Hypertension is a major cardiovascular risk factor, which seriously affects the quality of life of patients. Banxia Baizhu Tianma Decoction (BXD) is a Chinese herbal formula that is widely used to treat hypertension in China. This study aimed to evaluate the efficacy and potential mechanism of BXD for hypertension by meta-analysis and network pharmacology. Meta-analysis was performed to explore the efficacy and safety of BXD combined with conventional treatment for hypertension. Network pharmacology was used to explore the molecular mechanism of BXD in antihypertension. A total of 23 studies involving 2,041 patients were included. Meta-analysis indicated that compared with conventional treatment, combined BXD treatment was beneficial to improve clinical efficacy rate, blood pressure, blood lipids, homocysteine, endothelial function, inflammation, and traditional Chinese medicine symptom score. In addition, meta-analysis indicated that BXD is safe and has no obvious adverse reactions. Network pharmacology showed that the antihypertensive targets of BXD may be AKT1, NOS3, ACE, and PPARG. The antihypertensive active ingredients of BXD may be naringenin, poricoic acid C, eburicoic acid, and licochalcone B. Due to the poor methodological quality of the Chinese studies and the small sample size of most, the analysis of this study may have been affected by bias. Therefore, the efficacy and safety of BXD for hypertension still need to be further verified by high-quality clinical studies.Systematic Review Registration:https://www.crd.york.ac.uk/prospero/, identifier CRD42022353666 Hypertension is a clinical syndrome characterized by increased systemic arterial blood pressure, which may be accompanied by functional or organic damage of the heart, brain, kidney, and other organs [Hypertension was defined as systolic blood pressure (SBP) ≥ 140 mmHg or diastolic blood pressure (DBP) ≥ 90 mmHg] . HypertePinellia ternata (Thunb.) Makino [Araceae] (Banxia), Atractylodes macrocephala Koidz. [Asteraceae] (Baizhu), Citrus × aurantium L. [Rutaceae] (Chenpi), Glycyrrhiza uralensis Fisch. ex DC. [Fabaceae] (Gancao), Gastrodia elata Blume [Orchidaceae] (Tianma), Wolfiporia cocos (F.A. Wolf) Ryvarden & Gilb. 1984 (Fulin). BXD has the effects of eliminating dampness and phlegm, dispelling pathogenic wind and eliminating phlegm and is widely used in hypertension and its complications  . Hyperteications , howeverications , howeverications . TCM netications . In thishttps://www.crd.york.ac.uk/prospero/).This study was conducted and reported according to the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . The stuThe databases used in this study included PubMed, Cochrane, Embase, Wanfang database, China national knowledge infrastructure (CNKI), and China Science and Technology Journal Database (VIP). The retrieval time was set as the establishment of the database until August 2022. The search terms were MeSH terms combined with the keywords: “banxia baizhu tianma” and “hypertension”. The search strategy was shown in β-receptor blockers.The inclusion criteria for this study were: 1) The type of study is randomized controlled trials (RCTs). 2) Patients met the diagnostic criteria for hypertension . 3) The The exclusion criteria for this study were: 1) Duplicate published studies. 2) Studies with incorrect or incomplete data. 3) Unable to extract data for research. 4) Review or experiment articles.The primary outcome measures were: SBP, DBP, and clinical efficacy rate. The secondary outcome measures were: total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), homocysteine (Hcy), endothelial function, inflammatory biomarkers, and TCM symptom score.via email.Data were extracted independently from the included literature by SM and SY. A “basic information extraction table” was developed, and the information extracted included: the investigator, year of publication, number of cases, age, intervention, duration of intervention, and outcome measures. Any disputes were resolved through discussion with the third author (QW). When necessary, study details were requested from the corresponding authors The quality of the included studies was evaluated with the risk bias assessment by Cochrane collaboration’s tool , includiI2 ≤ 50%, a fixed-effects model was used, and if I2 > 50%, indicating greater statistical heterogeneity, a random-effects model was used. Both results were expressed using a forest plot. The publication bias was estimated by Egger’s test and funnel plot. It was regarded as a significant difference when p < 0.05.The Stata 17.0 software  was applied to statistical analysis. Standardized mean difference (SMD) was utilized for continuous outcomes. Risk ratio (RR) was utilized for dichotomous outcomes. All of them were expressed with a 95% confidence interval (CI). Heterogeneity was tested using the Q test, and if https://tcmspw.com) (http://swisstargetprediction.ch/). With “hypertension” as the keyword, the targets of hypertension were obtained through four databases. The four different databases and search criteria are as follows: GeneCards (https://www.genecards.org/) (https://ctdbase.org/) (https://www.disgenet.org) (http://cbcb.cdutcm.edu.cn/INPUT/)  , the actspw.com) . The tards.org/) , and these.org/) , and thenet.org) , and the/INPUT/) , and thehttps://string-db.org/) (https://david.ncifcrf.gov/) (https://hiplot-academic.com/) was used to visualize the results.PPI analysis of overlapping targets was conducted through STRING platform (db.org/) , and thedb.org/)  for netwrf.gov/) . It mainhttps://www.rcsb.org/). The structure of the key ingredients was obtained through the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Then used Pymol, Autodock Vina (https://plip-tool.biotec.tu-dresden.de/)   for moleA total of 1,747 related studies were retrieved. 1,003 studies remain after the elimination of duplicates by NoteExpress software and manual assistance. After reading the abstract and title, 101 studies remain. After reading the full text, 78 studies were excluded. Finally, a total of 23 studies were included . The floAlisma plantago-aquatica L. [Alismataceae] (Zexie), Neolitsea cassia (L.) Kosterm. [Lauraceae] (Guizhi), Arisaema heterophyllum Blume [Araceae] (Dannanxing), Conioselinum anthriscoides ‘Chuanxiong’ [Apiaceae] (Chuanxiong), Chrysanthemum × morifolium (Ramat.) Hemsl. [Asteraceae] (Juhua), Vitex trifolia L. [Lamiaceae] (Manjingzi), Acorus gramineus Aiton [Acoraceae] (Shichangpu)) were added or subtracted according to syndrome differentiation. The characteristics of the included studies were shown in A total of 23 RCTs involving 2,041 patients  were included. The publication period was from 2007 to 2022. The shortest treatment time was 4 weeks, and the longest treatment time was 12 weeks. Types of hypertension include H-type hypertension , senile All studies were randomized, and the majority of studies described specific randomization methods. One study  describeThe calculated results of all meta-analyses were shown in I2 = 0%, p < 0.05, 14 studies reported clinical efficacy rate. There were 604 patients in the treatment group and 598 in the control group. Meta-analysis indicated that BXD combined with conventional treatment had a higher clinical efficacy rate than conventional treatment . There were 143 patients in the treatment group and 143 in the control group. Meta-analysis indicated that compared to the control group, the treatment group had a better ability to increase NO  . Among tp < 0.05, p = 0.1383, p = 0.2814, SBP, DBP, and clinical efficacy rate were evaluated by publication bias. The funnel plot and Egger’s test suggested the possibility of publication bias in DBP , eburicoic acid (CID: 73402), licochalcone B (CID: 5318999) as binding ligands. The results showed that the docking energy was ≤−6 kcal·mol−1 (Through integrating data of al·mol−1  and the al·mol−1 .To the best of our knowledge, this is the first article that integrates meta-analysis and network pharmacology to evaluate the efficacy and potential pharmacological mechanisms of BXD in the treatment of hypertension. This article reviews 23 intervention studies of BXD combined with conventional treatment in patients with hypertension, aiming to reveal the clinical effects of BXD. The meta-analysis found that compared with conventional treatment, combined BXD treatment was beneficial to improve clinical efficacy rate, blood pressure, blood lipids, Hcy, endothelial function, and inflammation. Notably, the combined treatment with BXD was effective in improving TCM symptoms which were important for improving the quality of life of patients. The treatment of hypertension should not only lower blood pressure but also improve the patient’s symptoms. According to TCM theory, the appearance of symptoms is a response to the imbalance of the internal environment of the body. Through the treatment of BXD, the internal environment of the body can be changed to improve the disease. In addition, BXD is safe and has no obvious adverse reactions. In general, BXD can be used as a complementary and alternative therapy for patients with hypertension on the premise of TCM syndrome differentiation.Unfortunately, the overall quality of this study was not high. The methodological quality of RCTs was low, and the blinding, selective reporting of results and other biases were not described. In addition, the sample size of these RCTs was small, and they were all Chinese studies, and they were positive publications, which suggested a significant publication bias.Glycyrrhiza uralensis Fisch. ex DC. [Fabaceae], which has a strong anti-inflammatory, antioxidant capacity, and can inhibit the production of NO, IL-6, and PGE2 in LPS-induced macrophage cells (The pathological mechanism of hypertension is associated with endothelial dysfunction, increased vasoconstriction, and vascular changes characterized by arterial remodeling. The sympathetic nervous system, the renin-angiotensin-aldosterone system, and the immune system are all involved in the pathogenesis of hypertension . Using nge cells . Recent ge cells . In concIn addition, we also found that the key antihypertensive targets of BXD were AKT1, NOS3, ACE, PPARG, TNF, and PTGS2. AKT regulates cell proliferation and growth and is involved in cellular processes including apoptosis and glucose metabolism. A study showed that AKT regulated endothelial function in SHR rats . Cid-Sotvia iNOS/NOS2 and arginase 1 (2+levels. Disruption of Ca2+signaling circuits may contribute to endothelial dysfunction in hypertension (Finally, we also performed gene enrichment analysis for these targets. We found that these genes were mainly enriched in HIF-1 signaling pathway, fluid shear stress and atherosclerosis, calcium signaling pathway, cGMP-PKG signaling pathway. HIF-1 signaling pathway regulates oxygen homeostasis and plays an important role in the circulatory system. Evidence suggested that transcriptional changes in HIFs are an important molecular mechanism of hypertension . Studiesginase 1 . Intracertension . In summIn conclusion, meta-analysis indicated that BXD combined with conventional treatment for hypertension is effective and safe. BXD has the characteristics of multi-pathway, multi-component, and multi-target in the treatment of hypertension. The antihypertensive targets of BXD may be AKT1, NOS3, ACE, and PPARG. The antihypertensive active ingredients of BXD may be naringenin, poricoic acid C, eburicoic acid, and licochalcone B. However, the evidence of BXD for hypertension should be carefully interpreted due to the low methodological quality, small sample size, limited number of trials, and other unidentified risks of bias. The efficacy and safety of BXD for hypertension still need to be further proved by high-quality clinical and basic studies.


Planophila-clade . Some genera in this clade share similar morphology: cell packages forming branched pseudofilaments, uniseriate or sometimes biseriate filaments, often embedded in common mucilage. During a study on soil algal diversity in the temperate monsoon climate zone in Russia , we isolated a strain of sarcinoid green alga and examined it using an integrative approach. SSU and ITS rDNA sequence data, morphological characteristics, and life cycle features differentiated this strain from closely related genera of the order Ulotrichales and led us to describe it as Ulosarcina terrestrica gen. et sp. nov.Many filamentous and sarcinoid terrestrial or freshwater green algae that were previously assigned to the Chlorophyceae are members of lineages belonging to the class Ulvophyceae. One of these lineages is the  Planophila-clade in the Ulotrichales. Some genera in this clade, namely Sarcinofilum Darienko & Pröschold, Planophila Gerneck, Rhexinema Geitler, Hazenia H.C.Bold, Tupiella Darienko & Pröschold, and Vischerioclonium Darienko & Pröschold, share similar morphology: cell packages with different tendencies to form branched pseudofilaments, uniseriate or sometimes biseriate filaments, often embedded in a common mucilage envelope.One of the most successful groups of algae is the phylum Chlorophyta which inhabits the most contrasting ecosystems on Earth . Three cUlosarcina terrestrica.During a study on soil algal diversity in the temperate monsoon climate zone in the Russian Far East , a strain of sarcinoid green alga was isolated and examined using an integrative approach, including light and confocal laser scanning microscopy, and molecular data. It led us to differentiate this strain from other members of the order Ulotrichales and describe it as a new genus and new species, Ulosarcina A.A. Gontcharov, Sh.R. Abdullin, A.Yu. Nikulin, V.Yu. Nikulin, R.Z. Allaguvatova and V.B. Bagmet gen. nov.Diagnosis: Vegetative cells solitary or in sarcinoid-like two- or three-dimensional packages, embedded in common mucilage; cells spherical and hemispherical, often with vacuoles. Chloroplast parietal, with a distinct pyrenoid, covered with several starch grains. Nucleus single, not visible using light microscopy.Reproduction by vegetative division or by biflagellated zoospores without a cell wall. Each cell forms a single zoospore. Sexual reproduction was not observed.Ulosarcina terrestrica A.A. Gontcharov, Sh.R. Abdullin, A.Yu. Nikulin, V.Yu. Nikulin, R.Z. Allaguvatova and V.B. Bagmet, sp. nov.Type species (designated herein): Rhexinema, Sarcinofilum, Planophila, Hazenia (Chamaetrichon), Tupiella, Vischerioclonium) by SSU-ITS sequences and the following phenotypic characters: formation of sarcinoid-like three-dimensional packets by vegetative cell division rather than the development of filaments; biflagellated zoospores.Differs from related genera , in disturbed soil with ruderal vegetation.terrestrica“ is based on the habitat where it was found.Etymology: The species epithet “U. terrestrica was deposited in GenBank under accession no. OM700179.Holotype (designated herein): Exsiccatum number VLA-CA-0951, dried biomass of unialgal population was deposited in the Herbarium, Federal Scientific Center of East Asian Terrestrial Biodiversity, Vladivostok, Russia. Gene sequence: DNA sequence obtained from a clonal strain of Ulosarcina was poorly resolved in the Planophila-clade. Topologically, it branched basally following Gayralia sp. (ALC-2011), which was the first divergence in the clade and was the outgroup to the remaining Planophyla-clade. Overall resolution in the order Ulotrichales was weak in contrast to the well-resolved relationships in Ulvales clade that was characterized by significantly more divergent SSU rDNA sequences.Phylogenetic analyses of 86 SSU rDNA sequences representing major groups of the Ulvophyceae  placed tUlosarcina in the Ulotrichales , and this lineage was resolved as a sister (0.98 PP) to a strongly supported Rhexinema clade (95/1.00). ITS data along produced almost identical topology and supports  between ulotrichalean genera ranged from 0 to 0.56 ± 0.10% and from 0.23 ± 0.12 to 3.49 ± 0.43%, respectively (p-distances). ITS sequence of the new strain differed from those in other genera by more than 16%, which exceeded the difference between some genera: e.g., Monostroma and Collinsiella—14.75 ± 1.58%; Vischerioclonium and Tupiella—13.96 ± 1.32%; Hazenia and Ulothrix—10.91 ± 1.23%; Sarcinofilum and Ulothrix—6.85 ± 1.04%, etc. The results of the sequence comparisons support our conclusion that Ulosarcina is a new genus.Intrageneric and intergeneric ectively . The divU. terrestrica in the SSU rDNA-based tree reflected the presence of a significant number of autapomorphic substitutions in its sequence. To locate these substitutions in the SSU rDNA secondary structure and assess their effect on this structure, we compared SSU models of U. terrestrica and Ulothrix zonata  regions and did not alter common secondary structure. Overall, SSU rDNA structure of the new species was the same as in other Ulotrichales.The relatively long branch of Phenotypic features observed in the new algal strain isolated from soil in the temperate monsoon climate zone of the Russian Far East  such as sarcinoid habit, cell shape, parietal chloroplasts with a pyrenoid, and asexual reproduction by vegetative division and zoospores occur in many groups of green algae and do not allow unambiguous taxonomic assignment even at the class level. Packet-like colonies are known for most classes of green algae, although the number of genera characterized by this morphology is rather limited. Multiple independent origins of this habit in green algae are very likely. Phylogenetic analyses assigned our strain to the order Ulotrichales of the class Ulvophyceae. This assignment further extended the number of non-aquatic algae sharing similar morphology  in the class that mostly accommodated numerous marine taxa until recently .U. terrestrica) in its genera Planophila, Rhexinema, and Sarcinofilum. Ulosarcina terrestrica showed no affinity to generic clades sharing similar morphology to any other lineages; or occupied and unresolved position in the order according to rDNA sequence comparison results. In analyses based on a combined SSU and ITS rDNA sequence data, U. terrestrica was allied with paraphyletic Gayralia  is more plausible because these genera are more similar morphologically and occur in non-marine habitats. Rhexinema is characterized by very short (2 to 10 cells) filaments or two-dimensional cell packages embedded in common mucilage and reproduction by vegetative division or biflagellated zoospores with stigma. Similar two- and three-dimensional cell packages in common mucilage are also typical for U. terrestrica, but no filament formation was observed in this alga. Biflagellated zoospores differentiate Ulosarcina, Rhexinema, and Gayralia (see discussion above) from the rest of Ulotrichales because either quadriflagelated zoospores are known in other genera, or no data on zoospore morphology is available. However, in all phylogenetic analyses, Ulosarcina was significantly positioned outside the well-supported Rhexinema clade.The moderately supported affinity between −2s−1 in a 16:8 h light: dark cycle. The strain was maintained in the culture collection of the Laboratory of Botany in the Federal Scientific Center of East Asian Terrestrial Biodiversity, Russian Federation (strain number VCA-205).A soil sample was collected from the wasteland with ruderal vegetation in Vladivostok city  on 05 August 2018. Sampling was carried out using standard methods  at 20–22The morphology of vegetative and reproductive cells was examined using an Olympus BX 53 light microscope  equipped with Nomarski DIC optics and Olympus DP27 digital camera . Cultures were repeatedly examined throughout lifecycle stages, i.e., in cultures of different ages after transfer.For confocal laser scanning microscopy, 0.01% Triton X-100 was added to the culture of living algal cells to increase membrane permeability. Then cells were fixed in FAA  for 20 min, then rinsed twice and counterstained with DAPI  at a final concentration of 5 µg/mL. After another rinse of samples, fluorescence was detected with LSM 710 LIVE confocal laser scanning microscope  at the Instrumental Centre of Biotechnology and Gene Engineering of FSCEATB FEB RAS. DAPI fluorescence was detected at 410–497 nm, and autofluorescence of chloroplasts was recorded in the additional emission channel after 600 nm using Plan-Apochromat 63x/1.40 Oil DIC M27 objective with digital zoom. 3D files of the captured images were recorded and analyzed with ZEN microscope software.For DNA analysis, cultures were harvested during the exponential growth phase and concentrated by centrifugation. Total genomic DNA was extracted as described previously by Abdullin et al. . SSU andUlothrix zonata (SAG 38.86) as a template. Introns, if present, were excluded from the alignment. Alignment of the divergent spacer sequences  was guided by primary and secondary structure conservation [Monostroma sp. (Ush) and Rhexinema paucicellularis (SAG 463-1) proposed by Bast [http://www.unafold.org/mfold/applications/rna-folding-form.php; accessed on 10 November 2022; [U. terrestrica  was userestrica , that weOltmannsiellopsis-clade used as an outgroup; (ii) concatenated dataset of 40 SSU and ITS rDNA sequences (2291 bp); and (iii) ITS rDNA dataset of 39 sequences (539 bp) of the Ulotrichales and its sister lineage Acrosiphoniales [In order to clarify the phylogenetic position of the new genus, three datasets were used: (i) the SSU rDNA alignment, including 86 taxa and 1771 bp of representatives of the Ulvophyceae and equences 39 bp of http://embnet.vital-it.ch/raxml-bb/; accessed on 15 September 2022; [p-distances).Maximum likelihood (ML) analysis was carried out using PAUP 4.0b10 . Bayesiaer 2022; ). MEGA ver 2022;  was used


Ceratitiscapitata flies was carried out in different locations in Tunisia.Several nematode isolates were recovered, laboratory colonies were established,and their taxonomic identities were determined based on molecular methods. Amongall the recovered nematode isolates, two of them, Oscheiustipulae TC2 and OC2, were evaluated for their capacity to controlC. capitata flies and for their ability tokill and reproduce on Galleria mellonella larvae. Our resultsshow a great potential of these two isolates as biocontrol agents as they killC. capitata eggs and pupae and interferewith the metamorphosis of C. capitata larvae.More specifically, TC2 and OC2 nematodes killed 39 and 31% ofC. capitata eggs, respectively, impaired themetamorphosis of up to 77% and up to 67% of C.capitata larvae, respectively, and killed up to 66% and upto 58% of C. capitata pupae, respectively. Theefficacy of TC2 and OC2 nematodes was particularly high on C.capitata pupae, and significant insect mortalities wereobserved even at concentrations of 1 and 5 nematodes/pupae, respectively. Wealso found that TC2 and OC2 nematodes efficiently kill and reproduce inG. mellonella larvae, suggesting thatthese insects could be used for mass-multiplication of these nematodes. Theseresults reveal the potential of O. tipulae tocomplement integrated pest management programs against C.capitata flies.A survey to collect soil nematodes with potential to control  Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), theMediterranean fruit fly or medfly, is one of the most limiting insect pests forcitrus production [Clementine,Mandarin, Navel, Maltaise,Valencia Late and Double Fine oranges areattacked by C. capitata although to differentdegrees of intensities [The citrus agro-industry is one of the most important sectors for the economy ofTunisia. Citrus production is unfortunately hampered by the occurrence of differentdiseases and pests that considerably impair tree growth and crop yields.oduction .Severalensities ,3. This ensities . The conensities ,5. The uensities ,7.Steinernema(Travassos) (Nematoda: Rhabditida) and Heterorhabditis (Poinar)(Nematoda: Rhabditida), which are associated with bacteria of the genusXenorhabdus and Photorhabdus, respectively[Oscheius (Andrássy) (Nematoda: Rhabditidae) [From the great variety of biocontrol agents, entomopathogenic nematodes (EPNs) arehighly promising due to their capacity to efficiently control different insect pests–10. The ectively–17. Seveectively–20. Aparditidae) –30.Oscheius nematodes are soil-dwelling nematodes of the Rhabditidaefamily. Several of the species of this genus are considered scavengers, orbacteriophagous free-living nematodes, while several others have been shown to killinsects. Currently, there are about 30 recognized species in the genusOscheius, and more than 13 have the ability to kill insects[Oscheiusonirici kills the larvae of Galleria mellonella(Linnaeus) , Tenebrio molitor (Linnaeus)(Coleoptera: Tenebrionidae) and Drosophila suzukii (Matsumura)(Diptera: Drosophilidae); Oscheius carolinensis has potential as abiological control agent against Pieris rapae (Linnaeus)(Lepidoptera: Papilionoidae) and T. molitor;Oscheius gingeri was efficient against G.mellonella and Conogethes punctiferalis(Guenée) (Lepidoptera: Crambidae) larvae; and Oscheius tipulae andO. rugaoensis also kill G.mellonella larvae [O. onirici isolates were shown to killdifferent insect species, while a Swiss isolate was not harmful toG. mellonella [O. saproxylicus, and O.tereticorpus, whose ability to kill insects have not beentested, however, suggesting that this trait could be much more spread in the genusthan it is currently thought [Steinernema and Heterorhabditis nematodes tocontrol agricultural pests. insects,22. For a larvae ,23–30. Ta larvae . Howeverllonella ,33. Ther thought ,32–40. COscheius nematodes as biocontrolagents and aiming at increasing their availability to be used to controlC. capitata flies, we collected soil-dwellingnematodes at several locations in Tunisia using C.capitata pupae as baits, characterized them using moleculartools to determine their taxonomic identities, and selectedOscheius nematodes to specifically evaluate their abilities tokill C. capitata at different developmental stagesand temperatures. As C. capitata pupates in thesoil, it is one of the most suitable stages for biocontrol using soil-born nematodessuch as O. tipulae. The objective of our study wasto show the great biocontrol potential of two O.tipulae isolates against C.capitata flies. Hence, our study encourages to continuestudying the biology of O. tipulae nematodes andtheir interaction with C. capitata and otherinsect pests to determine their actual biocontrol potential and to explore thepossibility of incorporating them in biocontrol programs against agriculturalpests.Given the promising potential of Citrussinensis var. Maltaise) infested by C.capitata. Twenty soil samples per location were collectedat a depth of 0-30cm using a hand shovel. Samples were placed in plastic bagsand stored at 7°C for further analyses. Soil nematodes were recovered from thesoil samples using C. capitata pupae orgreater wax moth larvae as baits according to the procedures described byBedding and Akhurst [Ceratitis capitata pupae were included as baits to obtainnematodes closely associated to this pest. For each sample, 300g of soil wereplaced in 500ml plastic containers. Then, ten C.capitata pupae, reared in artificial diets as describedbelow, were added to the plastic containers. Soil was moistened using a watersprayer. Plastic containers were incubated at 22°C in the dark for 5 days. Afterthis period, all dead insects were collected, rinsed three times with distilledwater and incubated for 24 hours at ambient temperature. Then, fiveG. mellonella larvae were added to thesame soil container. Plastic containers were incubated at 22°C in the dark for 5days. All dead insects were collected, rinsed three times with distilled water,incubated for 24 hours at ambient temperature, transferred to White traps andincubated at 22°C for 8 days in darkness [C. capitata pupae and G.mellonella larvae, or were cultured in egg yolk media . In all cases, cultures weremaintained in darkness at 25°C. Progenies were collected and cultured in freshmedia/insects. After several cycles, the nematode cultures were inspected undera light microscope  to determinepotential mixture of different nematode species.Soil samples were collected, during the winter season of 2019 and 2020, at fivedifferent locations in Tunisia: Morneg, Takilsa, Kobba, Sidi-Saad, and Ouzra.Soil samples were taken from soils of citrus crops  . Adult f5’-TTGATTACGTCCCTGCCCTTT-3’ (forward), and 26S:5’-TTTCACTCGCCGTTACTAAGG-3’ (reverse) [5’-CCTTAGTAACGGCGAGTGAAA-3’ (forward) and 536:5’-CAGCTATCCTGAGGAAAC-3’(reverse). The 18S rRNA genewas amplified using primers NEM18SF:5’-CGCGAATRGCTCATTACAACAGC-3’ (forward) and NEM18SR:5’-GGGCGGTATCTGATCGCC-3’ (reverse) [Nematode genomic DNA was extracted from about five thousand nematodes at alldevelopmental stages using the genomic DNA isolation kit from NORGEN BioTEK(Cat. 24700) following the manufacturer’s instructions. Genomic DNA was used toamplify different regions of the rRNA genes by PCR. Briefly, ITS regions  were amplified using primers 18S:reverse) . The frareverse) . PCR cycThe evolutionary histories based on the different rRNA gene sequences wereinferred by using the Maximum Likelihood method based on the General TimeReversible model (18S and D2D3) or on the Tamura 3-parameter model (ITS) ,48. BestO. tipulaenematodes to control C. capitata, we evaluatedmetamorphosis and mortality of C. capitataeggs, larvae, and pupae exposed to different concentrations of twoO. tipulae isolates, TC2 and OC2, asdescribed below.To evaluate the potential of O. tipulae TC2and OC2 nematodes on C. capitata eggs wasevaluated based on the induced mortality of these latter. Fifty eggs wereplaced in sterile Petri plates (60 mm diameter) layered with two filterpaper sheets. Then, 5000 O. tipulae TC2 or5000 OC2 nematodes suspended in 2ml of sterile water were added to eachPetri plate. Controls were treated with water only. Then, all Petri plateswere sealed with Parafilm. Six Petri plates for each treatment wereevaluated (n = 6). Egg mortalities, measured as the number of eggs that didnot hatch, were recorded three days post-treatment. The bioassay wasmaintained at 22°C.The biocontrol potential of O. tipulae TC2and OC2 nematodes on C. capitata larvae waevaluated based on successful metamorphic transition of C.capitata larvae. Twenty third instar larvae were placedin sterile Petri plates (60 mm diameter) layered with two filter papersheets. Then, either 50, 125, 250 or 500 nematodes/larva suspended in 1 mldistilled water were added to each Petri plate. Controls were treated withpure water only. Then, all Petri plates were sealed with Parafilm. FivePetri plates for each treatment were evaluated (n = 5). Due to the rapidtransition from the larval to the pupal stage, the number of healthy andunhealthy pupae was recorded three days post-treatment. The bioassay wasmaintained at 25°C.The biocontrol potential of O. tipulae TC2and OC2 nematodes on C. capitata pupae wasevaluated based on the induced mortality of these latter. Six nematode doseswere used: 1, 5, 10, 50, 100 and 500 nematodes/pupa suspended in 1 ml ofwater. Controls were treated with pure water only. For each treatment, 5Petri plates (60 mm diameter) layered with two filter paper sheets and with20 pupae each were assayed. All Petri plates were sealed with Parafilm. Thebioassays were maintained at 25°C.The biocontrol potential of O. tipulae TC2 and OC2 nematodes, themortality of C. capitata pupae exposed todifferent nematode concentrations was evaluated at different temperatures.Four different nematode concentrations were used: 50, 125, 250 and 500nematodes/pupa suspended in 1 ml of distilled water. Controls were treatedwith pure water only. Five Petri plates (60 mm diameter) layered with twofilter paper sheets and with twenty 3- to 5-day-old pupae each were assayed.All Petri plates were sealed with Parafilm. The bioassays were maintained at20, 25, and 30°C.To evaluate the impact of temperature on the biocontrol potential ofO. tipulae TC2 andOC2 nematodes to kill and reproduce on G.mellonella, the mortality of G.mellonella larvae and the number of emerging nematodes fromG. mellonella larvae exposed to thesenematodes was evaluated in three independent experiments. In the firstexperiment, Petri plates (110mm diameter) were layered with two filter papersheets, then ten first-instar G. mellonellalarvae were placed in each plate and treated with either O.tipulae TC2 or OC2 nematodes at a concentration of 500nematodes/larva suspended in 1 ml of water. Four Petri plates per nematodestrain were used. In the second experiment, Petri plates (60 mm diameter) wereeach layered with two filter paper sheets, then one first-instarG. mellonella larva was placed in eachPetri plate and treated with either O. tipulaeTC2 or OC2 nematodes at a concentration of 500 nematodes/larva suspended in 1mlof water. Ten Petri plates per nematode strain were used. In the thirdexperiment, Petri plates (60 mm diameter) were layered with two filter papersheets, then ten first-instar G. mellonellalarvae were placed in each Petri plate and treated with eitherO. tipulae TC2 or OC2 nematodes at aconcentration of either 250 or 500 nematodes/larva suspended in 1 ml of water.Three Petri plates per nematode strain and concentration were used. In allexperiments, controls were treated with pure water only. Insect mortality wasrecorded every 24h for five days. To evaluate the reproductive potential ofnematodes, all dead larvae from the third experiment were rinsed with water andindividually placed in White traps [To evaluate the potential of te traps . EmerginC. capitata pupae wasassessed by three-way ANOVA with temperature, nematode isolate and nematodeconcentration as factors. Galleria mellonella mortalities wereanalyzed by two-way repeated measures ANOVA with time and nematode isolate asfactors. Nematode reproduction was evaluated by two-way ANOVA with nematodeisolate and nematode concentration as factors. Normality and equality ofvariance were verified using Shapiro–Wilk and Levene’s tests, respectively.Holm–Sidak post hoc tests were used for multiple comparisons. All statisticalanalyses were conducted using Sigma Plot 14.5 .Differences in egg mortalities were assessed by one-way ANOVA with nematodeisolate as a factor. Differences in the number of deformed pupae and pupalmortalities were assessed by two-way ANOVA with nematode isolate and nematodeconcentration as factors. The effect of temperature on nematode biocontrolpotential against O.tipulae , Caenorhabditis elegans(TG3), and Acrobeloides spp.    andS2.equences .Howeveridi-Saad .O. tipulae TC2 and OC2show clear symptoms of infestation , andtherefore are used as biocontrol agents against many insect pests . Howeverize EPNs . Severalsothovii , O. carolinensis , O.choningensis ,63,O. mcrovilli , O.myriiophilus , O. onir onirici , O.rugagaoensis –68,O. safricana  and O.ttipulae . Some spropsophi  and O. eancensis . ApparenO. tipulaeare capable of killing C. capitata andG. mellonella, in a manner that is comparableto highly efficient entomopathogenic nematodes such asHeterorhabditis and Steinernema [C. capitata pupae. Thus, we add more evidenceon the ability of free-living, bacteriophagous nematodes to kill insects, and theirpotential to complement pest management programs based on these biological controlagents [Our results show that two populations of inernema –73. Signlagents . ClearlyO.tipulae, O. onirici andAcrobeloides spp. have been found to naturally co-occur withEPNs in certain regions of the world and have been isolated in conjunction with EPNsfrom G. mellonella cadavers retrieved from baitedtraps in the soil [Heterorhabditis and Steinernema were found insoil samples, supporting this notion, and suggesting that free-living nematodesmight out compete EPNs or that certain edaphic or climatic parameters might favorthem over the EPNs.Several studies have shown that some free-living nematodes in Rhabditida orderinteract with EPNs ,58,74,75the soil ,76,77. Uthe soil ,58. In tOscheius species, can be considered EPNs[O.tipulae nematodes, and also that less than five individuals cancause significant mortality of C. capitata pupae,but we still do not know if this effect is caused by insect-pathogenic bacteria. Themode of action is therefore uncertain and clearly deserves more attention. Hence, itremains to be determined if O. tipulae can beconsidered an EPN, based on Dillman’s definition [O. chongmingensis and O.carolinensis are considered EPNs [O.tipulae is considered a facultative kleptoparasite that competewith EPNs for insects [There is still a lot of controversy on whether or not certain species of free-livingnematodes, including red EPNs. An EPN,red EPNs. Inour finition . Other mred EPNs ,62. Howered EPNs . Similar insects ,an ento insects , and oth insects . ClearlyC.capitata and G. mellonellaare highly susceptible to these nematodes. If they rely on closely-associatedbacteria to kill remains to be investigated. The entomopathogenic action of someOscheius species has been linked to Serratiabacteria which play an essential role during the infection and presumably cause thedeath of the host insect [O. carolinensis wasconsistently associated with four bacterial species, one of which, Serratiamarcescens, appears to be carried on the cuticle of the nematodes andthrough its association provides the worms with entomopathogenic potential [Independently of their ecological classification, we show that t insect . For exaotential . Anotherotential ,82. CleaO. tipulae against C.capitata. Under which circumstances and the exact mechanismshow these free-living bacteriophagous nematodes kill insect hosts remain to beinvestigated. Likewise, their real potential to be used as a biological controlagent in the future requires further studies such as extensive field trials.In conclusion, the present study shows the high control potential of two strains ofS1 FigPhylogenetic relationships based on the nucleotide sequences of the D2-D3expansion segments of the 28S rRNA gene were inferred by using the MaximumLikelihood method based on the General Time Reversible model. The tree withthe highest log likelihood (-6470.16) is shown. The percentage of trees inwhich the associated taxa clustered together is shown next to the branches.A discrete Gamma distribution was used to model evolutionary ratedifferences among sites ). The ratevariation model allowed for some sites to be evolutionarily invariable. The tree is drawn to scale, with branch lengthsmeasured in the number of substitutions per site. NCBI accession numbers ofthe sequences used for the analyses are shown.(PDF)Click here for additional data file.S2 FigPhylogenetic relationships based on the nucleotide sequences of the internaltranscribed spacer (ITS) region of the rRNA gene were inferred by using theMaximum Likelihood method based on the Tamura 3-parameter. The tree with thehighest log likelihood (-10573.30) is shown. The percentage of trees inwhich the associated taxa clustered together is shown next to the branches.A discrete Gamma distribution was used to model evolutionary ratedifferences among sites ). The ratevariation model allowed for some sites to be evolutionarily invariable. The tree is drawn to scale, with branch lengthsmeasured in the number of substitutions per site. NCBI accession numbers ofthe sequences used for the analyses are shown.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Mycobacterium tuberculosis. Increasingly, studies have shown that circular (circ)RNAs play regulatory roles in different diseases through different mechanisms. However, their roles and potential regulatory mechanisms in pulmonary TB remain unclear. In this study, we analyzed circRNA sequencing data from adjacent normal and diseased tissues from pulmonary TB patients and analyzed the differentially expressed genes. We then constructed machine learning models and used single-factor analysis to identify hub circRNAs. We downloaded the pulmonary TB micro (mi)RNA (GSE29190) and mRNA (GSE83456) gene expression datasets from the Gene Expression Omnibus database and performed differential expression analysis to determine the differentially expressed miRNAs and mRNAs. We also constructed a circRNA–miRNA–mRNA interaction network using Cytoscape. Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were used to predict the biological functions of the identified RNAs and determine hub genes. Then, the STRING database and cytoHubba were used to construct protein-protein interaction networks. The results showed 125 differentially expressed circRNAs in the adjacent normal and diseased tissues of pulmonary TB patients. Among them, we identified three hub genes associated with the development of pulmonary TB: hsa_circ_0007919 (upregulated), hsa_circ_0002419 (downregulated), and hsa_circ_0005521 (downregulated). Through further screening, we determined 16 mRNAs of potential downstream genes for hsa-miR-409-5p and hsa_circ_0005521 and established an interaction network. This network may have important roles in the occurrence and development of pulmonary TB. We constructed a model with 100% prediction accuracy by machine learning and single-factor analysis. We constructed a protein-protein interaction network among the top 50 hub mRNAs, with FBXW7 scoring the highest and SOCS3 the second highest. These results may provide a new reference for the identification of candidate markers for the early diagnosis and treatment of pulmonary TB.Pulmonary tuberculosis (TB) is a chronic infectious disease that is caused by respiratory infections, principally  Mycobacterium tuberculosis (MTB), which is spread by aerosols [Pulmonary tuberculosis (TB) is a contagious disease that is caused by the slow-growing aerosols . The priaerosols . TB infeaerosols . Pulmonaaerosols –6. Howevaerosols . Althougaerosols , 9. Lateaerosols . Therefoaerosols .Circular (circ)RNAs are a type of noncoding RNA molecule that lack a 5′ cap and 3′ tail and form a single-stranded circular structure by covalent bonds . CircRNAAdditionally, circRNAs can be used as micro (mi)RNA sponges to bind miRNAs through miRNA response elements, releasing the degradation or translational inhibition of downstream target mRNAs by disrupting the competitive endogenous (ce)RNA mechanism . CircRNATo summarize, many studies have shown that circRNAs are associated with the occurrence and development of a variety of respiratory and infectious diseases , 26–28. The Shanghai (Fudan University) Public Health Clinical Center provided all patient data used in this study. They performed positron emission tomography (PET) scans on the lungs of nine pulmonary TB patients and whole transcriptome sequencing of lung tissues with high PET (high metabolic activity) and normal PET (low metabolic activity) uptake. For raw sequencing data, please refer to PRJNA795290 (4∗2 samples) from the Sequence Read Archive (SRA) database and GSE158767 (5∗2 samples) from the Gene Expression Omnibus (GEO) database. All patients signed an informed consent form before obtaining tissue samples. RNA extraction, library construction, and sequencing were performed in the same way as previously described . In thisP value, and false discovery rate (FDR). The criteria for selecting differentially expressed circRNAs were ∣log2FC | >1 and P value < 0.05. Scatter plots, volcano plots, and heat maps were used to show the differently expressed circRNAs.First, the raw data were corrected, filtered, and normalized using the R package. Analyses of differentially expressed RNAs included fold change (FC), In machine learning applications, the number of features is often large, and there may be irrelevant features. With more features, the resulting model will be more complex. Feature selection can eliminate irrelevant or redundant features, reduce the number of features, improve model accuracy, reduce the running time, and simplify the model. We identified hub circRNAs using eight feature screening methods: CfsSubsetEval-BestFirst, PrincipalComponents-Ranker-T, CorrelationAttributeEval-Ranker-T, GainRatioAttributeEval-Ranker-T, InfoGainAttributeEval-Ranker-T, OneRAttributeEval-Ranker-T, ReliefFAttributeEval-Ranker-T, and SymmetricalUncertAttributeEval-Ranker-T.We used 13 algorithms to build the models: ZeroR, Logistic, SMO, IBK, AttributeSelectedClassifier, OneR, DecisionStump, HoeffdingTree, J48, LMT, RandomForest, RandomTree, and REPTree. Comparisons of the average accuracy of the machine learning models established by different feature selection methods were then conducted. We also performed statistical and univariate analyses of the number of occurrences of circRNAs in various models to identify hub circRNAs.http://starbase.sysu.edu.cn/). We screened the pulmonary TB miRNA gene chip (GSE29190) dataset from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) for differentially expressed miRNAs. The criteria for selecting differentially expressed miRNAs were ∣log2FC | >1 and P value < 0.05. Predictions of potential mRNAs downstream of miRNAs were made by the miRTarBase database (http://mirtarbase.mbc.nctu.edu.tw/php/index.php). Then, the GEO database of pulmonary TB mRNA gene chip (GSE83456) was used to screen for differentially expressed mRNAs. The criteria for selecting differentially expressed mRNAs were |log2FC | >1 and P value < 0.05. Finally, Cytoscape (http://www.cytoscape.org/) was used to construct the circRNA–miRNA–mRNA interaction network.Predictions of potential miRNAs targets of the circRNAs were made through the ENCORI website (https://david.ncifcrf.gov) for the differentially expressed mRNAs satisfying ∣log2FC | >1 and P value < 0.05. GO analysis is divided into three categories, cellular component (CC), molecular function (MF), and biological process (BP), and summarizes the biological functions, pathways, or cellular localization of gene enrichment. We used the KEGG database for pathway enrichment analysis.Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the DAVID database (https://string-db.org/) and uncovered information on the interactions of these proteins. Each node in the PPI network represents a protein. To identify the key nodes in the PPI network, we calculated the top 50 hub genes using the cytoHubba function in Cytoscape (http://www.cytoscape.org/). We then constructed a PPI network on the basis of these 50 hub genes.For the 372 mRNAs, we identified from the previous screen; we analyzed them using the STRING database  > 2. There were 125 circRNAs expressed in the adjacent normal and diseased tissues of pulmonary TB patients that satisfied P value < 0.05 and ∣log2FC | >1, among which 50 were upregulated and 75 were downregulated. There were more downregulated genes in normal tissues compared with diseased tissues . The CfsSubsetEval-BestFirst feature screening method was the next best, and its three algorithms  had a 100% accuracy rate. OneRAttributeEval-Ranker-T feature screening had two algorithms (SMO and IBK) that showed a 100% accuracy rate. CfsSubsetEval-BestFirst feature screening also had two algorithms with a 100% correct rate (Logistic and SMO). Only CorrelationAttributeEval-Ranker-T had a better accuracy rate than the model constructed with 125 circRNAs. Therefore, the CorrelationAttributeEval-Ranker-T feature screening method was more suitable for the 125 circRNAs . Among tTo further clarify the effect of circRNAs on pulmonary TB, we built machine learning models for 14 circRNAs and performed univariate analysis. We calculated the average of the models with correct rates of over 80%, with random seeds taken from 1 to 10 . The resTo identify hub genes in the development of pulmonary TB, we made Venn diagrams that included 20 DEGs and 14 univariate analysis genes using Jvenn. We uncovered three hub circRNAs: hsa_circ_0007919 (upregulated), hsa_circ_0002419 (downregulated), and hsa_circ_0005521 (downregulated). We identified these three circRNAs as hub genes in the development of pulmonary TB .P < 0.05). We screened out potential miRNAs and differentially expressed miRNAs by Jvenn, which identified hsa-miR-409-5p . We screened out 16 potential downstream mRNAs for hsa-miR-409-5p, and their differential expression was analyzed using Jvenn. These mRNAs included RPS4X, NBEA, GSK3B, RGL2, ZNF512B, SOD2, ZNF12, KPNA3, AKAP1, RPRD2, ACAP2, RSU1, FDXR, EIF4EBP2, SRRD, and ZBTB34 (We used the mRNA Gene Chip GSE83456 of pulmonary TB from the GEO database to screen out 9272 differentially expressed mRNAs (d ZBTB34 . Finallyd ZBTB34 .To study the effects of the identified mRNAs on pulmonary TB, we performed GO and KEGG analysis of the downstream mRNAs of the three hub circRNAs using the DAVID database. In BP analysis, DNA template, transcription, and positive regulation of transcription from RNA polymerase II promoter were the three terms with the highest enrichment . RegardiIn this study, we have uncovered potential mRNAs downstream of three hub circRNAs. Among them, there were 1429 downstream mRNAs of hsa_circ_0007919, 2201 downstream mRNAs of hsa_circ_0002419, and 5401 downstream mRNAs of hsa_circ_0005521. We hypothesized that mRNAs simultaneously regulated by the three circRNAs may play important roles in the development of pulmonary TB. Therefore, we screened out 372 overlapping mRNAs using Jvenn and conducted further analysis . To furtCurrent laboratory tests for pulmonary TB include immunological- and molecular-based assays. Traditional smear staining has the disadvantages of a low positive rate and a long culture period . With thWe further studied the effects of potential genes downstream of the hub circRNAs on pulmonary TB. According to GO and KEGG analysis, the genes were enriched in the PI3K-Akt signaling pathway, the proteoglycan pathway in cancer, and Ras signaling. Yang et al. showed that the inflammatory response of macrophage-like cells to MTB can be attenuated by modulating the PI3K/Akt/mTOR signaling pathway . Other svia dendritic cells [We uncovered 50 hub genes and then constructed a PPI network using the STRING database and cytoHubba. FBXW7 scored the highest, and SOCS3 was the next highest. We concluded that these genes may be associated with the development of pulmonary TB. Additionally, some of these genes have been reported to be involved in the development of pulmonary TB and other diseases. FBXW7 is an important tumor suppressor. Ni et al. found that miR-92a plays an oncogene role in nonsmall cell lung cancer by regulating FBXW7 . It has ic cells . Harlingic cells .However, this study had some limitations. First, expression levels of the DEGs need to be further verified by quantitative PCR. Second, the model with 100% prediction accuracy established in this study needs further parameter changes, optimization, and assessments before it can be widely used. Finally, the mechanisms of these DEGs in pulmonary TB need to be further explored by molecular experimentation.In conclusion, we screened three hub circRNAs using the adjacent normal and diseased tissues of pulmonary TB patients. GO and KEGG enrichment were used to identify pathways associated with pulmonary TB. We identified hub genes through a PPI network. This study may provide a reference for finding candidate markers for the early diagnosis of pulmonary TB and provide new directions for possible pulmonary TB therapeutic targets.


Malus sieversii) is an ancient relic; a plant with abundant genetic diversity and disease resistance. Several transcription factors were studied in response to different biotic and abiotic stresses on the wild apple. Basic/helix–loop–helix (bHLH) is a large plant transcription factor family that plays important roles in plant responses to various biotic and abiotic stresses and has been extensively studied in several plants. However, no study has yet been conducted on the bHLH gene in M. sieversii. Based on the genome of M. sieversii, 184 putative MsbHLH genes were identified, and their physicochemical properties were studied. MsbHLH covered 23 subfamilies and lacked two subfamily genes of Arabidopsis thaliana based on the widely used classification method. Moreover, MsbHLH exon–intron structures matched subfamily classification, as evidenced by the analysis of their protein motifs. The analysis of cis-acting elements revealed that many MsbHLH genes share stress- and hormone-related cis-regulatory elements. These MsbHLH transcription factors were found to be involved in plant defense responses based on the protein–protein interactions among the differentially expressed MsbHLHs. Furthermore, 94 MsbHLH genes were differentially expressed in response to pathogenic bacteria. The qRT-PCR results also showed differential expression of MsbHLH genes. To further verify the gene function of bHLH, our study used the transient transformation method to obtain the overexpressed MsbHLH155.1 transgenic plants and inoculated them. Under Valsa canker infection, the lesion phenotype and physiological and biochemical indexes indicated that the antioxidant capacity of plants could increase and reduce the damage caused by membrane peroxidation. This study provides detailed insights into the classification, gene structure, motifs, chromosome distribution, and gene expression of bHLH genes in M. sieversii and lays a foundation for a better understanding disease resistance in plants, as well as providing candidate genes for the development of M. sieversii resistance breeding.Xinjiang wild apple ( It has members) , Dendrobmembers) , Malus dmembers) , Oryza smembers) , Raphanumembers) , Prunus members) , Cucumisthaliana 2 membersmembers) , and Popomestica 8 membersM. sieversii) is a relic from the third century. The M. sieversii is highly likely to be the ancestor of modern cultivated apples (M. domestica) [M. sieversii forest is mainly distributed in the TianShan Mountains of Kazakstan and Kyrgyzstan of central Asia and Xinjiang, China, and is considered a wild apple gene pool with important scientific research value [M. sieversii is an ideal material for improving the disease resistance of apples in scion breeding [M. sieversii populations in Xinjiang have been shrinking [Hsp20 (12 members) [Hsp90 (8 members) [WRKY (112 members) [M. sieversii. However, the screening identification of functional gene resources in M. sieversii is not sufficient. As one of the largest transcription factors in plants, bHLH genes play important roles in plant growth and development [M. sieversii and M. domestica, the identification of bHLH gene families in M. sieversii will contribute to understanding of important functional genes.The Xinjiang wild apple (mestica) . The M. ch value . In addibreeding . Howeverhrinking ,22. Hencmembers) , Hsp90 (members) , and WRKmembers)  gene famelopment , materiaelopment ,28, and elopment ,30. In aCmbHLH87 gene in tobacco may increase resistance to powdery mildew, bacterial wilt, and scab [Glycine max) plays a crucial role in defense against Phytophthora root rot [Xanthomonas gardneri, thereby participating in the resistance pathway [GhbHLH171 in cotton (Gossypium hirsutum) activated jasmonic acid (JA) biosynthesis and signaling pathways and improved the plant resistance to Verticillium wilt [The bHLH transcription factors play important roles in plant disease resistance . For exaand scab . The bHLroot rot . It was root rot . Two bHL pathway . Overexpium wilt . Researcium wilt . Rice MAium wilt . Studiesium wilt .M. siseversii. We identified the bHLH gene family in M. sieversii and investigated its expression pattern in response to Valsa canker infection. In addition, M. sieversii cultured seedlings overexpressing MsbHLH155.1 under V. mali infection were tested in a gene-resistant functional identification system based on transient transformation. In order to improve M. sieversii’s disease resistance, this study provides an essential theoretical basis and high-quality candidate genes for molecular breeding and improving the strain.However, no research has been reported for the bHLH family in MsbHLH family genes in M. sieversii, 201 putative MsbHLH protein sequences were obtained. By using the Simple Modular Architecture Research Tool (SMART) database and CD-Search (Conserved Domains) database to identify candidate genes with “HLH” domains and removing redundant sequences, we identified 184 sequences as genes in the bHLH family of M. sieversii. According to their sequence similarity and phylogenetic relationship with a single AtbHLH protein, they were named MsbHLH001-MsbHLH162.6. Since one AtbHLH gene may have multiple homologous MsbHLH genes, the group of 184 bHLH genes was named “MsbHLH001–MsbHLH162.6”. Sequence analysis revealed that the lengths of the MsbHLH protein sequences varied from 92 to 879 aa and had predicted molecular weights of 28837.95–90283.67 kDa. The predicted isoelectric points (pIs) ranged from 4.6 to 10.37. The instability indexes of nine MsbHLH proteins were less than 40, suggesting that these putative MsbHLH proteins have good stability. The instability indexes of the remaining bHLH proteins were more than 40, suggesting unstable proteins. The subcellular localization prediction results showed that MsbHLH transcription factors were mainly located in the nucleus, and nuclear localization signals also showed that 138 MsbHLH proteins had nuclear localization signals, of which 54 were monomolecular nuclear localization signals (MN), 29 were bimolecular nuclear localization signals (BN), and 55 were MN and BN types . In order to further understand the phylogenetic relationship, we evaluated the number of representative genes in bHLH subgroups of A. thaliana and M. sieversii. There were no members of the S1 and S15 in M. sieversii, indicating that these genes may be missing during evolution. The largest MsbHLH subfamily was S25, with 20 members, whereas the smallest subfamily, S20, had only 2 members.To understand the evolutionary relationship of bHLH transcription factors among species, a neighbor-joining phylogenetic tree was constructed using 159 equences . The genM. sieversii , MsbHLH genes contain abiotic-stress-response elements . Among them, there are 29 hormone-response elements in the promoter region of MsbHLH035.1. The promoter region of MsbHLH084.3 contains 17 abiotic-stress-response elements. Among the results of the study, family members play a key role in hormone action pathways and stress response to abiotic stress. Moreover, 165 MsbHLH promoters contained biological stress-related components , accounting for 89.7% of the total bHLH genes; 143 MsbHLH promoter regions were rich in JA-related cis-elements; and 82 MsbHLH promoter regions were rich in SA-related elements. In addition, 104 MsbHLH promoter regions contained a W-box; 85 MsbHLH promoter regions contained WER3 elements; and 102 MsbHLH promoter regions contained a WUN-motif, indicating that they may be involved in a series of biological stress responses and defenses . Interestingly, MYC2 is involved in the jasmonic-acid-mediated pathway in this protein network when interacting with multiple JASMONATE-ZIMDOMAIN (JAZ) proteins. Moreover, MsbHLH155.1 was found to interact with LBA1 (AT5G47010) and multiple cell-wall-related proteins (AT1G05310) by predicting its protein network  network generated with STRING was used to predict the interaction relationship of 94 putative MsbHLH proteins A. Many M network B. These MsbHLH genes in response to V. mali infection, the expression pattern of differentially expressed MsbHLH genes was analyzed from the transcriptome data [V. mali resulted in the quantification of 94 MsbHLH genes by mapping reads  (MsbHLH genes) were selected for qRT-PCR verification. As a result, all genes except MsbHLH092.1 exhibited differential expression patterns that were consistent with transcriptome data  on the 3rd day (−1ꞏFW)  in the leaves of MsbHLH155.1 transgenic plants and was significantly decreased after infection with V. mali  C. The H2 V. mali F. As a rA. thaliana and O. sativa were used in previous phylogenetic analyses of bHLH proteins in plants [In plants, the bHLH transcription factor family is the second largest family of eukaryotic transcription factors after MYBs. The genome sequences of n plants ,43,44,45MsbHLH genes were identified and characterized. A larger number of MsbHLHs were found in Arabidopsis (162 members) [M. domestica (188 members) [M. sieaersii might have been the ancestor of M. domestica. The 23 subgroups of MsbHLHs were identified based on phylogenetic analysis  , but fewmembers) . Due to analysis . Within entified . This co domains . For exaomestica . A phyloanalysis . Thus, tM. sieversii and 159 AtbHLH proteins in A. thaliana, a phylogenetic tree was constructed. Twenty-three subfamilies were found in M. sieversii, but not subfamilies S2 and S15, indicating that there is a difference in evolutionary processes between M. sieversii and Arabidopsis. It was observed that the numbers of MsbHLH transcription factors were amplified in many subfamilies, such as S8, S10, and S23. The expansion of numbers of the bHLH gene family could improve the environmental adaptability of M. sieversii. The MYC2 transcription factor, as a direct inhibitor of the jasmonate ZIM domain (JAZ), plays a role in regulating the JA signaling pathway in multiple biological processes [A. thaliana [M. sieversii lives in a relatively cold environment and suffers from Valsa spp. infection, biotic- and abiotic-stress-related genes may be amplified more in the evolutionary process, improving its adaptability.In order to explore the evolutionary relationship between 184 MsbHLH proteins in rocesses ,47. In tthaliana ,49. AtGLthaliana ,51. In tthaliana ,53. A nuthaliana . AdaptatVerticillium wilt [Verticillium’s wilt resistance. When stress signals are present, JA accumulates, resulting in the degradation of JA-ZIM (JAZ), which then activates the transcription factors inhibited by JAZ, allowing for defense response and development to occur [Increasing plant resistances to powdery mildew , bacteriium wilt , and whiium wilt  are depeium wilt . For exato occur .M domestica, the transcription factor MdERF100 physically interacts with the MdbHLH92 transcription factor to mediate powdery mildew resistance and enhance plant resistance by regulating the JA and SA signaling pathways [MsbHLH gene family has not been studied. The bHLH genes may also regulate SA or JA in M. sieversii, which requires experimental confirmation with more precise tests to explain why M sieversii is resistant to Valsa canker.Some pathogen-related (PR) genes controlled by the SA signaling pathway were markedly down-regulated in rice RNA sequences from OsbHLH61-RNAi plants , suggestpathways . Howevercis-elements in their promoters. Consequently, determining functional cis-acting elements in promoters is an essential step to understanding gene function experiments [cis-acting elements in the genome, but little is known about their cis-activity, including on the W-box [cis-element analysis, MsbHLH-promoter regions contain elements related to JA, SA, W-box, and WER3, suggesting that they may be involved in the biotic stress response and defense. Additionally, interaction networks were predicted for 94 MsbHLH transcription factors. Several factors have been implicated in the promotion of pathogen-induced callose deposition, including MsbHLH028 (homologous with AtMYC2), SA biosynthesis, PR1 expression, and SA response. In addition, MYC2 is required for the JA-mediated defense against the necrotrophic pathogen Botrytis cinerea [M. sieversii in response to V. mali infection. Furthermore, MsbHLH028 (homologous with MYC2) might be a key transcription factor of the JA signaling pathway in plants. During JA signaling rest, a group of JAZ proteins physically recruit transcriptional co-repressor (TOPLESS) to form a repression complex, inhibiting the expression of JA-responsive genes in MYC2 and its close homologs [MsbHLH genes that may respond to biotic stress, which is of great significance for mining disease resistance gene resources in M. sieversii.Plant defense responses are initiated by pathogens or pathogen-derived inducers, which lead to extensive transcriptional changes ,57. Durieriments . Pathogehe W-box , S-box [he W-box , and GCChe W-box . Some ofhe W-box . As show cinerea . As a co cinerea . It is ihomologs . MYC2 mahomologs . AccordiMsbHLH genes in plant-disease resistance, we analyzed transcriptome sequencing results under pathogen infection conditions. It was found that nearly half of the MsbHLH genes responded to pathogen infection. Based on the results, it was found that these differentially expressed MsbHLH genes had relatively complex expression patterns in response to V. mali. We found that MsbHLH155.1 (homologous to MdbHLH155) participates in the JA pathway to regulate anthocyanin synthesis [2O2 levels), the MDA and H2O2 contents were significantly reduced with the overexpression of MsbHLH155.1 compared with the control. It was demonstrated that MsbHLH155.1 presented disease-resistance functions through scavenging reactive oxygen species. In MsbHLH155.1-transient-overexpression plants, the average areas of lesions in the leaves significantly decreased by suppressing the pathogen’s proliferation. Based on the above results, MsbHLH155.1 can enhance plant disease resistance. It improves the ability to scavenge ROS, reduces plant cell membrane damage, and inhibits pathogen growth. In summary, these results provide a good reference for further research into the functions of the MsbHLH gene family.To investigate the role of ynthesis . Based oynthesis . With thArabidopsis from the plant transcription factor database v4.0 (http://planttfdb.cbi.pku.edu.cn/ (accessed on 22 January 2023)). The hidden Markov model (HMM) file corresponding to the bHLH domain (PF00010) was downloaded from the Pfam protein family database (http://pfam.sanger.ac.uk/ (accessed on 22 January 2023)). Then, HMMER 3.0 software was used to build a hidden Markov model, and the existence of HLH core sequences was verified using the PFAM and SMART programs (http://smart.embl-heidelberg.de/ (accessed on 22 January 2023)). The 184 MsbHLH genes were identified in the M. sieversii genome. The M. sieversii genome (version JAHTLV010000000) was retrieved from the NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 22 January 2023)) databases [bHLH genes of M. sieversii  were identified using the ExPasy (http://web.expasy.org/protparam/ (accessed on 22 January 2023)).We downloaded the bHLH transcription factor protein sequences of atabases . BLASTp https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi (accessed on 22 January 2023)) were then used to adjust the bHLH structure domain while using the deduced amino acid sequences. The online program MEME (http://meme.nbcr.net/meme/intro.Html (accessed on 22 January 2023)) was used to analyze the motifs of the MsbHLH proteins. Then, the motifs, CDS, and UTR areas of MsbHLH genes were analyzed using TBtools [To analyze the characteristic domain of MsbHLH proteins, ClustalW was used to generate multi-sequence alignment with default parameters. Mega software (version 6.0) and ESPript 3.0 ).A total of 184  TBtools . The 200Arabidopsis were downloaded from TAIR (https://www.arabidopsis.org/ (accessed on 22 January 2023)). The full-length amino acid sequences of Arabidopsis and M. sieversii were aligned with Clustur W software, and then MEGA 6.0 software was used to obtain a tree using the neighbor-joining method with a bootstrap of 1000 replicates. Finally, the system’s evolutionary tree was beautified using iTOL (https://itol.embl.de (accessed on 22 January 2023)).The 159 AtbHLH proteins of https://cn.string-db.org (accessed on 22 January 2023)) website. The bHLH protein sequences in A. thaliana were chosen as the references, and then the minimum required interaction score (0.4000) was set to middle confidence. Following blast analysis, the orthologous genes with the highest scores were used to construct the network. Genes with no interactions with other genes were removed. Using the blast results, functional annotations were manually pasted.The 94 differentially expressed MsbHLH protein sequences were submitted to the STRING  in a natural Wild Reserve Forest in Yili, Xinjiang. Following a 75% medical alcohol sterilization, the twigs were injured with a sterilized pattern wheel (2 cm in diameter) and inoculated with a mycelial plug (5 mm) from a 3-day-old canker pathogen, Valsa mali (isolate EGI1). After 0, 1, 2, and 5 dpi, barks of twigs near the canker were separately harvested and frozen for RNA extraction. Each sample contained three biological replicates. Bark samples from 0 dpi timepoints were collected for RNA extraction as controls. The differentially expressed MsbHLH genes were identified in the transcriptome of M. sieversii infected by V. mali [2 (fold change)|≥1 with the Q-value < 0.05. The expression pattern heat map was generated using FPKM values of genes by TBtools software [Twigs of  V. mali . The gensoftware .V. mali infection, the differentially expressed MsbHLH genes were selected for qRT-PCR. By blasting primer sequences against the NCBI database, primer specificity for qRT-PCR was evaluated using Primer5 software.(EF-α) gene was used as an internal reference [−ΔΔCt method [Under software.. The mRNeference . The 2−Δt method  was usedAgrobacterium tumefaciens EHA105. The infection process of transient transformation according to the method of Liu et al. [M. sieversii leaves were transiently expressed with MsbHLH155.1 and p1307-flag (empty vector). The leaves inoculated with V. mali isolate EGI1 were photographed at 1, 2, and 3 dpi. The lesion areas were calculated by ImageJ software. The area ratios of the diseased lesion were analyzed by Excel. Determination of the concentrations of H2O2, MDA, and Bacteroidetes in the samples was performed. The plants transformed with empty p1307-flag or p1307-myc were used as the controls. Statistical analysis was performed using SPSS 26.0  software. Three independent biological replicates were performed to ensure the accuracy of the analyses. Each biological replicate contained three technical replicates. The value showed the expression level as the mean ± standard error (n = 9). The data were submitted for analysis using Duncan’s test, following one-way ANOVA at the p ≤ 0.05 and p ≤ 0.01 levels.The intact coding sequences (CDS) of the studied transcription factors were fused in frame with the C-terminus of 3× flag tags under the control of the CaMV 35S promoter in the p1307-flag plant expression vector. The intact CDS of the studied functional genes replaced 5× Myc tags and cloned into the 1307-myc vector under the control of the CaMV 35S promoter to generate the plant expression vectors. All constructs were confirmed by DNA sequencing and transferred into u et al. . The M. M. sieversii, the bHLH transcription factor family was identified and characterized. According to the phylogenetic analysis, 184 members of the MsbHLH family were divided into 23 subfamilies, with conservative gene structures and motifs. It was predicted that the MsbHLH domain has certain functional characteristics. Additionally, the transcriptomic data of the MsbHLH gene revealed various expression profiles after pathogen infection, which may contribute to a better understanding of the resistance function of the gene. MsbHLH155.1 was identified as a disease-resistant gene, through inhibiting pathogen growth and scavenging ROS to reduce damage to plant cell membranes. In conclusion, this study provides new insight into the resistance function and regulatory mechanism of the MsbHLH protein in M. sieversii.Using the whole genome of


TPS) gene family in Brassica napus. In this study, in total, 26 TPS genes in B. napus (BnTPS genes) were identified and classified into two groups. In each group, the BnTPS genes showed relatively conserved gene structures. The protein–protein interaction (PPI) network and enrichment analysis indicated that BnTPS genes were involved in the glycolysis/gluconeogenesis, fructose and mannose metabolism, galactose metabolism, pentose phosphate pathway, carbohydrate transmembrane transport, trehalose–phosphatase activity, etc. The expression of BnTPS genes varied greatly across different tissues, while most of the BnTPS genes showed a considerable improvement in expression under different abiotic stresses, indicating that BnTPS genes were significantly responsive to the abiotic treatments. In addition, the association mapping analysis revealed that eight BnTPS genes were potential regulators of particular agronomic traits. Among them, the gene BnTPS23 was significantly associated with the primary flowering time (PFT), full flowering time (FFT1), and final flowering time (FFT2), suggesting that BnTPS genes may play an important role in regulating key agronomic traits in B. napus. In summary, our research provides a better understanding of BnTPS genes, facilitates the breeding of superior B. napus varieties, and paves the way for future functional studies.Trehalose and trehalose-6 phosphate played important roles in floral organ development, embryonic development, cell morphogenesis, and signal transduction under abiotic stress. However, little is known about the trehalose-6-phosphate synthase ( Trehalose  is a structurally stable non-reducing disaccharide that can help biological cells maintain nucleic acid, protein, and biological membrane activity as well as protect cellular structures under adverse conditions. It consists of two glucose molecules linked by α, α, 1, and 1-glycosidic bonds ,3. It coTPS genes was noticeably higher in the leaves and roots . In. InBnTPSequences . On averp I (21) . There w BnTPS24 , suggestnt trait . The geno groups .BnTPS genes are involved in trehalose synthesis and stress resistance in many plants [TPS gene family has been found in numerous plants [A. thaliana [TPS gene family in B. napus has not been extensively studied. The Brassica genus underwent a genome triplication after divergence from the Arabidopsis lineage. Subsequently, a natural cross between B. rapa and B. oleracea occurred about ~7000 years ago to form the allotetraploid B. napus [A. thaliana gene is projected to discover up to six homolog genes in B. napus. According to our results, B. napus contains only 26 TPS genes, while A. thaliana has 11 TPS genes  homologous to AtTPS1  of AtTPS1 contained the N-terminal extension, indicating that the other one (BnTPS3) may have lost its N-terminal extension during evolution  for the cultivar ‘Darmor-bzh’ of B. napus [BnTPS genes, the TPS  and TPP  domains of Hidden Markov Models (HMMs) were downloaded from the Pfam database  [BnTPS candidate genes, including typical TPS and TPP domains, in B. napus with the setting e-value of 1 × 10−5. Then, all candidate BnTPS genes were subjected to the NCBI-CDD (Conversed Domain Database)  [http://smart.embl.de/, accessed on 28 September 2022) [BnTPS genes were subjected to the online software ProtParam  [http://cello.life.nctu.edu.tw/, accessed on 28 September 2022) [Reference genome sequence and gene annotation files were obtained from the BRAD Database (Available online: B. napus . To idener 2022) . The HMMer 2022)  and the er 2022)  to verifer 2022)  to predier 2022) .bzh’ reference genome, the chromosomal locations of TPS genes were determined. BLASTP [−10, and MCScan X [BnTPS gene pairs and the duplication patterns. Chromosomal locations and paralogous BnTPS gene pairs were visualized by the Circos software [BnTPS gene family, the ratio of non-synonymous substitution to synonymous substitution (Ka/Ks) of paralogous BnTPS gene pairs was calculated by Tbtools [Based on the annotation of the ‘Darmor-. BLASTP  was usedMCScan X  was usedsoftware . To dete Tbtools .https://meme-suite.org/meme/, accessed on 10 October 2022) was used to identify the conserved motifs in the BnTPS proteins sequence [cis-acting regulatory elements via online software PlantCARE5  [BnTPS genes were also displayed by Tbtools software [The online software MEME  (Available online: sequence  with tensequence . Promoteer 2022) . The chrsoftware .TPS genes, multiple sequence alignments of BnTPS and AtTPS protein sequences were performed by ClustalW software [https://itol.embl.de/, accessed on 10 October 2022) [To reveal the evolutionary relationships of software . We thensoftware . Furtherer 2022)  was usedA. thaliana, the protein–protein interaction networks of the BnTPS proteins were predicted. The software Cytoscape [https://www.string-db.org/, accessed on 11 October 2022) [Based on the homologous genes in ytoscape  was useder 2022) . All of er 2022) .BnTPS genes were calculated with the software Stringtie [There were five tissues  and four stress conditions’  transcriptome data from previous studies ,24. The tringtie  after altringtie  and thenB. napus core collection germplasm was used to investigate the potential effects of BnTPS genes on regulating important traits. All the SNPs in BnTPS gene region were annotated by the software SnpEff [p value (0.05/N) was set as the significant threshold.A worldwide 324 diverse e SnpEff . PFT, FFe SnpEff . The rMVe SnpEff . The p vBnTPS genes were identified and investigated. These BnTPS genes were grouped into two groups. BnTPS genes from the same groups have similar structures and motifs. Many important cis-acting regulatory elements responsible for plant growth and stress response were detected in the promoters of BnTPS genes. The protein interaction analysis showed that BnTPS genes played an important role in development and stress resistance. The various expression patterns of BnTPS genes were revealed in different tissues and under abiotic stresses. Additionally, the association mapping results also showed that the BnTPS genes potentially influence agronomic traits in B. napus. In summary, these results offered comprehensive knowledge about BnTPS genes, which provided a basis for further functional research and genetic improvement for the breeding of superior B. napus varieties.In this study, 26


Leptospira-secreted exotoxins that mediate the molecular and cellular pathogenesis of severe and fatal leptospirosis. If confirmed experimentally, this concept could revolutionize the treatment, diagnosis, prognosis, and vaccine-mediated prevention of leptospirosis by enabling a novel array of targeted interventions. VM proteins, as with other bacterial-secreted protein exotoxins, mediate their virulence effects by attaching to eukaryotic cells, competing with other microorganisms for limited resources in environmental niches, directly intoxicating target cells, and disrupting their function in the mammalian host. In contrast with the most pathogenic group of Lept ospira, particularly L. interrogans, whose genomes contain 12–15 PF07598 paralogs, strains of the livestock and human pathogen L. borgpetersenii have two PF07598 paralogs. Given the possible non-environmentally mediated transmission of some L. borgpetersenii strains and the much smaller number of VM proteins in this species, their role in infection and disease may well differ from other leptospiral species. Comparison of VM proteins from different clades of pathogenic Leptospira may deepen our understanding of leptospirosis’s pathogenesis, leading to novel approaches to ameliorating Leptospira infection in humans and animals.Leptospirosis is an emerging infectious disease, with increasing frequency and severity of outbreaks, a changing epidemiology of populations at risk, and the emergence of new strains, serovars, serogroups, and species. Virulence-modifying (VM) proteins encoded by the PF07598 gene family are hypothesized to be  Leptospira, which has 64 genomic species and 300 serovars identified to date [Leptospira in their urine. Humans are accidental hosts and acquire the infection from either direct or indirect sources such as contaminated water, like rivers, puddles, and rice paddies [Leptospira species, including L. borgpetersenii serovar Hardjo and L. interrogans serovar Pomona [Leptospira [Human deaths due to infectious diseases remain the leading scourge of humanity . Many of to date . A wide  paddies ,9,10,11. paddies . Bovinesr Pomona ,14. Infeptospira . The serptospira . To achiLeptospira [Leptospira and the pathobiology of leptospirosis by a comparative analysis of this gene family in L. interrogans and L. borgpetersenii.Fundamental biological insights are essential for understanding how to prevent infectious disease transmission and intervene against disease in both humans and animals. The recently discovered virulence-modifying (VM) proteins, encoded by the PF07598 gene family, are found only in group 1 pathogenic ptospira ,17,18. TA human being who comes into contact with urine-infected animals or contaminated environments, whether directly or indirectly, is considered at risk for leptospiral infection . FindingL. borgpetersenii serovar Hardjo (type Hardjo-bovis) and L. interrogans serovar Hardjo (type Hardjo-prajitno) [L. borgpetersenii serovar Hardjo type Hardjo-bovis is most often isolated from cattle [L. interrogans serovar Lai VM proteins protected mice from any clinical manifestations of the disease. The authors observed that this led to ~3–4 log10 reduction in bacterial load in the liver and kidney, two key organs in the pathogenesis of leptospirosis and transmission of Leptospira, respectively. Hence, recombinant protein vaccines may be key to global immunization against pathogenic leptospirosis, eliminating kidney colonization and spread in the environment [With few outwardly observable clinical signs, cattle are a chronic host of serologically identical but genetically distinct members of serovar Hardjo, namely rajitno) . Cattle rajitno) ,31. In Nm cattle ,31. Vaccm cattle . Informam cattle  found thironment . HoweverLeptospira still has several unknown aspects. Fouts et al. [Leptospira and the exotoxin PF07598 paralogous gene family. Members of the PF07598 gene family are expressed and upregulated to various extents, as shown in vivo in a hamster model of acute leptospirosis. It has been established that group 1 (pathogenic Leptospira) includes L. interrogans serovars Icterohaemorrhagiae and Copenhageni, which are the strains most often associated with severe and fatal outcomes [Leptospira [Virulence in s et al.  describeoutcomes , which soutcomes ,34,35. Tptospira .Leptospira, and they have been more intensely studied in interrogansL.  than in borgpeterseniiL.  [Virulence factors are often the focus of characterization since a bacterin vaccine that exposes an immunized host to more known virulence factors are likely to produce improved protection ,34,35. Ttersenii ,33. The Leptospira [Leptospira pathogenesis and, perhaps, in enabling Leptospira to persist in the hostile and competitive environmental microbial world [Leptospira.The large novel gene family PF07598, previously of unknown function, encodes the VM proteins found uniquely in group 1 pathogenic ptospira ,18,36. Tal world . Here weLeptospira borgpetersenii and Leptospira interrogans, which, combined, cause most cases of leptospirosis [L. borgpetersenii [L. borgpetersenii associated with a switch to a symbiotic lifestyle that causes protein production changes, although they remain as virulent as L. interrogans, which has more VM proteins [Two of the largest phylogenetically distinct pathogenic species are spirosis . Althougtersenii . After ttersenii . Overallproteins ,13,14,15L. borgpetersenii and L. interrogans survival in water at 20 °C, the former lost >90% viability within 48 h whereas L. Interrogans retained 100% viability over the same period [L. interrogans retained 30% viability over a 3-week incubation, by which time no viable L. borgpetersenii were detected (R.L.Z. unpublished data) [L. borgpetersenii does not tolerate nutrient deprivation and does not survive passage through water [L. borgpetersenii has a limited capacity to acquire nutrients and survive in environments external to a mammalian host [Leptospira species requires some specificity and that horizontal gene transfers may be one of the methods allowing fast adaptation to these hosts [Leptospira species and host dynamics.In a study comparing e period . L. inteed data) . The autgh water . L. borgian host . Althougse hosts . Adherense hosts . In somese hosts . This leLeptospira and is not found in either intermediate or saprophytic Leptospira [in L. interrogans serovar Lai (12), L. interrogans serovar Copenhageni (13), L. borgpetersenii serovar Hardjo (4), and several others using system biology and “pathogenomic” approaches [L. borgpetersenii have four VM proteins? There may be one protein and three isomers, or there may be four different proteins. Perhaps there have also been mildly deleterious mutations or, similarly, new insertions of intrinsic elements that might facilitate intragenomic recombination.Our body of work has shown that the PF07598 gene family is restricted to group 1 pathogenic ptospira ,18,36,41proaches . This raL. borgpetersenii serovar Hardjo, one hypothesis is that the bacteria are undergoing a process of insertion sequence (IS)-mediated genome reduction [L. borgpetersenii and genome expansion in L. interrogans, which reflects differences in the environments during transmission between hosts. That is one theory that could support the evolution of L. borgpetersenii. Based on several comparisons [L. interrogans Copenhageni (LIC) apart from LIC_10639, and each protein from L. borgpetersenii has copies inside the species. Answering the general question about why this happens is a work in progress.A tenet of evolution is that alleles that are under negative selection are often deleterious and confer no evolutionary advantage; these are removed from the gene and are eventually extinguished from the population . Converseduction , leadingparisons ,17,18,43L. interrogans Lai cause severe leptospirosis in humans and L. borgpetersenii Hardjo-bovis causes fetal loss in cattle, we focused on the phylogenetic comparison between the VM proteins from these species and serovars  [L. borgpetersenii serovar Hardjo-bovis strain Sponselee showed 90% identity with Q04V07. Based on this finding, L. borgpetersenii serovar Hardjo-bovis presents four VM proteins, and further experimental validations could be used to develop a potential vaccine candidate for cattle and other animals worldwide.Comparative analysis of protein content suggests that different protein profiles are expressed by d 37 °C) . For theL. borgpetersenii serovar Hardjo-bovis M6BFG1 was clustered with Q04V07 with a bootstrap value of 100%, and with Q04NE0 and Q04T47 with a bootstrap value of 99%  and L. borgpeterseni serovar Hardjo-bovis  proteins are still a novel approach to discovering more about leptospirosis pathogenesis, presenting an opportunity for new treatments and prevention protocols. The use of in silico and in vitro approaches have started to show how these proteins can be an asset in combatting and preventing leptospirosis in humans and, in the future, animals. While previous mouse immunization experiments suggest that VM proteins are important virulence factors in vivo, the proposed mechanism of protective immunity in the animal system remains unknown. Demonstrating that immunization with VM proteins from one Leptospira species and serogroup cross-protects against challenge infection containing a different serogroup would be an important advancement in the field. Such research would form the basis for leptospirosis vaccine development for several species.The unique “lifestyle” of


Mitochondrial DNA changes can contribute to both an increased and decreased likelihood of cancer. This process is complex and not fully understood. Polymorphisms and mutations, especially those of the missense type, can affect mitochondrial functions, particularly if the conservative domain of the protein is concerned. This study aimed to identify the possible relationships between brain gliomas and the occurrence of specific mitochondrial DNA polymorphisms and mutations in respiratory complexes III, IV and V. The investigated material included blood and tumour material collected from 30 Caucasian patients diagnosed with WHO grade II, III or IV glioma. The mitochondrial genetic variants were investigated across the mitochondrial genome using next-generation sequencing (MiSeq/FGx system—Illumina). The study investigated, in silico, the effects of missense mutations on the biochemical properties, structure and functioning of the encoded protein, as well as their potential harmfulness. The A14793G (MTCYB), A15758G, (MT-CYB), A15218G (MT-CYB), G7444A (MT-CO1) polymorphisms, and the T15663C (MT-CYB) and G8959A (ATP6) mutations were assessed in silico as harmful alterations that could be involved in oncogenesis. The G8959A (E145K) ATP6 missense mutation has not been described in the literature so far. In light of these results, further research into the role of mtDNA changes in brain tumours should be conducted.  The respiratory chain consists of four protein complexes located in the inner mitochondrial membrane and an ATP synthase called the V complex. The high efficiency of energy production is ensured by arranging carriers according to the increasing oxygen reduction potential. The first four complexes are involved in the transport of electrons to the oxygen molecule. Complex I oxidises NADH (nicotinamide adenine dinucleotide), complex II oxidises succinate to fumarate, transferring the resulting electrons to ubiquinol, which carries electrons to complex III, and then, by cytochrome c, to complex IV. Complex V uses the transmembrane proton gradient  to produce ATP. Complex I is the largest component of the mitochondrial respiratory chain, and its dysfunction is the main contributor to conditions related to the dysfunction of oxidative phosphorylation . One of MT-CYB encoding cytochrome b is fairly susceptible to human mitochondrial DNA changes associated with a range of conditions   .http://pfam.xfam.org/search#tabview=tab1; accesed on 29 September 2020]  .http://agadir.crg.es/protected/academic/calculation4.jsp; accessed 11 September 2020] [AGADIR—theoretical prediction of the helix percentage in a given segment of the protein [er 2020] .http://www.cbs.dtu.dk/services/TMHMM/; accessed on 4 July 2020] [TMHMM 2.0—assessment of the presence of transmembrane segments in a given protein with a specific sequence of amino acids [ly 2020] .http://www.ncbi.nlm.nih.gov/Class/Structure/pssm/ pssm_viewer; acccesed 20 October 2020] [PSSM Viewer—assessment of the incidence of amino acids in each alignment of a protein sequence [er 2020] .https://sift.bii.a-star.edu.sg/www/SIFT_seq_submit2.html; accessed on 2 November 2020]  .https://mitimpact.css-mendel.it/; accessed on 15 October 2020]  .https://consurfdb.tau.ac.il/index.php; accesed on 9 October 2020] [ConSurf-Database—analysis of amino acid conservation in a given protein alignment. Score on a 1–9 scale  and normalised points  [er 2020] ,51.The detected missense polymorphisms and mutations were analysed in silico using the following bioinformatics software: