly reduced intracellular reactive oxygen species (ROS) levels, indicating a protective effect. In vitro wound-healing assays demonstrated enhanced scratch closure in keratinocyte monolayers. RT-qPCR analysis revealed modulation of antioxidant-related genes (CAT, SOD1, HMOX1, NQO1, GPX, GSR), while mRNA sequencing suggested selective stress-adaptive responses, involving extracellular matrix (ECM)-, metabolic-, and tissue-repair/aging-associated pathways. Overall, HEE exhibits antioxidant and cytoprotective effects in keratinocytes and is associated with transcriptional changes linked to cellular stress responses and wound closure. These findings support its potential relevance for dermatological, pharmaceutical, and cosmeceutical applications, while further studies are required to establish the underlying molecular mechanisms.

Since the outbreak of the COVID-19 pandemic, extensive efforts including vaccine and drug development have been accentuated to address the emergence of SARS-CoV-2 variants. The rising variants and subvariants made early discoveries in effective treatment strategies less relevant and potent. For instance, multiple therapeutics like Evusheld are no longer effective against current variants and therefore have their emergency use authorizations (EUA), a regulatory mechanism allowing emergency use of medical products, revoked until further notice. Similarly, sotrovimab (STV), a human neutralizing monoclonal antibody (MAB), received EUA in May 2021 and was later granted authorization for COVID-19 treatment in Singapore. However, mutations presented in XBB, a subvariant of Omicron, substantially reduced the potency of STV at the currently approved dose. To retain STV as a part of the treatment strategy for COVID-19 infected patients, it is important to carefully design and prioritize drugs that may further enhance the potency of STV when paired in combinations. In this study, IDentif.AI, an artificial intelligence (AI)-derived drug combination optimization platform, was harnessed to rapidly pinpoint STV-based combinations that may substantially improve the efficacy and potency of STV against XBB, which was predominantly spreading worldwide in 2023. IDentif.AI-pinpointed STV/EIDD-1931 and STV/GS-441524 combinations were able to interact synergistically to enhance efficacy against XBB and importantly, substantially reduce STV's in vitro EC50 (half maximal effective concentration) by up to 7-fold. This optimization platform represents a strategic approach to rapidly repurpose existing or previously failed therapeutics and subsequently restore their efficacy against a disease indication by pairing them with the correct drugs in combinations.

The examination of molecular markers confirms hidden diversity and reveals a new species within the Nyctixalus pictus (Peters, 1871) complex. Three genetically distinct clades were recovered: one from the Thai-Malay Peninsula; one from Sumatra, Indonesia; and one from Sarawak, Borneo, East Malaysia. Voucher specimens from each region were compared, and significantly different mean values were found through morphological analyses. The populations also differ significantly in morphospace, as determined by PCA, DAPC, and MFA analyses. The Thai-Malay Peninsula individuals have a distinct, rounded and slightly elongated snout shape compared to the other two clades. Based on these combined data, these three clades are recognized as distinct species. The name N. anodon is reinstated for those from Sumatra, Indonesia. The type locality of Sarawak, East Malaysia for N. pictus restricts the distribution of the species to Borneo. The type specimen of N. pictus was lost (pers. communication, G. Doria), and no lectotype or syntypes were described; thus, a specimen from Sarawak, Borneo, in the collection of the Field Museum of Natural History, Chicago (FMNH), is designated here as the neotype of N. pictus.

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White's Skinks, Liopholis whitii (Lacépède 1804) are widely distributed throughout rocky habitats of temperate south-eastern Australia, with a highly disjunct population occurring in Mutawintji National Park in arid far western New South Wales. Based on an analysis of genome-wide nuclear single-nucleotide polymorphisms (SNPs) and an assessment of variation in morphology, we provide an appraisal of the group's evolutionary history and re-evaluate the taxonomic status of candidate lineages. We reveal the presence of three major genetic lineages, including two lineages from temperate south-eastern Australia, and another representing the isolated arid population from Mutawintji National Park. We herein apply the name Liopholis whitii to the temperate "southern" lineage which occurs in South Australia, Victoria and Tasmania; resurrect the name Liopholis compressicauda (Quoy & Gaimard 1824) for the temperate "northern" lineage from southern Queensland, New South Wales, Australian Capital Territory and north-eastern Victoria; and describe the arid lineage from Mutawintji National Park as Liopholis mutawintji sp. nov. Liopholis mutawintji sp. nov. is of particular conservation concern and likely eligible for listing as Critically Endangered under multiple IUCN Red List Criteria.

The genus Limnocoris is the only genus in the New World subfamily Limnocorinae (Naucoridae) and is predominantly neotropical in distribution. Presented here is the fourth and final contribution in a series to revise the genus; the species of eastern South America are revised, including the descriptions of three new species from Brazil: L. diamantinensissp. nov., L. hirsutussp. nov., and L. valimisp. nov. Also, Limnocoris montandoni La Rivers stat. restit. is resurrected from synonymy with L. abbreviatus La Rivers and its species status restored. Further, supplemental descriptions are provided for the previously described species: L. abbreviatus, L. aculabrum La Rivers, L. acutalis La Rivers, L. asper Nieser & Lopez-Ruf, L. brasiliensis De Carlo, L. decarloi Nieser & Lopez-Ruf, L. espinolai Nieser & Lopez-Ruf, L. insignis Stål, L. machrisi Nieser & Lopez-Ruf, L. melloleitaoi De Carlo, L. minutus De Carlo, L. montandoni, L. pauper Montandon, L. rotundatus De Carlo, L. saphis Nieser & Lopez-Ruf, L. sattleri De Carlo, L. siolii (De Carlo), L. submontandoni La Rivers, and L. volxemi (Lethierry). The following synonymies are proposed, with junior synonyms in brackets: L. espinolai [L. porphyros Nieser & Lopez-Ruf n. syn.], L. insignis [L. caraceae Nieser & Lopez-Ruf n. syn.], and L. pauper [L. subpauper Nieser & Lopez-Ruf n. syn.]. An illustrated taxonomic key, distribution maps, and a checklist for all species in the genus also are presented.

Reviving extinct animals offers a crucial opportunity to recover lost or unknown genetic resources, yet cloning methods are unsuitable because they depend on intact donor nuclei and abundant oocytes or recipients from closely related species. To overcome these constraints, we explored a chromosome level revival strategy. Blood cells from rat carcasses stored at - 30 °C for over one year were introduced into enucleated mouse oocytes, where the rat nuclei underwent premature chromosome condensation. Microtubule polymerization inhibition enabled dispersion of rat chromosomes within the ooplasm, allowing isolation of individual chromosomes by micromanipulation. Each chromosome was subsequently transferred into an intact mouse oocyte, followed by intracytoplasmic sperm injection using GFP-transgenic mouse sperm. Embryos were cultured to the blastocyst stage, yielding 17 ES cell lines, two of which carried 41 chromosomes. Spectral karyotyping confirmed the presence of rat chromosome 9 alongside a full set of normal mouse chromosomes. These ES cells generated chimeric mice exhibiting GFP based chimerism across multiple organs. Histological analyses further demonstrated expression of numerous genes located on rat chromosome 9 within chimera mouse. This study demonstrated that a single chromosome from a frozen extinct species can be functionally revived and its transcriptional activity assessed within an interspecies oocyte.