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Severe acute respiratory syndrome-related Coronavirus 2 (SARS-CoV-2) has infected more than 762 million people to date and has caused approximately 7 million deaths all around the world, involving more than 187 countries. Although currently available vaccines show high efficacy in preventing severe respiratory complications in infected patients, the high number of mutations in the S proteins of the current variants is responsible for the high level of immune evasion and transmissibility of the virus and the reduced effectiveness of acquired immunity. In this scenario, the development of safe and effective drugs of synthetic or natural origin to suppress viral replication and treat acute forms of COVID-19 remains a valid therapeutic challenge. Given the successful history of flavonoids-based drug discovery, we developed esters of substituted cinnamic acids with quercetin to evaluate their in vitro activity against a broad spectrum of Coronaviruses. Interestingly, two derivatives, the 3,4-methylenedioxy 6 and the ester of acid 7, have proved to be effective in reducing OC43-induced cytopathogenicity, showing interesting EC50s profiles. The ester of synaptic acid 7 in particular, which is not endowed with relevant cytotoxicity under any of the tested conditions, turned out to be active against OC43 and SARS-CoV-2, showing a promising EC 50 . Therefore, said compound was selected as the lead object of further analysis. When tested in a yield reduction, assay 7 produced a significant dose-dependent reduction in viral titer. However, the compound was not virucidal, as exposure to high concentrations of it did not affect viral infectivity, nor did it affect hCoV-OC43 penetration into pre-treated host cells. Additional studies on the action mechanism have suggested that our derivative may inhibit viral endocytosis by reducing viral attachment to host cells.

A series of 1-(4-sulfamoylbenzoyl)piperidine-4-carboxamides deriving from substituted piperazines/benzylamines was designed, synthesized, and tested on human carbonic anhydrase (hCA). The inhibitory activity of the new sulfonamides was analyzed using acetazolamide (AAZ) as a standard inhibitor against hCA I, II, IX, and XII. Several sulfonamides showed both inhibitory activity at low nanomolar concentrations and selectivity against the cytosolic hCA II isoform, and the same trend was observed on the tumor-associated hCA IX and XII. The benzenesulfonamido carboxamides 11 and 15 were the most potent of the piperazino- and benzylamino-based series, respectively. Docking and molecular dynamics studies related the high selectivity of compound 11 toward the tumor-associated hCA isoforms to its capability to participate in favorable interactions within hCA IX and hCA XII active sites, whereas no such interactions were detected within both hCA I and hCA II isoforms., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Background: The National Surgical Quality Improvement Program (NSQIP) Surgical Risk Calculator from the American College of Surgeons is a widely available tool for peri-operative risk prediction. This study investigates its predictive performance in an Australian setting., Methods: A single-centre retrospective external validation study was conducted at a tertiary referral centre in New South Wales, Australia. Data from a general surgical cohort in a 2-year period from 2020 to 2021 was collected from the NSQIP database and entered into the NSQIP calculator. The predictive performance of the calculator was analysed across the standard 14 NSQIP postoperative outcome measures at 30 days., Results: There were 2121 patient records analysed using tests of accuracy and in the discrimination and calibration domains. The overall predictive performance of the NSQIP calculator was reasonable. There was greater accuracy at lower-risk predictions. At higher-risk predictions, Readmission, Death, and Discharge to Nursing or Rehab Facility, and Length of Stay were overestimated, whilst other outcomes were underestimated., Conclusion: This study demonstrates reasonable overall performance of the NSQIP calculator in the context of this cohort and provides data to support the need for locally adapted and validated risk prediction tools for use by Australian perioperative physicians., (© 2023 Royal Australasian College of Surgeons.)

In this update paper, we present the latest developments in the OMA browser knowledgebase, which aims to provide high-quality orthology inferences and facilitate the study of gene families, genomes and their evolution. First, we discuss the addition of new species in the database, particularly an expanded representation of prokaryotic species. The OMA browser now offers Ancestral Genome pages and an Ancestral Gene Order viewer, allowing users to explore the evolutionary history and gene content of ancestral genomes. We also introduce a revamped Local Synteny Viewer to compare genomic neighborhoods across both extant and ancestral genomes. Hierarchical Orthologous Groups (HOGs) are now annotated with Gene Ontology annotations, and users can easily perform extant or ancestral GO enrichments. Finally, we recap new tools in the OMA Ecosystem, including OMAmer for proteome mapping, OMArk for proteome quality assessment, OMAMO for model organism selection and Read2Tree for phylogenetic species tree construction from reads. These new features provide exciting opportunities for orthology analysis and comparative genomics. OMA is accessible at https://omabrowser.org., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many proteins exist in the so-called "twilight zone" of sequence alignment, where low pairwise sequence identity makes it difficult to determine homology and phylogeny. 1 , 2 As protein tertiary structure is often more conserved, 3 recent advances in ab initio protein folding have made structure-based identification of putative homologs feasible. 4 , 5 , 6 We present a pipeline for the identification and characterization of distant homologs and apply it to 7-transmembrane-domain ion channels (7TMICs), a protein group founded by insect odorant and gustatory receptors. Previous sequence and limited structure-based searches identified putatively related proteins, mainly in other animals and plants. 7 , 8 , 9 , 10 However, very few 7TMICs have been identified in non-animal, non-plant taxa. Moreover, these proteins' remarkable sequence dissimilarity made it uncertain whether disparate 7TMIC types (Gr/Or, Grl, GRL, DUF3537, PHTF, and GrlHz) are homologous or convergent, leaving their evolutionary history unresolved. Our pipeline identified thousands of new 7TMICs in archaea, bacteria, and unicellular eukaryotes. Using graph-based analyses and protein language models to extract family-wide signatures, we demonstrate that 7TMICs have structure and sequence similarity, supporting homology. Through sequence- and structure-based phylogenetics, we classify eukaryotic 7TMICs into two families (Class-A and Class-B), which are the result of a gene duplication predating the split(s) leading to Amorphea (animals, fungi, and allies) and Diaphoretickes (plants and allies). Our work reveals 7TMICs as a cryptic superfamily, with origins close to the evolution of cellular life. More generally, this study serves as a methodological proof of principle for the identification of extremely distant protein homologs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The development of drugs for the treatment of advanced prostate cancer (PCA) remains a challenging task. In this study we have designed, synthesized and tested twenty-nine novel HDAC inhibitors based on three different zinc binding groups (trifluoromethyloxadiazole, hydroxamic acid, and 2-mercaptoacetamide). These warheads were conveniently tethered to variously substituted phenyl linkers and decorated with differently substituted pyrrolo-pyrimidine and purine cap groups. Remarkably, most of the compounds showed nanomolar inhibitory activity against HDAC6. To provide structural insights into the Structure-Activity Relationships (SAR) of the investigated compounds, docking of representative inhibitors and molecular dynamics of HDAC6-inhibitor complexes were performed. Compounds of the trifluoromethyloxadiazole and hydroxamic acid series exhibited promising anti-proliferative activities, HDAC6 targeting in PCA cells, and in vitro tumor selectivity. Representative compounds of the two series were tested for solubility, cell permeability and metabolic stability, demonstrating favorable in vitro drug-like properties. The more interesting compounds were subjected to migration assays, which revealed that compound 13 and, to a lesser extent, compound 15 inhibited the invasive behaviour of androgen-sensitive and -insensitive advanced prostate cancer cells. Compound 13 was profiled against all HDACs and found to inhibit all members of class II HDACs (except for HDAC10) and to be selective with respect to class I and class IV HDACs. Overall, compound 13 combines potent inhibitory activity and class II selectivity with favorable drug-like properties, an excellent anti-proliferative activity and marked anti-migration properties on PCA cells, making it an excellent lead candidate for further optimization., 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 © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The main protease (M pro ) plays a pivotal role in the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is considered a highly conserved viral target. Disruption of the catalytic activity of M pro produces a detrimental effect on the course of the infection, making this target one of the most attractive for the treatment of COVID-19. The current success of the SARS-CoV-2 M pro inhibitor Nirmatrelvir, the first oral drug for the treatment of severe forms of COVID-19, has further focused the attention of researchers on this important viral target, making the search for new M pro inhibitors a thriving and exciting field for the development of antiviral drugs active against SARS-CoV-2 and related coronaviruses.

Vaccines have been hailed as one of the most remarkable medical advancements in human history, and their potential for treating cancer by generating or expanding anti-tumor T cells has garnered significant interest in recent years. However, the limited efficacy of therapeutic cancer vaccines in clinical trials can be partially attributed to the inadequacy of current preclinical mouse models in recapitulating the complexities of the human immune system. In this study, we developed two innovative humanized mouse models to assess the immunogenicity and therapeutic effectiveness of vaccines targeting human papillomavirus (HPV16) antigens and delivering tumor antigens to human CD141 + dendritic cells (DCs). Both models were based on the transference of human peripheral blood mononuclear cells (PBMCs) into immunocompromised HLA-A*02-NSG mice (NSG-A2), where the use of fresh PBMCs boosted the engraftment of human cells up to 80%. The dynamics of immune cells in the PBMC-hu-NSG-A2 mice demonstrated that T cells constituted the vast majority of engrafted cells, which progressively expanded over time and retained their responsiveness to ex vivo stimulation. Using the PBMC-hu-NSG-A2 system, we generated a hyperplastic skin graft model expressing the HPV16-E7 oncogene. Remarkably, human cells populated the skin grafts, and upon vaccination with a DNA vaccine encoding an HPV16-E6/E7 protein, rapid rejection targeted to the E7-expressing skin was detected, underscoring the capacity of the model to mount a vaccine-specific response. To overcome the decline in DC numbers observed over time in PBMC-hu-NSG-A2 animals, we augmented the abundance of CD141 + DCs, the specific targets of our tailored nanoemulsions (TNEs), by transferring additional autologous PBMCs pre-treated in vitro with the growth factor Flt3-L. The Flt3-L treatment bolstered CD141 + DC numbers, leading to potent antigen-specific CD4 + and CD8 + T cell responses in vivo , which caused the regression of pre-established triple-negative breast cancer and melanoma tumors following CD141 + DC-targeting TNE vaccination. Notably, using HLA-A*02-matching PBMCs for humanizing NSG-A2 mice resulted in a delayed onset of graft-versus-host disease and enhanced the efficacy of the TNE vaccination compared with the parental NSG strain. In conclusion, we successfully established two humanized mouse models that exhibited strong antigen-specific responses and demonstrated tumor regression following vaccination. These models serve as valuable platforms for assessing the efficacy of therapeutic cancer vaccines targeting HPV16-dysplastic skin and diverse tumor antigens specifically delivered to CD141 + DCs.

COVID-19 now ranks among the most devastating global pandemics in history. The causative virus, SARS-CoV-2, is a new human coronavirus (hCoV) that spreads among humans and animals. Great efforts have been made to develop therapeutic agents to treat COVID-19, and among the available viral molecular targets, the cysteine protease SARS-CoV-2 M pro is considered the most appealing one due to its essential role in viral replication. However, the inhibition of M pro activity is an interesting challenge and several small molecules and peptidomimetics have been synthesized for this purpose. In this work, the Michael acceptor cinnamic ester was employed as an electrophilic warhead for the covalent inhibition of M pro by endowing some peptidomimetic derivatives with such a functionality. Among the synthesized compounds, the indole-based inhibitors 17 and 18 efficiently impaired the in vitro replication of beta hCoV-OC-43 in the low micromolar range (EC 50 = 9.14 μM and 10.1 μM, respectively). Moreover, the carbamate derivative 12 showed an antiviral activity of note (EC 50 = 5.27 μM) against another hCoV, namely hCoV-229E, thus suggesting the potential applicability of such cinnamic pseudopeptides also against human alpha CoVs. Taken together, these results support the feasibility of considering the cinnamic framework for the development of new M pro inhibitors endowed with antiviral activity against human coronaviruses.

Suitable methods to assess in vivo immunogenicity and therapeutic efficacy of cancer vaccines in preclinical cancer models are critical to overcome current limitations of cancer vaccines and enhance the clinical applicability of this promising immunotherapeutic strategy. In particular, availability of methods allowing the characterization of T cell responses to endogenous tumor antigens is required to assess vaccine potency and improve the antigen formulation. Moreover, multiparametric assays to deeply characterize tumor-induced and therapy-induced immune modulation are relevant to design mechanism-based combination immunotherapies. Here we describe a versatile multiparametric flow cytometry method to assess the polyfunctionality of tumor antigen-specific CD4 + and CD8 + T cell responses based on their production of multiple cytokines after short-term ex vivo restimulation with relevant tumor epitopes of the most common mouse strains. We also report the development and application of two 21-color flow cytometry panels allowing a comprehensive characterization of T cell and natural killer cell exhaustion and memory phenotypes in mice with a particular focus on preclinical cancer models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Moi, Zeng, Minnie, Bhatt, Wood, Sester, Mazzieri and Dolcetti.)

SARS-CoV-2 M pro is a chymotrypsin-like cysteine protease playing a relevant role during the replication and infectivity of SARS-CoV-2, the coronavirus responsible for COVID-19. The binding site of M pro is characterized by the presence of a catalytic Cys145 which carries out the hydrolytic activity of the enzyme. As a consequence, several M pro inhibitors have been proposed to date in order to fight the COVID-19 pandemic. In our work, we designed, synthesized and biologically evaluated MPD112 , a novel inhibitor of SARS-CoV-2 M pro bearing a trifluoromethyl diazirine moiety. MPD112 displayed in vitro inhibition activity against SARS-CoV-2 M pro at a low micromolar level (IC 50 = 4.1 μM) in a FRET-based assay. Moreover, an inhibition assay against PL pro revealed lack of inhibition, assuring the selectivity of the compound for the M pro . Furthermore, the target compound MPD112 was docked within the binding site of the enzyme to predict the established intermolecular interactions in silico. MPD112 was subsequently tested on the HCT-8 cell line to evaluate its effect on human cells' viability, displaying good tolerability, demonstrating the promising biological compatibility and activity of a trifluoromethyl diazirine moiety in the design and development of SARS-CoV-2 M pro binders.

A small series of hydrazonobenzenesulfonamides was designed, synthesized and studied for their human carbonic anhydrase (hCA) inhibitory activity. The synthesized compounds were evaluated against hCA I, II, IX and XII isoforms using acetazolamide (AAZ) as the standard inhibitor. Various hydrazonosulfonamide derivatives showed inhibitory activity at low nanomolar levels with selectivity against the cytosolic hCA II isoform, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. The most potent and selective hydrazones 8 , 9 , 10 , 11 , 19 and 24 were docked into isoforms I, II, IX and XII to better understand their activity and selectivity for the different CA isoforms.

A series of 6- and 6,8-halocoumarin derivatives have been investigated as potential antiproliferative compounds against a panel of tumor and normal cell lines. Cytotoxic effects were determined by the MTT method. To investigate the potential molecular mechanism involved in the cytotoxic effect, apoptosis assay, cell cycle analysis, reactive oxygen species (ROS), and reduced glutathione analysis were performed. Among the screened compounds, coumarins 6,8-dibromo-2-oxo-2 H -chromene-3-carbonitrile 2h and 6,8-diiodo-2-oxo-2 H -chromene-3-carbonitrile 2k exhibited the most antiproliferative effect in thyroid cancer-derived cells TPC-1. The apoptosis assay showed that both 2h and 2k induced apoptosis in TPC-1 thyroid cancer cells. According to these experiments, both coumarins induced a slight increase in TPC-1 cells in the G2/M phase and a decrease in the S phase. A significant increase in ROS levels was observed in TPC-1 treated with diiodocoumarin 2k , while the dibromocoumarin 2h induced a decrease in ROS in a dose and time-dependent manner.

The straightforward, continuous-flow synthesis of cyclopropyl carbaldehydes and ketones has been developed starting from 2-hydroxycyclobutanones and aryl thiols. This acid-catalyzed mediated procedure allows access to the multigram and easily scalable synthesis of cyclopropyl adducts under mild conditions, using reusable Amberlyst-35 as a catalyst. The resins, suitably ground and used for filling steel columns, have been characterized via TGA, ATR, SEM and BET analyses to describe the physical-chemical properties of the packed bed and the continuous-flow system in detail. To highlight the synthetic versatility of the arylthiocyclopropyl carbonyl compounds, a series of selective oxidation reactions have been performed to access sulfoxide and sulfone carbaldehyde cyclopropanes, oxiranes and carboxylic acid derivatives.

Here we report a small library of hydrazinocarbonyl-ureido and thioureido benzenesulfonamide derivatives, designed and synthesized as potent and selective human carbonic anhydrase inhibitors (hCAIs). The synthesized compounds were evaluated against isoforms hCA I, II, IX and XII using acetazolamide (AAZ) as standard inhibitor. Several urea and thiourea derivatives showed inhibitory activity at low nanomolar levels with selectivity against the cytosolic hCA II isoform, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. The thiourea derivatives showed enhanced potency as compared to urea analogues. Additionally, eight compounds 5g , 5m , 5o , 5q , 6l , 6j , 6o and 6u were selected for docking analysis on isoform I, II, IX, XII to illustrate the potential interaction with the enzyme to better understand the activity against the different isoforms.

Sexual reproduction consists of genome reduction by meiosis and subsequent gamete fusion. The presence of genes homologous to eukaryotic meiotic genes in archaea and bacteria suggests that DNA repair mechanisms evolved towards meiotic recombination. However, fusogenic proteins resembling those found in gamete fusion in eukaryotes have so far not been found in prokaryotes. Here, we identify archaeal proteins that are homologs of fusexins, a superfamily of fusogens that mediate eukaryotic gamete and somatic cell fusion, as well as virus entry. The crystal structure of a trimeric archaeal fusexin (Fusexin1 or Fsx1) reveals an archetypical fusexin architecture with unique features such as a six-helix bundle and an additional globular domain. Ectopically expressed Fusexin1 can fuse mammalian cells, and this process involves the additional globular domain and a conserved fusion loop. Furthermore, archaeal fusexin genes are found within integrated mobile elements, suggesting potential roles in cell-cell fusion and gene exchange in archaea, as well as different scenarios for the evolutionary history of fusexins., (© 2022. The Author(s).)

Histone deacetylase (HDAC) inhibitors are highly involved in the regulation of many pharmacological responses, which results in anti-inflammatory and anti-cancer effects. In the present work, chemoinformatic analyses were performed to obtain two potent and selective aminotriazoloquinazoline-based HDAC6 inhibitors. We unexpectedly obtained an aminotriazole from a water-driven ring opening of the triazoloquinazoline scaffold. Both compounds were evaluated as HDAC6 inhibitors, resulting in subnanomolar inhibitory activity and high selectivity with respect to class I HDAC1 and HDAC8. Importantly, the compounds were about 3- and 15-fold more potent compared to the reference compound trichostatin A. Additionally, the predicted binding modes were investigated with docking. Considering that the aminotriazole scaffold has never been embedded into the chemical structure of HDAC6 inhibitors, the present study suggests that both the aminotriazoloquinazoline and aminotriazole classes of compounds could be excellent starting points for further optimization of potential anticancer compounds, introducing such novel groups into a relevant and new area of investigation., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Protein posttranslational modifications add great sophistication to biological systems. Citrullination, a key regulatory mechanism in human physiology and pathophysiology, is enigmatic from an evolutionary perspective. Although the citrullinating enzymes peptidylarginine deiminases (PADIs) are ubiquitous across vertebrates, they are absent from yeast, worms, and flies. Based on this distribution PADIs were proposed to have been horizontally transferred, but this has been contested. Here, we map the evolutionary trajectory of PADIs into the animal lineage. We present strong phylogenetic support for a clade encompassing animal and cyanobacterial PADIs that excludes fungal and other bacterial homologs. The animal and cyanobacterial PADI proteins share functionally relevant primary and tertiary synapomorphic sequences that are distinct from a second PADI type present in fungi and actinobacteria. Molecular clock calculations and sequence divergence analyses using the fossil record estimate the last common ancestor of the cyanobacterial and animal PADIs to be less than 1 billion years old. Additionally, under an assumption of vertical descent, PADI sequence change during this evolutionary time frame is anachronistically low, even when compared with products of likely endosymbiont gene transfer, mitochondrial proteins, and some of the most highly conserved sequences in life. The consilience of evidence indicates that PADIs were introduced from cyanobacteria into animals by horizontal gene transfer (HGT). The ancestral cyanobacterial PADI is enzymatically active and can citrullinate eukaryotic proteins, suggesting that the PADI HGT event introduced a new catalytic capability into the regulatory repertoire of animals. This study reveals the unusual evolution of a pleiotropic protein modification., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The design of multi-target drugs acting simultaneously on multiple signaling pathways is a growing field in medicinal chemistry, especially for the treatment of complex diseases, such as cancer. Histone deacetylase 6 (HDAC6) is an established anticancer drug target involved in tumor cells transformation. Being an epigenetic enzyme at the interplay of many biological processes, HDAC6 has become an attractive target for polypharmacology studies aimed at improving the therapeutic efficacy of anticancer drugs. For example, the molecular chaperone Heat shock protein 90 (Hsp90) is a substrate of HDAC6 deacetylation, and several lines of evidence demonstrate that simultaneous inhibition of HDAC6 and Hsp90 promotes synergistic antitumor effects on different cancer cell lines, highlighting the potential benefits of developing a single molecule endowed with multi-target activity. This review will summarize the complex interplay between HDAC6 and Hsp90, providing also useful hints for multi-target drug design and discovery approaches in this field. To this end, crystallographic structures of HDAC6 and Hsp90 complexes will be extensively reviewed in light of discussing binding pockets features and pharmacophore requirements and providing useful guidelines for the design of dual inhibitors. The few examples of multi-target inhibitors obtained so far, mostly based on chimeric approaches, will be summarized and put into context. Finally, the main features of HDAC6 and Hsp90 inhibitors will be compared, and ligand- and structure-based strategies potentially useful for the development of small molecular weight dual inhibitors will be proposed and discussed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Histone deacetylase 6 (HDAC6) is an established drug target for cancer treatment. Inhibitors of HDAC6 based on a hydroxamic acid zinc binding group (ZBG) are often associated with undesirable side effects. Herein, we describe the identification of HDAC6 inhibitors based on a completely new 3-hydroxy-isoxazole ZBG. A series of derivatives decorated with different aromatic or heteroaromatic linkers, and various cap groups were synthesised and biologically tested. In vitro tests demonstrated that some compounds are able to inhibit HDAC6 with good potency, the best candidate reaching an IC 50 of 700 nM. Such good potency obtained with a completely new ZBG make these compounds particularly attractive. The effect of the most active inhibitors on the acetylation levels of histone H3 and α- tubulin and their anti-proliferative activity of DU145 cells were also investigated. Docking studies were performed to evaluate the binding mode of these new derivatives and discuss structure-activity relationships.

Since the approval of three hydroxamic acid-based HDAC inhibitors as anticancer drugs, such functional groups acquired even more notoriety in synthetic medicinal chemistry. The ability of hydroxamic acids (HAs) to chelate metal ions makes this moiety an attractive metal binding group-in particular, Fe(III) and Zn(II)-so that HA derivatives find wide applications as metalloenzymes inhibitors. In this minireview, we will discuss the most relevant features concerning hydroxamic acid derivatives. In a first instance, the physicochemical characteristics of HAs will be summarized; then, an exhaustive description of the most relevant methods for the introduction of such moiety into organic substrates and an overview of their uses in medicinal chemistry will be presented., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Gene duplications and novel genes have been shown to play a major role in helminth adaptation to a parasitic lifestyle because they provide the novelty necessary for adaptation to a changing environment, such as living in multiple hosts. Here we present the de novo sequenced and annotated genome of the parasitic trematode Atriophallophorus winterbourni and its comparative genomic analysis to other major parasitic trematodes. First, we reconstructed the species phylogeny, and dated the split of A. winterbourni from the Opisthorchiata suborder to approximately 237.4 Ma (±120.4 Myr). We then addressed the question of which expanded gene families and gained genes are potentially involved in adaptation to parasitism. To do this, we used hierarchical orthologous groups to reconstruct three ancestral genomes on the phylogeny leading to A. winterbourni and performed a GO (Gene Ontology) enrichment analysis of the gene composition of each ancestral genome, allowing us to characterize the subsequent genomic changes. Out of the 11,499 genes in the A. winterbourni genome, as much as 24% have arisen through duplication events since the speciation of A. winterbourni from the Opisthorchiata, and as much as 31.9% appear to be novel, that is, newly acquired. We found 13 gene families in A. winterbourni to have had more than ten genes arising through these recent duplications; all of which have functions potentially relating to host behavioral manipulation, host tissue penetration, and hiding from host immunity through antigen presentation. We identified several families with genes evolving under positive selection. Our results provide a valuable resource for future studies on the genomic basis of adaptation to parasitism and point to specific candidate genes putatively involved in antagonistic host-parasite adaptation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

OMA is an established resource to elucidate evolutionary relationships among genes from currently 2326 genomes covering all domains of life. OMA provides pairwise and groupwise orthologs, functional annotations, local and global gene order conservation (synteny) information, among many other functions. This update paper describes the reorganisation of the database into gene-, group- and genome-centric pages. Other new and improved features are detailed, such as reporting of the evolutionarily best conserved isoforms of alternatively spliced genes, the inferred local order of ancestral genes, phylogenetic profiling, better cross-references, fast genome mapping, semantic data sharing via RDF, as well as a special coronavirus OMA with 119 viruses from the Nidovirales order, including SARS-CoV-2, the agent of the COVID-19 pandemic. We conclude with improvements to the documentation of the resource through primers, tutorials and short videos. OMA is accessible at https://omabrowser.org., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The insect chemosensory repertoires of Odorant Receptors (ORs) and Gustatory Receptors (GRs) together represent one of the largest families of ligand-gated ion channels. Previous analyses have identified homologous 'Gustatory Receptor-Like' (GRL) proteins across Animalia, but the evolutionary origin of this novel class of ion channels is unknown. We describe a survey of unicellular eukaryotic genomes for GRLs, identifying several candidates in fungi, protists and algae that contain many structural features characteristic of animal GRLs. The existence of these proteins in unicellular eukaryotes, together with ab initio protein structure predictions, provide evidence for homology between GRLs and a family of uncharacterized plant proteins containing the DUF3537 domain. Together, our analyses suggest an origin of this protein superfamily in the last common eukaryotic ancestor., Competing Interests: RB, CD, DM No competing interests declared, (© 2020, Benton et al.)

Phylogenetic profiling is a computational method to predict genes involved in the same biological process by identifying protein families which tend to be jointly lost or retained across the tree of life. Phylogenetic profiling has customarily been more widely used with prokaryotes than eukaryotes, because the method is thought to require many diverse genomes. There are now many eukaryotic genomes available, but these are considerably larger, and typical phylogenetic profiling methods require at least quadratic time as a function of the number of genes. We introduce a fast, scalable phylogenetic profiling approach entitled HogProf, which leverages hierarchical orthologous groups for the construction of large profiles and locality-sensitive hashing for efficient retrieval of similar profiles. We show that the approach outperforms Enhanced Phylogenetic Tree, a phylogeny-based method, and use the tool to reconstruct networks and query for interactors of the kinetochore complex as well as conserved proteins involved in sexual reproduction: Hap2, Spo11 and Gex1. HogProf enables large-scale phylogenetic profiling across the three domains of life, and will be useful to predict biological pathways among the hundreds of thousands of eukaryotic species that will become available in the coming few years. HogProf is available at https://github.com/DessimozLab/HogProf., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Preclinical radiotherapy applications require dedicated irradiation systems which are expensive and not widely available. In this work, a clinical dual source 137 Cs cell irradiator was adapted to deliver 1-cm diameter preclinical treatment beams using a lead and stainless steel custom-made collimator to treat one or two mice at a time., Methods: The dosimetric characteristics of all the different components of the system (including collimator, phantoms, and radiation sources) have been simulated with EGSnrc Monte Carlo methods. The collimator was constructed based on these simulations and the calculated results were verified against dosimetric measurements with MOSKin detectors, GAFchromic films, and dosimetric gels., Results: The comparisons showed an agreement, in terms of full width half maximum values, between the simulated and the measured dose cross profiles at the midline within 4% for both gel dosimetry and GAFchromic films. Out of beam dose, measured in air at the collimator midplane with MOSFET detectors was between 6% and 10% of the beam axis dose. The dimensions of the beam are constant along the vertical axis of the collimator and also the simulated and measured Percentage Depth Dose (PDD) curves show an agreement within 1%., Conclusions: The collimator design developed in this work allows the creation of a beam with the necessary characteristics for ablative radiotherapy treatments on small animals using a standard clinical cell irradiator. This collimator design will make advanced preclinical studies with ablative beams possible for all those institutions which do not have collimated preclinical irradiators available., (© 2020 American Association of Physicists in Medicine.)

Herein we report on a new series of hydrazidoureidobenzensulfonamides investigated as inhibitors of the cytosolic human (h) hCA I and II isoforms, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. The reported derivatives contain a 4-substituted piperidine fragment in which the hydrazidoureido linker has been involved as spacer between the benzenesulfonamide fragment which binds the zinc ion from the active site, and the tail of the inhibitor. Depending on the substitution pattern at the piperidine ring, low nanomolar inhibitors were detected against hCA II, hCA IX and hCA XII, making the new class of sulfonamides of interest for various pharmacologic applications., 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 © 2020 Elsevier Inc. All rights reserved.)

In the search of multifunctional compounds we designed benzimidazole derivatives endowed with phenolic hydroxy groups and a hydrazone moiety as potential radical-scavenger and the antioxidant agents. The target molecules have been prepared by a simple synthetic procedure and tested for their antioxidant activity by DPPH, FRAP, and ORAC test, for photoprotective activity against UV rays and for antiproliferative activity against Colo-38 melanoma cells. Furthermore, two different dermocosmetic formulations were prepared with the compounds endowed with the best antioxidant and photoprotective profile and their release from formulation evaluated using Franz Cells system. High antioxidant activity is related to the presence of at least two hydroxy groups on arylidene moiety of benzimidazoles. Structure activity analysis revealed that the position of hydroxy groups is crucial for antioxidant activity as well as the presence of a 2-hydroxy-4-(diethylamino)arylidene group. The same correlation pattern was found to be related to photoprotective activity resulting in an UVA Protection Factor better than the commercial solar filter PBSA and antiproliferative activity against melanoma cells without producing cytotoxicity on normal keratinocytes. The release analysis indicated that high antioxidant activities are achieved with limited release at concentration compatible with the use as UV sunscreen filter., Competing Interests: The authors declare no conflict of interest

A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies., Competing Interests: Declaration of interests The authors declare no competing interests other than the following: The avelumab work was conducted under investigator-initiated contract with Merck KGaA. The costs of the experiments were paid for by Merck KGaA and avelumab supplied by Merck. This work utilizes technology subject to the following patents: 2015 WO2014063206-A1 “classifying epidermal growth factor receptor positive tumor into subtype eg epidermal growth factor receptor antagonist sensitive subtype, involves analyzing ligand-induced epidermal growth factor receptor internalization status of tumor.” Simpson, F., Saunders, N.A.; 2015 WO2014063205-A1 “composition useful in kit for treating tumor, preferably cell surface antigen positive tumor eg cancerous tumors, comprises antibody that binds to cell surface antigen of tumor and inhibitor of receptor mediated endocytosis” Simpson, F., Saunders, N.A., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We report here a thorough structure-activity relationship (SAR) with piperazinylureido sulfamates as inhibitors of human (h) carbonic anhydrase (CA, EC 4.2.1.1). A SAR investigation over the structure of reported anti-cancer zinc-binder CAIs such as SLC-0111 and S4 was carried out by including the urea outer nitrogen atom into a substituted piperazine ring reducing the linker flexibility. The derivatives were assessed for the inhibition of CA I, II and IV (off-target isoforms) and the tumor-associated CA IX (anticancer drug target). CA I and IV were not effectively inhibited, whereas many low nanomolar inhibitors were evidenced against CA II (K I s in the range of 1.0-705.5 nM), and IX (K I s in the range of 0.91-155.9 nM). Interestingly, a subset of CA II/IX selective inhibitors was detected which might represent interesting lead for the development of new anticancer strategies., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Three series of arylbenzimidazole derivatives 3-40, 45 have been simply synthesized and tested for their antioxidant capacity. The 2-arylbenzimidazoles were tested against various radicals by the DPPH, FRAP and ORAC tests and showed different activity profiles. It has been observed that the number and position of the hydroxy groups on the 2-aryl portion and the presence of a diethylamino group or a 2-styryl group are related to a good antioxidant capacity. Furthermore, benzimidazoles showed satisfactory SPF values ​​in vitro compared to the commercial PBSA filter, proving to have a good photoprotective profile. In particular, 2-arylbenzimidazole-5-sulphonic acids 15 and 38, the 2-styryl-benzimidazole 45 showed broad spectrum solar protection against UVA and UVB rays. The antiproliferative effect of the benzimidazoles was tested on human skin melanoma Colo-38 cells. The styrylbenzimidazole 45 exhibited antiproliferative effect at low micromolar concentration against Colo-38 cells and very low antiproliferative activity on normal HaCat keratinocyte cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Two new piperazinyl-ureido single ring aryl sulfamate-based inhibitor series were designed against the emerging oncology drug target steroid sulfatase (STS), for which there are existing potent steroidal and non-steroidal agents in clinical trials. 4-(Piperazinocarbonyl)aminosulfamates (5-31) were obtained by reacting 4-hydroxyarylamines with phenylchloroformate, subsequent sulfamoylation of the resulting hydroxyarylcarbamates and coupling of the product with 1-substituted piperazines. Pyrimidinyl-piperazinourea sulfamates (35-42) were synthesized by pyrimidine ring closure of 4-Boc-piperazine-1-carboxamidine with 3-(dimethylamino)propenones, deprotection and coupling with the sulfamoylated building block. Target ureidosulfamates 5-31 and 35-42 were evaluated both as STS inhibitors in vitro using a lysate of JEG-3 human placenta choriocarcinoma cell line and in a whole cell assay. SAR conclusions were drawn from both series. In series 35-42 the best inhibitory activity is related to the presence of a benzofuryl on the pyrimidine ring. In series 5-31 the best inhibitory activity was shown by the ureas bearing 4-chlorophenyl, 3,4-dichlorophenyl groups or aliphatic chains at the piperazino 4-nitrogen displaying IC 50 in the 33-94 nM concentration range. Final optimization to the low nanomolar level was achieved through substitution of the arylsulfamate ring with halogens. Four halogenated arylsulfamates of high potency were achieved and two of these 19 and 20 had IC 50 values of 5.1 and 8.8 nM respectively and are attractive for potential in vivo evaluation and further development. We demonstrate the optimization of this new series to low nanomolar potency, employing fluorine substitution, providing potent membrane permeant inhibitors with further development potential indicating piperazinyl-ureido aryl sulfamate derivatives as an attractive new class of STS inhibitors., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Four new series of aromatic sulfamates were synthesized and investigated for the inhibition of four human (h) isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA I, II, IX, and XII. The reported derivatives, obtained by a sulfamoylation reaction of the corresponding phenolic precursors, bear 3,5-diarylpyrazoline moieties as spacers between the benzenesulfamate fragment which binds the zinc ion from the active site, and the tail of the inhibitor. Pyrazolines are biologically privileged scaffolds, endowed with versatile biological activity, such as an anti-proliferative action. The derivatives were tested for the inhibition of the cytosolic, hCA I and II (off target isoforms) and the trans-membrane, tumor-associated hCA IX and XII enzymes (anticancer drug targets). Generally, hCA I was not effectively inhibited, whereas many low nanomolar inhibitors were evidenced against hCA II (KIs in the range of 0.42-90.1 nM), IX (KIs in the range of 0.72-63.6 nM), and XII (KIs in the range of 0.88-85.2 nM). The best substitution fragments at the pyrazoline ring included for CA II a 4-sulfamic group on the 3-aryl and halogens on the 5-aryl or a methoxy group on the 3-aryl and a 4-sulfamate group on the 5-aryl; for CA IX and CA XII they included the sulfamic group on the 3- or 4-position of the 5-aryl and an electronwithdrawing group on the 4-postion of the 3-aryl ring., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)