The impact of AlphaFold2 on experimental structure solution.

AlphaFold2 is a machine-learning based program that predicts a protein structure based on the amino acid sequence. In this article, we report on the current usages of this new tool and give examples from our work in the Coronavirus Structural Task Force. With its unprecedented accuracy, it can be utilized for the design of expression constructs, de novo protein design and the interpretation of Cryo-EM data with an atomic model. However, these methods are limited by their training data and are of limited use to predict conformational variability and fold flexibility; they also lack co-factors, post-translational modifications and multimeric complexes with oligonucleotides. They also are not always perfect in terms of chemical geometry. Nevertheless, machine learning-based fold prediction is a game changer for structural bioinformatics and experimentalists alike, with exciting developments ahead.

Probiotic potential of γ-aminobutyric acid (GABA)-producing yeast and its influence on the quality of cheese.

Kazakh cheese is a traditional dairy product in Xinjiang, China. To study the function and potential probiotic characteristics of yeast in Kazakh cheese and its contribution to cheese fermentation, we screened the γ-aminobutyric acid (GABA)-producing yeasts Pichia kudriavzevii 1-21, Kluyveromyces marxianus B13-5, Saccharomyces cerevisiae DL6-20, and Kluyveromyces lactis DY1-10. We investigated the potential probiotic properties of these strains and their use in cheese fermentation (cheeses designated CSP, CSM, CSS, and CSI, respectively); a control with no added yeast was designated CS. The results showed that the 4 yeast strains all showed high self-polymerization (2- and 24-h autoaggregation capacity of >80 and 90%, respectively), hydrophobicity (40-92% variation, low hydrophobicity in xylene, but within the range of probiotics), and the ability to survive the gastrointestinal tract (survival rate >75% after simulation), indicating the probiotic ability of the strains in vitro. The GABA production capacity of the CSM cheese increased (to 95.6 mg/100 g), but its protein content did not change significantly, and amino acid degradation was obvious. The GABA production capacity of the CSS cheese decreased (to 450 mg/kg); its protein content declined, and its amino acid content increased. Except for water and protein, we found no obvious differences in most physical and chemical indicators. Kluyveromyces marxianus B13-5 helped to form the desired texture. Multivariate statistical analysis showed that fermentation of the cheese with the 4 yeasts improved the production of esters and alcohols. The CSS cheese had good aroma production performance, because S. cerevisiae DL6-20 produced high concentrations of isoamyl alcohol, hexanoic acid ethyl ester, benzyl alcohol, octanoic acid ethyl ester, 3-hydroxy-2-butanone, and hexanoic acid; the content of 2-methyl-propanoic acid was low. Compared with the CSP cheese, the CSI and CSM cheeses had a fruitier aroma and a milder odor, but the CSI and CSM cheeses had high concentrations of ethyl acetate, butanoic acid, ethyl ester, 3-methyl-1-butanol-acetate, ethyl hexanoate, ethyl octanoate, acetic acid 2-phenylethyl ester, and ethyl lactate; concentrations of 3-methyl-butanoic acid, propanoic acid, acetic acid, and butanoic acid were low. The CSP cheese had stronger acid-producing ability. The order of fragrance production performance was CSS > CSI, CSM > CSP > CS. Research into the fermentation mechanisms of GABA-producing yeast in cheese will provide a theoretical basis for the quality control and industrial production of Kazakh cheese.

IGF2BP1 promotes LPS-induced NFκB activation and pro-inflammatory cytokines production in human macrophages and monocytes.

BACKGROUND: Lipopolysaccharide (LPS)-induced macrophage/monocyte activation and pro-inflammatory cytokines production are important mediators for periodontitis progression. The current study tested the potential role of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in the process. METHODS: THP-1 human macrophages and primary human peripheral blood mononuclear cells (PBMCs) were treated with LPS. mRNA and protein expression of IGF2BP1 were tested by qPCR and Western blotting assay. IGF2BP1 expression was altered by shRNAs or CRISPR/Cas-9 gene editing methods. LPS-induced cytokine production was tested by ELISA assay. Cytokine mRNA expression was tested by the quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay. RESULTS: In THP-1 human macrophages and PBMCs, treatment with LPS induced mRNA and protein expression of IGF2BP1. IGF2BP1 silencing (by targeted shRNAs) or CRISPR/Cas-9 knockout largely inhibited LPS-induced production of multiple pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6. Conversely, forced over-expression of IGF2BP1 facilitated LPS-induced pro-inflammatory cytokines production in THP-1 cells. For the mechanism study, we show that IGF2BP1 co-immunoprecipitated with p65-p52 nuclear factor kappa B (NFκB) complex in nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced p65-p52 nuclear translocation and NFκB activation were inhibited by IGF2BP1 silencing or CRISPR/Cas-9 knockout. CONCLUSION: IGF2BP1 promotes LPS-induced NFκB signalling and transcriptional activation in human macrophages and monocytes.

3D Micromachined Polyimide Mixing Devices for in Situ X-ray Imaging of Solution-Based Block Copolymer Phase Transitions.

Advances in modern interface- and material sciences often rely on the understanding of a system's structure-function relationship. Designing reproducible experiments that yield in situ time-resolved structural information at fast time scales is therefore of great interest, e.g., for better understanding the early stages of self-assembly or other phase transitions. However, it can be challenging to accurately control experimental conditions, especially when samples are only available in small amounts, prone to agglomeration, or if X-ray compatibility is required. We address these challenges by presenting a microfluidic chip for triggering dynamics via rapid diffusive mixing for in situ time-resolved X-ray investigations. This polyimide/Kapton-only-based device can be used to study the structural dynamics and phase transitions of a wide range of colloidal and soft matter samples down to millisecond time scales. The novel multiangle laser ablation three-dimensional (3D) microstructuring approach combines, for the first time, the highly desirable characteristics of Kapton (high X-ray stability with low background, organic solvent compatibility) with a 3D flow-focusing geometry that minimizes mixing dispersion and wall agglomeration. As a model system, to demonstrate the performance of these 3D Kapton microfluidic devices, we selected the non-solvent-induced self-assembly of biocompatible and amphiphilic diblock copolymers. We then followed their structural evolution in situ at millisecond time scales using on-the-chip time-resolved small-angle X-ray scattering under continuous-flow conditions. Combined with complementary results from 3D finite-element method computational fluid dynamics simulations, we find that the nonsolvent mixing is mostly complete within a few tens of milliseconds, which triggers initial spherical micelle formation, while structural transitions into micelle lattices and their deswelling only occur on the hundreds of milliseconds to second time scale. These results could have an important implication for the design and formulation of amphiphilic polymer nanoparticles for industrial applications and their use as drug-delivery systems in medicine.

Targeting mTOR by CZ415 Inhibits Head and Neck Squamous Cell Carcinoma Cells.

BACKGROUND/AIMS: mTOR is an important therapeutic target for human head and neck squamous cell carcinoma (HNSCC). The current study tested the anti-HNSCC cell activity by a mTOR kinase inhibitor CZ415. METHODS: HNSCC cells were treated with CZ415. Cell death was tested by lactate dehydrogenase (LDH) assay and MTT assay. Cell proliferation was tested by BrdU ELISA assay and [H3] thymidine incorporation assay, with apoptosis assayed by the TUNEL staining. A Western blotting assay was applied to test autophagy-associated proteins, mTOR and signalings. The nude mice xenograft model was established to study CZ415-mediated anti-tumor activity. RESULTS: In established (SCC-9, SQ20B and A253 lines) and primary human HNSCC cells, CZ415 efficiently inhibited cell survival and proliferation. CZ415 blocked mTORC1/2 activation and inhibited ERK in HNSCC cells. CZ415 provoked feedback autophagy activation. Conversely, autophagy inhibitors (3-methyladenine and chloroquine) or Beclin-1 shRNA sensitized CZ415-induced HNSCC cell death. In vivo, CZ415 gavage inhibited SCC-9 tumor growth in nude mice, showing higher efficiency against Beclin-1-silenced tumors. CONCLUSION: CZ415 inhibits HNSCC cell growth in vitro and in vivo. Inhibition of autophagy can further sensitize CZ415 against HNSCC cells.

Design and Evaluation of Hydrophilic Matrix System Containing Polyethylene Oxides for the Zero-Order Controlled Delivery of Water-Insoluble Drugs.

The aim of this study was to design a polyethylene oxide (PEO) binary hydrophilic matrix controlled system and investigate the most important influence(s) on the in vitro water-insoluble drug release behavior of this controlled system. Direct-compressed PEO binary matrix tablets were obtained from a variety of low viscosity hydrophilic materials as a sustained agent, using anhydrous drugs as a model drug. Water uptake rate, swelling rate, and erosion rate of matrices were investigated for the evaluation of the PEO hydrophilic matrix systems. The effect of the dose, the solubility of water-insoluble drug, and the rheology of polymers on in vitro release were also discussed. Based on the in vitro release kinetics study, three optimized PEO binary matrices were selected for further research. And, these PEO binary matrices had shown the similar release behavior that had been evaluated by the similarity factor f 2. Further study indicated that they had identical hydration, swelling, and erosion rate. Moreover, rheology study exhibited the similar rheological equation of Herschel-Bulkley and their viscosity was also within the same magnitude. Therefore, viscosity plays the most important role to control drug release compared to other factors in PEO binary matrix system. This research provides fundamental understanding of in vitro drug release of PEO binary hydrophilic matrix tablets and helps pharmaceutical workers to develop a hydrophilic controlled system, which will effectively shorten the process of formulation development by reducing trial-and-error.

Preparation and pharmacokinetics study on gastro-floating sustained-release tablets of troxipide.

The purpose of this research aimed at preparing gastro-floating sustained-release tablets of troxipide and a further study on in vitro release and in vivo bioavailability. Under the circumstances of direct powder compression, the floating tablets were successfully prepared with HPMC as main matrix material, Carbopol as assistant matrix material, octadecanol as floating agent and sodium bicarbonate as foaming agent to float by gas-forming. The floating time and accumulative release amount as evaluation indexes were utilized to perform pre-experiment screening and single-factor test, respectively, while central composite design response surface method was applied for formulation optimization, followed by in vivo pharmacokinetic study in beagles after oral administration for floating tablets and commercial tablets used as the control. The results indicated that the floating sustained-release tablets held a better capability for floating and drug release and more satisfactory pharmacokinetic parameters, such as a lower Cmax, a prolonged Tmax, but an equivalent bioavailability calculated by AUC0-24 compared to commercial tablets. So a conclusion was finally drawn that the floating sustained-release tablets possessing a good release property could be suitable for demands of design.

Wekemo Bioincloud: A user-friendly platform for meta-omics data analyses.

The increasing application of meta-omics approaches to investigate the structure, function, and intercellular interactions of microbial communities has led to a surge in available data. However, this abundance of human and environmental microbiome data has exposed new scalability challenges for existing bioinformatics tools. In response, we introduce Wekemo Bioincloud-a specialized platform for -omics studies. This platform offers a comprehensive analysis solution, specifically designed to alleviate the challenges of tool selection for users in the face of expanding data sets. As of now, Wekemo Bioincloud has been regularly equipped with 22 workflows and 65 visualization tools, establishing itself as a user-friendly and widely embraced platform for studying diverse data sets. Additionally, the platform enables the online modification of vector outputs, and the registration-independent personalized dashboard system ensures privacy and traceability. Wekemo Bioincloud is freely available at https://www.bioincloud.tech/.

A study on the factors influencing the intention to receive booster shots of the COVID-19 vaccine in China based on the information frame effect.

Introduction: In the response to and prevention and control of the Novel coronavirus pneumonia, the COVID-19 vaccine does not provide lifelong immunity, and it is therefore important to increase the rate of booster shots of the COVID-19 vaccine. In the field of information health science, research has found that information frames have an impact in changing individual attitudes and health behaviors. Objective: This study focuses on the effects of different influencing factors on the public's willingness to receive the booster shots of the COVID-19 vaccine under two information frameworks. Methods: An online questionnaire was conducted to explore the effects of demographic characteristics, personal awareness, social relationships, risk disclosure, perceived booster vaccination protection rate, and duration of protection under the assumption of an information framework. T test and one-way analysis were used to testing the effect of variables. Results: (1) The persuasion effect under the gain frame is higher than that under the loss frame (B = 0.863 vs. B = 0.746); (2) There was no significant difference in subjects' intention of booster vaccination in terms of gender, age, income, occupation, educational background and place of residence. Whether family members received booster vaccination was strongly correlated with their intention of vaccination under the loss framework (p = 0.017, M = 4.63, SD = 0.664). (3) The higher the understanding of COVID-19, the higher the degree of compliance with the government's COVID-19 prevention and control measures, and the higher the willingness to strengthen vaccination; (4) Risk disclosure has a significant impact on people's willingness to receive COVID-19 booster shots (M = 2.48, under the loss framework; M = 2.44, under the gain framework); (5) Vaccine protection rate and duration of protection have an impact on people's willingness to vaccinate. Increased willingness to vaccinate when the protection rate of booster vaccine approaches 90% (M = 4.76, under the loss framework; M = 4.68, under the gain framework). When the vaccine protection period is 2 years, people are more willing to receive a booster vaccine; and the willingness to receive a booster shot is stronger under the loss framework (M = 4.60, SD = 0.721, p = 0.879). Conclusion: The impact of the information framework on COVID-19 vaccination intentions is different, and the disclosure of relevant health information should focus on the impact of the information framework and content on the public's behavior toward strengthening vaccination. Therefore, in the face of public health emergencies, public health departments, healthcare institutions, and other sectors can consider adopting the Gainful Information Framework tool to disseminate health information to achieve better persuasion and promote public health behavior change enhancing public health awareness, and promoting universal vaccination.

[4 + 2] Cyclization or Lossen Rearrangement: Rhodium-Catalyzed Divergent Synthesis of Carboline Derivatives with Anticancer Activity.

An unusual rhodium-catalyzed C-H activation/Lossen rearrangement/oxa-Michael addition tandem cyclization has been achieved along with a tunable well-known C-H activation/[4 + 2] annulation, leading to regio-, chemo-, and diastereoselective access to diverse pentacyclic α-carbolines and ß-carboline-1-one derivatives in moderate to good yields with significant anticancer activity.

The Microbiome Protocols eBook initiative: Building a bridge to microbiome research.

The Microbiome Protocols eBook (MPB) serves as a crucial bridge, filling gaps in microbiome protocols for both wet experiments and data analysis. The first edition, launched in 2020, featured 152 meticulously curated protocols, garnering widespread acclaim. We now extend a sincere invitation to researchers to participate in the upcoming 2nd version of MPB, contributing their valuable protocols to advance microbiome research.

Errors in structural biology are not the exception.

During the COVID-19 pandemic, the structural biology community swung into action quickly and efficiently, and many urgent questions were solved by macromolecular structure determination. The Coronavirus Structural Task Force evaluated all structures from SARS-CoV-1 and SARS-CoV-2, but errors in measurement, data processing and modelling are present beyond these structures and throughout the structures deposited in the Protein Data Bank. Identifying them is only the first step; in order to minimize the impact that errors have in structural biology, error culture needs to change. It should be emphasized that the atomic model which is published is an interpretation of the measurement. Furthermore, risks should be minimized by addressing issues early and by investigating the source of a given problem, so that it may be avoided in the future. If we as a community can do this, it will greatly benefit experimental structural biologists as well as downstream users who are using structural models to deduce new biological and medical answers in the future.

Morphology and genetic characterization of Physaloptera sibirica Petrow & Gorbunov, 1931 (Spirurida: Physalopteridae), from the hog-badger Arctonyx collaris Cuvier (Carnivora: Mustelidae), with molecular phylogeny of Physalopteridae.

BACKGROUND: Nematodes of the family Physalopteridae (Spirurida: Physalopteroidea) commonly parasitize the alimentary canal of all major vertebrate groups. However, many physalopterid species are not adequately described, especially regarding the detailed morphology of the cephalic end. The current genetic database for Physaloptera species is still very limited, which seriously hampers molecular-based species identification. Additionally, the systematic status of some genera and the evolutionary relationships of the subfamilies in the Physalopteridae remain under debate. METHODS: New morphological data for Physaloptera sibirica was gathered using light and scanning electron microscopy based on newly collected specimens from the hog badger Arctonyx collaris Cuvier (Carnivora: Mustelidae) in China. Six different genetic markers, including nuclear small ribosomal DNA (18S), large ribosomal DNA (28S) and internal transcribed spacer (ITS), mitochondrial cytochrome c oxidase subunit 1 (cox1) and subunit 2 (cox2), and the 12S small subunit ribosomal RNA gene of P. sibirica were sequenced and analyzed for the first time to our knowledge. Additionally, to construct a basic molecular phylogenetic framework for the Physalopteridae, phylogenetic analyses were performed based on the cox1 and 18S + cox1 genes using maximum likelihood (ML) and Bayesian inference (BI) methods. RESULTS: Scanning electron microscopy (SEM) observation displayed the details of the cephalic structures, deirids, excretory pore, caudal papillae, vulva, phasmids and egg of P. sibirica for the first time to our knowledge. Pairwise comparison of the sequences obtained for P. sibirica did not reveal intraspecific divergence regarding the 18S, 28S, cox1 and 12S genetic markers and a low level of divergence in the ITS (0.16%) and cox2 (2.39%) regions. Maximum likelihood and Bayesian inference analyses showed that the representatives of Physalopteridae formed two major clades (species of Physalopterinae + Thubunaeinae parasitic in terrestrial vertebrates and Proleptinae only occurring in marine or freshwater fishes). Turgida turgida was found nested among representatives of Physaloptera. Physaloptera sibirica clustered together with P. rara. Physalopteroides sp. (Thubunaeinae) formed a sister relationship to the physalopterine Abbreviata caucasica. CONCLUSIONS: Physaloptera sibirica was redescribed, which is the fourth nematode parasite reported from the hog badger A. collaris, and A. collaris represents a new host for P. sibirica. The phylogenetic results challenged the validity of the subfamily Thubunaeinae and of the genus Turgida and supported dividing the family Physalopteridae into two subfamilies, Physalopterinae and Proleptinae. However, we do not make any immediate systematic changes in the Physalopteridae, because a more rigorous study with broader representation of the Physalopteridae is required. These present findings contribute to morphologically identifying P. sibirica more accurately and provide new insights into the systematics of the Physalopteridae.

mtDNA CR Evidence Indicates High Genetic Diversity of Captive Forest Musk Deer in Shaanxi Province, China.

Forest musk deer (Moschus berezovskii) are endangered ruminants whose adult males secrete musk. China has been breeding forest musk deer artificially since the 1950s in an effort to restore wild populations, with Shaanxi and Sichuan provinces as the two main sites for captive breeding. Genetic diversity is a significant indicator that determines the long-term viability and status of a population, particularly for species at risk of extinction. In this study, we analyzed the current genetic makeup of seven captive forest musk deer populations in the Shaanxi province, using the mitochondrial DNA (mtDNA) control region (CR) as the molecular marker. We sequenced 604 bp of mtDNA CR, with an average content of A+T higher than G+C. We observed 111 variable sites and 39 different haplotypes from 338 sequences. The nucleotide diversity (Pi) and haplotype diversity (Hd) were 0.02887 and 0.908, respectively. Genetic differentiation between these populations was not significant, and the populations might not have experienced rapid growth. By combining our sequences with previous ones, we identified 65 unique haplotypes with 26 rare haplotypes and estimated a total of 90 haplotypes in Shaanxi province captive populations. The Shaanxi province and Sichuan province obtained 88 haplotypes, the haplotypes from the two populations were mixed together, and the two populations showed moderate genetic differentiation. Our findings suggested that captive forest musk deer populations in the Shaanxi province had high genetic diversity, with a rich founder population of about 90 maternal lines. Additionally, managers could develop genetic management plans for forest musk deer based on the haplotype database. Overall, our study will provide insights and guidelines for the conservation of genetic diversity in captive forest musk deer populations in the Shaanxi province.

A novel loop-mediated isothermal amplification-lateral flow dipstick method for Helicobacter pylori detection.

Introduction: To eradicate Helicobacter pylori (H. pylori) and reduce the risk of gastric cancer, a sensitive, specific, convenient, and simple detection method is needed. This study aimed to establish a novel loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for H. pylori detection. Methods: LAMP primer design software was used to design primers for the conserved sites of the H. pylori ureB gene. UreB-FIP-labeled biotin was used for LAMP amplification, and FAM-labeled probes were specifically hybridized with LAMP amplification products, which were then detected by LFD. In addition, a clinical study was conducted to assess LAMP-LFD in 20 fecal samples. Results: The results of the optimization indicated that H. pylori could be specifically detected by LFD without cross-reaction with other non-H. pylori bacteria when the LAMP was performed at 65°C for 60 min. The lower limit of the detection method was 102 copies/µL, which was 100 times the sensitivity of polymerase chain reaction (PCR). H. pylori-positive fecal samples were detected by LAMP-LFD in 13/20 patients. Discussion: In conclusion, a new LAMP-LFD assay has been fully established and confirmed for H. pylori detection. The entire process can be completed in approximately 1.5 h, with the advantages of strong specificity, high sensitivity, and simple operation. This study provides a novel potential method for the detection of H. pylori in the clinical settings of primary hospitals and low-resource countries.

How information processing and risk/benefit perception affect COVID-19 vaccination intention of users in online health communities.

Objective: To investigate the relationship among information processing, risk/benefit perception and the COVID-19 vaccination intention of OHCs users with the heuristic-systematic model (HSM). Methods: This study conducted a cross-sectional questionnaire via an online survey among Chinese adults. A structural equation model (SEM) was used to examine the research hypotheses. Results: Systematic information processing positively influenced benefit perception, and heuristic information processing positively influenced risk perception. Benefit perception had a significant positive effect on users' vaccination intention. Risk perception had a negative impact on vaccination intention. Findings revealed that differences in information processing methods affect users' perceptions of risk and benefit, which decide their vaccination intention. Conclusion: Online health communities can provide more systematic cues and users should process information systematically to increase their perceived benefits, consequently increase their willingness to receive COVID-19 vaccine.

Rapid access to C2-quaternary 3-methyleneindolines via base-mediated post-Ugi Conia-ene cyclization.

Highly efficient synthesis of diverse 2,2-disubstituted 3-methyleneindoline derivatives through a one-pot base-promoted post-Ugi 5-exo-dig "Conia-ene"-type cyclization has been disclosed. The mechanism study indicates that an intramolecular hydrogen bond may play a vital role in this process. The antiproliferative evaluation of cancer cell lines reveals that this protocol provides practical use in the green synthesis of bioactive compound libraries.

EasyAmplicon: An easy-to-use, open-source, reproducible, and community-based pipeline for amplicon data analysis in microbiome research.

It is difficult for beginners to learn and use amplicon analysis software because there are so many software tools to choose from, and all of them need multiple steps of operation. Herein, we provide a cross-platform, open-source, and community-supported analysis pipeline EasyAmplicon. EasyAmplicon has most of the modules needed for an amplicon analysis, including data quality control, merging of paired-end reads, dereplication, clustering or denoising, chimera detection, generation of feature tables, taxonomic diversity analysis, compositional analysis, biomarker discovery, and publication-quality visualization. EasyAmplicon includes more than 30 cross-platform modules and R packages commonly used in the field. All steps of the pipeline are integrated into RStudio, which reduces learning costs, keeps the flexibility of the analysis process, and facilitates personalized analysis. The pipeline is maintained and updated by the authors and editors of WeChat official account "Meta-genome." Our team will regularly release the latest tutorials both in Chinese and English, read the feedback from users, and provide help to them in the WeChat account and GitHub. The pipeline can be deployed on various platforms, and the installation time is less than half an hour. On an ordinary laptop, the whole analysis process for dozens of samples can be completed within 3 h. The pipeline is available at GitHub (https://github.com/YongxinLiu/EasyAmplicon) and Gitee (https://gitee.com/YongxinLiu/EasyAmplicon).

Detecting ice artefacts in processed macromolecular diffraction data with machine learning.

Contamination with diffraction from ice crystals can negatively affect, or even impede, macromolecular structure determination, and therefore detecting the resulting artefacts in diffraction data is crucial. However, once the data have been processed it can be very difficult to automatically recognize this problem. To address this, a set of convolutional neural networks named Helcaraxe has been developed which can detect ice-diffraction artefacts in processed diffraction data from macromolecular crystals. The networks outperform previous algorithms and will be available as part of the AUSPEX web server and the CCP4-distributed software.

Phylogenetic Analyses Support the Monophyly of the Genus Lispe Latreille (Diptera: Muscidae) with Insights into Intrageneric Relationships.

Lispe Latreille (Diptera: Muscidae) are a widespread group of predatory flies that inhabit semi-aquatic environments. Previous studies on this genus have mainly focused on morphological classification, so molecular data are entirely lacking, and there has been no attempt at a phylogenetic placement of the genus or the resolution of intragenic relationships. To address the phylogenetic placement of Lispe and to fill its gap in the Tree of Life Web Project, 58 Lispe spp. (covering 11 out of 13 acknowledged Lispe species groups) were selected to reconstruct a phylogeny using Maximum likelihood (ML) estimates, Maximum Parsimony (MP) analyses, and Bayesian inference (BI) based on two mitochondrial protein-coding genes (cytochrome c oxidase subunit I (COXI) and cytochrome b gene (CYTB)) and one nuclear gene (a fragment of the carbamoyl phosphate synthetase region of the CAD gene). The phylogenetic analyses indicated that the monophyletic Lispe is the sister group of the monophyletic Limnophora, together forming the tribe Limnophorini under the subfamily Coenosiinae. Three generic categories are proven obsolete: Chaetolispa Malloch, Lispacoenosia Snyder, and Xenolispa Malloch. Within the genus, the validity of 11 species groups is clarified by both molecular and morphological data. This study provides a sound basis for continuing intergeneric and intrageneric research into this fascinating and widespread genus.