This review discusses the main methods of breeding material development, the current state, problemsand prospects for medicinal and essential oil plants breeding. The relevance of this area has especially increaseddue to the sanctions, the resulting shortage of medicinal plants and their low quality, which does not meet the requirements of the pharmaceutical industry. To produce a stable plant raw material base, it is necessary to activelydevelop a breeding process to create new highly productive varieties of medicinal plants resistant to biotic andabiotic environments. In breeding with the use of modern molecular biological methods, related species and generic complexes of the All-Russian Research Institute of Medicinal and Aromatic Plants (VILAR) collection can beinvolved, where there is extensive original genetic material of medicinal, essential oil, rare and endangered species. In the breeding of medicinal and essential oil crops, traditional methods of individual and individual-familyselection, polyploidy, chemical mutagenesis and a combination of methods to obtain original breeding materialare still promising. VILAR has created more than 90 varieties of medicinal and essential oil crops, most of whichhave been approved for use throughout the Russian Federation. Medicinal plant raw material is either freshor dried plants, or their parts  used for the production of herbal medicines. Herbalmedicines include fatty oils, essential oils, resins, balms,extracts, tinctures, aqueous extracts, and individual bioactivesubstances (BS) or their mixtures2. These drugs are recommended for the treatment and prevention of almost the entirespectrum of diseases.Currently, about 350,000 species of flowering plants have beendescribed, but not every one of them is sufficiently studied tobe called medicinal. According to the definition of the GreatMedical Encyclopedia, medicinal plants are plants used as asource of medicinal plant raw material and medicinal productsof natural originhttps://xn--90aw5c.xn--c1avg/The Great Medical Encyclopedia. Ed. by B.V. Petrovsky. 3rd edn. Vol. 12.Available at: URL:https://pharmacopoeia.ru/ofs-1-5-1-0001-15-lekarstvennoe-rastitelnoe-syre/Russian State Pharmacopoeia. 14 edn. GMP.1.5.1.0001.15. Medicinal plantraw materials. Available at: URL: The chemical composition of medicinal plants and plantderived BS is extremely complex. In order to estimate whichgroup of BS has a particular effect, All-Russian ResearchInstitute of Medicinal and Aromatic Plants (VILAR) carriesout a complex of chemical and pharmacological studies onextraction, fractionation, purification, isolation of BS fromeach studied object and determination of their specific activity.It is important that the species of medicinal plants growing indifferent botanical and geographical zones may contain thesame groups of BS allowing in the absence of some necessaryspecies to use alternative medicinal plants and obtain targetsubstances. For example, the flavonoid rutin (vitamin P)which has been shown to strengthen the capillaries is found invarying quantities in the aerial part of the buckwheat (Fagopyrum esculentum Moench), the fruits of the chokeberry (Aronia melanocarpa (Michx.) Elliott) and black currant (Ribesnigrum L.), buds and fruits of Japanese sophora (Styphnolobium japonicum (L.) Schott), flowers and fruits of blood-redhawthorn , different species ofrose hips (Rosa L.), in the wood of the lower part trunk ofSiberian larch and Gmelin larch  Kuzen). Another group of plant-derived BSare tannins, which are contained in the rhizomes of Bergeniacrassifolia (L.) Fritsch, Bistorta off icinalis Delarbre, varioustypes of potentilla (Potentilla L.), Sanguisorba off icinalis L.,in the bark of various oak species (Quercus L.), viburnum(V iburnum opulus L.), bird cherry (Prunus padus L.), blueberry (Vaccinium myrtillus L.), multiple fruits of Alnus glutinosa (L.) Gaertn., leaves of Cotinus coggygria Scop. andRhus coriaria L. The ability to accumulate cardiac glycosidesof cardenolide and bufadienolide nature in tissues has beenfound in 20 species of medicinal plants belonging to 10 different families . The scientific program “From plant biochemistry to humanbiochemistry”, developed by VILAR, allows to study thebiosynthesis of plant BS and purposefully use them for theimprovement of people’s health, which is consistent with thegoal of the state drug policy: to provide the population withaffordable and high-quality medicines, including plant-derivedones, in a timely manner .Currently, their proportion occupies about 30 % of all drugs. And in this aspect, making a sustainableresource base of medicinal plants for pharmaceutical industryis of current interest. Among the set of tasks to solve this issue,the creation of new high-yielding varieties of medicinal andaromatic plants, resistant to the effects of biotic and abioticfactors, and the development of agricultural technologies fortheir cultivation is the most important.Biotechnology and molecular biology are used along with traditional breeding methods to create new varieties of medicinalplants. In such a case, the main goal is to increase both theyield of medicinal plant raw material and the content of certainsecondary metabolites. Improved genotypes are important toincrease the profitability of the production of high-qualitymedicinal plant materials.Information on the genetic diversity and inheritance of theselected traits is one of the conditions for effective plant breeding . The development of molecular biology methods opens many new possibilities for plant breedersto solve complex problems they face in traditional breedingprocess. Most modern research on medicinal plants focuseson the study of their genetic variability using DNA markers. New high-yielding cultivar of Perilla frutescens L. was created using data of genome-wide sequencing and SNP analysis . At the same time,very little is known regarding the approaches to medicinalplants improving based on the molecular mechanisms ofmetabolite biosynthesis . For example, severalstructural genes associated with the biosynthesis of flavonoidsin gentian were isolated and characterized . Wagner et al. (2005) showed thephenomenon of monogenic inheritance of the (–)-α-bisabololand chamazulene content in chamomile. The greatest successhas been achieved in Artemisia annua L. breeding, which produces the important sesquiterpene lactone called artemisinin.In vitro tissue cultures are often used in the breeding of medicinal plants . Effective systems for cultivationand regeneration of tissues including the cultivation of callus,anthers, and protoplasts, have been created for some speciessuch as Echinacea purpurea (L.) Moench, Dendrobium candidum Wall., Aristolochia contorta Bunge, Centella asiatica L.and Curcuma wenyujin Y.H. Chen. . Biotechnology techniques play an important role in the conservation of some medicinal plants – in particular endangered species. In spite of the number of problems, there are significantprospects for future development of this field of research.The institute has collected and created an original and uniquegenetic material base of medicinal and aromatic (includingrare and endangered) plant species. However, until recently,this material has not been studied properly. Morphotypes,closely related species and generic complexes can be involvedin breeding using modern methods. For these purposes, collections of the genera Digitalis L., Echinacea Moench, Origanum[Tourn.] L., Atropa L., Tanacetum L. and Mentha L. havebeen set up in VILAR in the last decade. The developmentof modern approaches using cytological and molecular biological methods is very promising for the study of medicinalplants. Studies using various modern methods of chromosomalanalysis at the early stages of ontogenesis  make it possible to establishthe cytogenetic characteristics of promising plant lines, whichcan then be included in the breeding process. Many species ofmedicinal plants have small chromosomes (up to 3 microns).The methods of selection and application of chromosomalmarkers were tested on Potentilla alba L. . In a paper by Samatadze et al. (2018) it was shownthat the chromosomes of P. alba 2n = 28 are very small (0.88to 1.7 μm) and have a similar morphology (based of monochrome staining).In the breeding of medicinal and essential oil plants successis often achieved by a combination of methods for obtainingoriginal breeding material .Selection of polyploid forms obtained as a result of 0.2 %colchicine solution exposure has been successfully used onchamomile (Matricaria chamomilla L.). Among the three varieties recommended for cultivation in the Russian Federation,the variety Podmoskovnaya is an autotetraploid (2n = 36),and Nasten’ka and Sibirskaya bisabololnaya  arediploids (2n = 18) . Polyploid variety Podmoskovnaya is distinguished from others by largerinflorescences, which are 1.5 times bigger than those in thestandard (variety Azulena), an elongated peduncle and weakfoliage – important factors for mechanized harvesting. At thesame time, the effect of the same mutagen on seedlings ofDatura stramonium L. did not cause a change in the ploidylevel but led to a unique mutation: the absence of thorns incapsule fruits , which simplifies the harvestingof seeds and does not injure hands of plant collectors .Polyploids were also used at the initial stage of peppermintbreeding (Mentha×piperita L.) to obtain fertile plants andtheir generative offspring: when exposed to 0.025 % colchicinesolution, a fertile allopolyploid was obtained (2n = 144). Inmint breeding, a targeted selection of fertile forms with valuable traits  used for hybridization or toobtain generative offspring from free pollination was carriedout. In this case, vegetative propagation and clonal selection were used at the stage of assessment and reproductionof elite plants selected in hybrid offspring. In interspecifichybridization, other species of mint  were used to increase the winterhardiness of hybrids: this is how the Prilukskaya 6, Yantarnaya, Kubanskaya 6, Lekarstvennaya 1, Lekarstvennaya 4,Moskvichka and Medichka varieties, which are widespreadin industry, were created . Due to winter hardinessand high productivity in various natural climatic conditions,these varieties were zoned for all regions of the Russian Federation and recommended for complex use, including for theproduction of essential oil and menthol . TheAromatnaya mint variety was also isolated by clonal selection from a hybrid population; the essential oil of this varietyhas a mild taste and delicate aroma due to its low mentholcontent – an order of magnitude lower than that in the mostother varieties .In the breeding study carried out by Glazunova et al. (2020),chemical mutagenesis was also used to obtain polyploids of Polemonium coeruleum L. Onthe 2nd year of life polyploids were differed from diploid onesby sight : tetraploids were undersized compactplants with a large number of peduncles. Moderate growthof the aboveground part contributes to the accumulation ofactive substances in the aboveground and underground parts;the volume and mass of the rhizome also increased.The breeding based on the species created by mutagenesis was carried out for Calendula off icinalis L. .The most effective mutagens for C. off icinalis were 0.05 %diethyl sulfate and 0.08 % dimethyl sulfate. By selection formorphological characters, productivity and biologically activesubstances (in M1) and assessment for uniformity, distinctnessand stability (in M2–3), new marigold varieties Zolotoe more and Rajskij sad  were developed. Theyield of raw plant materials was 30–39 % higher compared tothe standard variety Kal’ta, the content of extractives and totalflavonoids content were increased by 13–21 and 29–43 %,respectively . The proportion of fractions suitable for mechanized sowing in the seed yield wasincreased: the fraction of hook-shaped seeds (up to 86 %), thefraction of ring-shared seeds  .In the breeding of medicinal and aromatic plants, traditionalmethods of individual and individual-family selection are stillpromising, since most of these species were introduced intoculture recently and are characterized by a high degree of polymorphism. Revealing the level of phenotypic variability andcorrelation of morphological and economically useful traitsmakes it possible to select the most productive morphotypesbased on visual traits, easily taken into account. Breeding forproductivity is carried out both for increasing the yield of rawplant materials and the content of BSIt should be noted that for the medicinal plants, the relationship between the yield of raw materials and the content of BSis a negative correlation value – this is because substancesuseful to humans are secondary metabolites used by plantsfor growth, development and adaptation to external factors.Therefore, sequential separate selection is mainly performed: initially, productive morphotypes are selected according toa complex of external characteristics ; after that, morphotypes with a highcontent of BS are selected. In accordance with the goals ofbreeding and the biological characteristics of particular plants,one or another method of selection and reproduction is used.For example, the breeding material of Digitalis lanata Ehrh.was created using forced self-pollination under the conditionsof mechanical isolation  and subsequent multiplesystematic individual selection in 1st–3rd self-pollinated offspring . Based on one of self‑pollinatedoffspring, the variety Ritm was created . Incomparison with the standard variety Spectr, the yield of theraw material (leaves) of the new variety was 25–30 % higherand the adaptability to mechanized harvesting was improveddue to the vertical deflection of the rosette leaves. The morphotypes were also differed in lanatoside content ranged from 0.22to 0.65 %. As a result of repeated self-pollination, the originalform of D. lanata was obtained. It was characterized by whiteflowers  with a decorativeness as good as thewidely known decorative varieties of D. purpurea L.; it evenexceeds these varieties in flowering duration (41–47 days).For breeding aromatic perennial herb oregano (Origanumvulgare L.), vegetative propagation by division of the rhizomeswas used to select clones. In the development of young plants,two reproductive phases were noted ; plantsformed seeds in the current growing season and could be usedin breeding and for preservation ex situ biological collection.Using the method of individual selection, we isolated thesamples of oregano clones with high yields of raw materialsand the content of essential oil . Theselection was carried out according to the height of the plantsand the color of the flowers . Tall forms includingvariety Raduga  were characterized by the maximum yield of raw plant materials. However, the maximumessential oil total harvest was possible from plants of mediumheight and low-growing plants due to the increased content ofessential oil being between 0.8 and 2.4 times higher, whichindicates their value for cultivation .With repeated successive self-pollination of O. vulgare,the original creeping form was obtained ,which does not form a typical rhizome with the aerial partapproximately 10–12 cm tall and consists of three hundred ormore thin succulent shoots. The content of essential oils corresponded to that in the initial form. Overall, this form couldbe recommended for food or decorative purposes.The phytochemical study of essential oil samples of oregano(O. vulgare) varieties revealed that sesquiterpenes predominated in all varieties and their maximum contentwas found in the variety Zima . Theidentification (or creation) of intraspecific chemotypes by thecomposition of the essential oil is relevant in connection withtheir specific pharmacological activity . The contentof monoterpenes in the variety Slavnitsa was 6 and 15 times higher than that invarieties Raduga and Zima, respectively. The highest contentof phenolic compounds  was found in the variety Raduga .Supportive selection. Traditional breeding methods usedin VILAR help in continuous breeding improvement with theinvolvement of already created varieties and primary seedproduction. However, multiple reproductions of the cultivatedvariety lead to accumulation of the low-value morphotypes resulting in decreased or lost stability of varietal indicators. Dueto the instability of meteorological indicators, the frequencyof drought, freezing, and soaking increases. Older varietiesmay not be adapted to such non-typical growing conditions.Breeding of introduced species. With regard to introducedspecies, the aim of breeding is to increase not only valuable agronomic indicators ,but also indicators showing adaptation to regional conditions. For example, only long-term acclimatization andmass selection in a cultivated population of Echinacea purpurea (L.) Moench made it possible to obtain high-qualityseeds of local reproduction and subsequently to create nationalvarieties, distribute and cultivate a new crop in Russia. Breeding of E. purpurea has been carried out in VILAR since 1996and resulted in the first national variety Tanyusha ;in specific regional conditions of the North Caucasian branchof VILAR, the variety Yuzhanka was created .By individual selection using vegetative reproduction  and self-pollination (within the family), we obtainedbreeding material with stable productivity and adaptabilitywhich became the basis for a new variety Severyanka developed for the Non-Chernozem zone  .Plant breeders of the VILAR and its branches have createdmore than 90 varieties of medicinal and aromatic plants usingselection methods, intraspecific and interspecific hybridization, experimental polyploidy and mutagenesis for more than70 years. A total 62 varieties, of which 17 varieties are protected by patents of the Russian Federation, were included inthe “State Register of Breeding Achievements” in 2020 andapproved for use on the territory of Russia. Collections ofseeds and vegetative plants have been created and maintainedto preserve varietal material.Long-term studies have shown that in the breeding ofmedicinal and aromatic plants, the most promising is thecomplex study and use of the natural intraspecific variabilityof the crops. In recent years, there have been large-scalechanges in molecular biology and information technologyrelated to the study of genomes, transcriptomes, proteomes,small RNAs, epigenetics, gene editing and synthetic biology. Modern methods of breeding can involve morphotypes,closely related species and generic complexes. Collections ofspecies promising for breeding and introduction are currentlybeing formed at VILAR for these purposes. Therefore, theduration of the breeding cycle – which traditionally required5–6 years for annual and biennial medicinal and aromaticcrops, and 7–10 years for perennial crops – can be reducedif the studies are carried out year-round in laboratory and ingreenhouses, making it less dependent on the duration of thegrowing season.The authors declare no conflict of interest.Glazunova A., Khazieva F., Samatadze T. Effect of Colchicine treatment on the cariology and morphology signs of Polemonium caeruleum L. In: Int. Sci.-Pract. Conference “Agriculture and FoodSecurity: Technology, Innovation, Markets, Human Resources”(FIES 2019). BIO Web Conf. 2020;17:00210. DOI 10.1051/bioconf/20201700210.Graham I.A., Besser K., Blumer S., Branigan C.A., Czechowski T.,Elias L., Guterman I., Harvey D., Isaac P.G., Khan A., Larson T.,Li Y., Pawson T., Penfield T., Rae A., Rathbone D., Reid S., Ross J.,Smallwood M., Bowles D. The genetic map of Artemisia annua L.identifies loci affecting yield of the antimalarial drug artemisinin. Science. 2010;327(5963):328-331. DOI 10.1126/science.1182612.Hazieva F.M., Korotkikh I.N., Osipov V.I. Composition of essential oilsof Origanum vulgare L. varieties from VILAR collection. VoprosyBiologicheskoi, Meditsinskoi i Farmatsevticheskoi Khimii = Problems of Biological, Medical and Pharmaceutical Chemistry. 2019;22(7):38-49. DOI 10.29296/25877313-2019-07-06. (in Russian)Karpuk V.V. Pharmacognosy. Minsk, 2011. (in Russian)Khazieva F.M., Bykova O.A., Anikina A.Yu., Korobov Yu.P., Svistunova N.Yu., Samatadze T.A. Comparison of varieties and populations Matricaria chamomilla L. on biochemical and kariologicalindicators. Voprosy Biologicheskoy, Meditsinskoy i Farmatsevticheskoy Khimii = Problems of Biological, Medical and PharmaceuticalChemistry. 2017;20(9):46-51. (in Russian)Khazieva F.M., Svistunova N.Yu., Samatadze T.E. Effect of chemical mutagens on morphological and cytogenetic changes Calendula officinalis L. Voprosy Biologicheskoy, Meditsinskoy i Farmatsevticheskoy Khimii = Problems of Biological, Medical andPharmaceutical Chemistry. 2016;8(19):37-43. (in Russian)Konon N.T., Khazieva F.M., Stanishevskaya I.E., Gryaznov M.Yu.,Totskaya S.A., Korotkikh I.N. The results of 80‑year-old breedingwork with medicinal and aromatic cultures. Voprosy Biologicheskoy,Meditsinskoy i Farmatsevticheskoy Khimii = Problems of Biological, Medical and Pharmaceutical Chemistry. 2012;1:17-22. (in Russian)Korotkikh I.N., Babaeva E.Yu., Burova A.E. Breeding results ofEchinacea purpurea (L.) Moench in Moscow province. Trudy poPrikladnoy Botanike, Genetike i Selektsii = Proceedings on Applied Botany, Genetics and Breeding. 2018;179(4):111-118. DOI10.30901/2227-8834-2018-4-111-118. (in Russian)Korotkikh I.N., Khazieva F.M., Sidelnikov N.I. Breeding of Digitalis lanata Ehrh. Voprosy Biologicheskoy, Meditsinskoy i Farmatsevticheskoy Khimii = Problems of Biological, Medical and Pharmaceutical Chemistry. 2013;8:8-10. (in Russian)Korotkikh I.N., Khazieva F.M., Totskaya S.A. Morphotypical varietiesof Origanum vulgare L. Kartofel i Ovoshchi = Potato and Vegetables. 2015;4:24-25. (in Russian)Máthé Á. (Ed.). Medicinal and Aromatic Plants of the World: Scientific,Production, Commercial and Utilization Aspects. Springer, 2015.DOI 10.1007/978-94-017-9810-5.Morozov A.I. Mint breeding for different specific purposes. VestnikRossiyskoy Sel’skokhozyaistvennoy Nauki = Vestnik of the RussianAgricultural Science. 2018;5:52-55. DOI 10.30850/vrsn/2018/5/52-55. (in Russian)Morozov A.I., Dmitrachkova N.S., Bykova O.A. New cultivar ofMentha piperita for pharmaceutical leaves production. VoprosyBiologicheskoy, Meditsinskoy i Farmatsevticheskoy Khimii = Problems of Biological, Medical and Pharmaceutical Chemistry. 2012;7:21-23. (in Russian)Muravenko O.V., Amosova A.V., Samatadze T.E., Popov K.V., Poletaev A.I., Zelenin A.V. 9-aminoacridin: an efficient reagent to improve human and plant chromosome banding patterns and to standardize chromosome image analysis. Cytometry. 2003;51(1):52-57.DOI 10.1002/cyto.a.10002.Nakatsuka T., Sato K., Takahashi H., Yamamura S., Nishihara M. Cloning and characterization of the UDP-glucose: anthocyanin 5-O-glucosyltransferase gene from blue-flowered gentian. J. Exp. Bot. 2008;59:1241-1252. DOI 10.1093/jxb/ern031.Run Q., Jun-Hui Z., Jian Y., Lu-Qi H., Yuan Y. Study progress onmolecular marker-assisted breeding of Chinese medicinal materials. Zhongguo Zhong Yao Za Zhi. 2020;45(20):4812-4818. DOI10.19540/j.cnki.cjcmm.20200710.602.Samatadze T.E., Zoshchuk S.A., Amosova A.V., Khomik A.S., Svistunova N.Yu., Suslina S.N., Hazieva F.M., Yurkevich O.Yu., Muravenko O.V. Molecular cytogenetic characterization, leaf anatomyand ultrastructure of the medicinal plant Potentilla alba L. Genet.Resour. Crop Evol. 2018;65(6):1637-1647. DOI 10.1007/s10722-018-0640-7.Shen Q., Zhang D., Sun W., Zhang Y.J., Shang Z.W., Chen S.L. Medicinal plant DNA marker assisted breeding (II) the assistant identification of SNPs assisted identification and breeding research of highyield Perilla frutescens new variety. Zhongguo Zhong Yao Za Zhi.2017;42(9):1668-1672. DOI 10.19540/j.cnki.cjcmm.20170418.001.Shimada N., Nakatsuka T., Nakano Y., Kakizaki Y., Abe Y., Hikage T.,Nishihara M. Identification of gentian cultivars using SCAR markers based on intron-length polymorphisms of flavonoid biosynthetic genes. Sci. Hortic. 2009;119(3):292-296. DOI 10.1016/j.scienta.2008.08.001.Shirokova I. The market of herbal medicines: trends, challenges andforecasts. Remedium. 2013;4(194):26-33. (in Russian)Townsend T., Segura V., Chigeza G., Penfield T., Rae A., Harvey D.,Bowles D., Graham I.A. The use of combining ability analysis toidentify elite parents for Artemisia annua F1 hybrid production.PLoS One. 2013;8(4):e61989. DOI 10.1371/journal.pone.0061989.Ulumbekova G.E., Kalashnikova A.V. Analysis of the pharmaceuticalmarket in the Russian Federation. Vestnik VSHOUZ = Bulletin ofthe Higher School of Health Care Management. 2018;4(14):53-75.(in Russian)Wagner C., Friedt W., Marquard R.A., Ordon F. Molecular analyseson the genetic diversity and inheritance of (–)-α-bisabolol andchamazulene content in tetraploid chamomile (Chamomilla recutita (L.) Rausch.). Plant Sci. 2005;169(5):917-927. DOI 10.1016/j.plantsci.2005.06.010.Wang W., Xu J., Fang H., Li Z., Li M. Advances and challenges in medicinal plant breeding. Plant Sci. 2020;298:110573. DOI 10.1016/j.plantsci.2020.110573.


Pseudophilautus. Except for one small clade of four species in Peninsular India, these cool-wet adapted frogs, numbering some 59 extant species, are distributed mainly across the montane and lowland rain forests of the island. With species described primarily by morphological means, the diversification has never yet been subjected to a molecular species delimitation analysis, a procedure now routinely applied in taxonomy. Here we test the species boundaries of Pseudophilautus in the context of the phylogenetic species concept (PSC). We use all the putative species for which credible molecular data are available  to build a well resolved phylogeny, which is subjected to species delimitation analyses. The ABGD, bPTP, mPTP and bGMYC species delimitation methods applied to the 16S rRNA frog barcoding gene , 12S rRNA and Rag-1 nDNA grouped P. procax and P. abundus; P. hallidayi and P. fergusonianus; P. reticulatus and P. pappilosus; P. pleurotaenia and P. hoipolloi; P. hoffmani and P. asankai; P. silvaticus and P. limbus; P. dilmah and P. hankeni; P. fulvus and P. silus.. Surprisingly, all analyses recovered 14 unidentified potential new species as well. The geophylogeny affirms a distribution across the island’s aseasonal ‘wet zone’ and its three principal hill ranges, suggestive of allopatric speciation playing a dominant role, especially between mountain masses. Among the species that are merged by the delimitation analyses, a pattern leading towards a model of parapatric speciation emerges–ongoing speciation in the presence of gene flow. This delimitation analysis reinforces the species hypotheses, paving the way to a reasonable understanding of Sri Lankan Pseudophilautus, enabling both deeper analyses and conservation efforts of this remarkable diversification. http://zoobank.org/urn:lsid:zoobank.org:pub:DA869B6B-870A-4ED3-BF5D-5AA3F69DDD27.Sri Lanka is an amphibian hotspot of global significance. Its anuran fauna is dominated by the shrub frogs of the genus  As a part of the Sri Lanka-Western Ghats biodiversity hotspot, Sri Lanka features an exceptional species richness and endemism in amphibians , 2. At pThe family Rhacophoridae, spread across tropical to sub-tropical Asia and Africa, is a diverse group of frogs containing some 432 species, 6% of the world’s anuran fauna . Their tPseudophilautus, characterized by terrestrial direct development  suggests a possible pattern of parapatric speciation through restricted gene flow. Here, P. cf. schmarda and P. dilmah are distributed at similar elevations in different parts of the central mountains , whereas P. fergusonianus had a wide range spanning Monaragala, Ginigathhena, Laggala, Pallegama, Deniyaya, Pitadeniya, Gannoruwa to Peradeniya, including both the lowlands as well as mid-elevations of the central and Knuckles hills.Further, the delimitation failure of Pseudophilautus cf. silus 2 in clade C seem to be species found at the edge of the distribution range of P. silus in the Central Hills; this lends confidence to our treating these populations as belonging to a single species. The recovery of P. fulvus and P. silus in clade C, as a single OTU, can be understood in the context of limited gene flow occurring among the populations of the two adjacent mountain regions, Knuckles and Central Hills, respectively, which are separated by the deep valley carved by the Mahaweli River.Pseudophilautus.Our delimitation analysis relies primarily on the 16s rRNA gene fragment, which is the most widely used barcoding marker in frogs . HoweverP. hoffmani and P. asankai (p-dist = 1.04%) for the 16S rRNA amphibian barcoding gene. This is perhaps close to the 16S threshold for pairwise species differentiation. Therefore, we propose an uncorrected 16S pairwise distance of 1.5% as a barcoding benchmark for species boundaries in new-species descriptions for Pseudophilautus, and also perhaps for rhacophorids in general.Further, it is useful to have a standardized molecular benchmark for delimiting species for this group, in order to objectively validate species. Among the synonymized species the highest uncorrected p-distance was recovered among Ideally, however, we suggest testing species boundaries using combinations of delimitation methods and criteria in the context of the PSC. We recommend that future new-species diagnoses and descriptions be based on multiple criteria, including both molecular and morphological approaches, in the context of the general lineage concept . ClearlyPseudophilautus. For almost all species assessed using the IUCN Red List Criteria, it is criterion B (restricted distribution of a species), where Extent of Occurrence  and Area of Occupancy  plays a major role in the assignment of threat categories, which lead to prioritization for conservation. Hence, a clearer understanding of operational taxonomic units provides a framework for targeted conservation action.Molecular species delimitation is a useful tool to refine the assessment of species hypotheses, which here we test in the case of Sri Lankan S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.


Geocenamus brevidens and Quinisulcius capitatus, were recovered from the potato growing regions of southern Alberta, described and characterized based on integrative taxonomy. Morphometrics, distribution, and host associations of both species are discussed. The Canadian populations of both species displayed minor variations in morphometrical characteristics  from the original descriptions. The populations of G. brevidens and Q. capitatus species examined in this study are proposed as standard and reference populations for each respective species until topotype specimens become available and molecularly characterized. Phylogenetic analyses, based on partial 18S, 28S, and ITS sequences, placed both species with related stunt nematode species. The present study updates the taxonomic records of G. brevidens and Q. capitatus from a new location, southern Alberta, Canada, and will aid in the decision whether these stunt nematodes should be included in nematode management programs.Two stunt nematode species,  Pratylenchus penetrans is a known contributor to PED, along with the fungal wilt pathogen Verticillium dahliae and to a lesser extent V. albo-atrum. The possible role of other plant-parasitic nematodes in PED development is unknown, although several studies noted the co-occurrence of stunt nematodes and root-lesion nematodes . The presence of the insert-containing plasmids in transformed E. coli cells was confirmed by PCR. Plasmid DNA was isolated and purified according to the manufacturer’s instructions (Omega Biotek). The DNA inserts were sequenced at Genewiz, Inc  using primers matching the flanking vector sequence.DNA samples were prepared from nematodes according to http://molevol.cmima.csic.es/castresana/Gblocks_server.html) using options for a less stringent selection .Sequenced genetic markers from the nematodes examined in the present study , along with several stunt nematode sequences obtained from the GenBank database, were used in the phylogenetic reconstruction. Outgroup taxa for each dataset were selected based on previously published studies . Multipl6 generations. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for each analysis. After discarding burn-in samples of 10% and evaluating convergence, the remaining samples were retained for more in-depth analyses. The topologies were used to generate a 50% majority-rule consensus tree. Posterior probabilities (PP) are given on appropriate clades. Trees from all analyses were edited using FigTree software V.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).Phylogenetic analyses of the sequence datasets were conducted based on Bayesian inference (BI) using MRBAYES 3.2.7a . The besGeocenamus brevidens  1.5 to 3.0 µm posterior to basal knobs. Median bulb spherical to oval, well-developed, central valve plates bean-shaped slightly anteriorly. Isthmus slender encircled with nerve ring. Deirids were present (observed in few specimens). Excretory pore anterior to the basal esophageal bulb. Hemizonid 2 to 3 body annuli long situated slightly anterior to the excretory pore. Cardia rounded, intestine densely globular. Ovaries outstretched, vulva with small epiptygma, which mostly appear as protruding lips, vagina inclined anteriorly covering half of the corresponding body diameter. Spermatheca rounded, scarcely filled with sperm. Tail subcylindrical, gradually tapering to a smooth broadly rounded or truncated terminus. Hyaline region of tail prominent 3.2–5.5 µm long. Phasmids near or slightly posterior to mid-tail.Not found.Geocenamus brevidens  of Vulcan county, whereas the second one was discovered in the rhizosphere of grass growing on the headland (uncultivated field margin)  of Taber Rural Municipality, Alberta, Canada.Two Quinisulcius capitatus  Siddiqi, 1971 .Body open C-shaped, appeared concave at vulval level. Cuticle annulated, lateral field with five incisures. Cephalic region continuous with slight depression at the junction of lip and body, broadly rounded with a few indistinct lip annuli. Stylet robust, 15 to 20 µm long with rounded basal knobs. DGO 2.5 to 4.0 µm posterior to basal knobs. Median bulb rounded, well developed with conspicuous central valve plates. Isthmus slender, encircled with nerve ring. Deirids not seen. Excretory pore at the middle of basal esophageal bulb. Hemizonid inconspicuous, 2 to 3 body annuli long, situated 3 to 4 annuli anterior to excretory pore. Cardia rounded, intestine densely globular. Ovaries outstretched, vulva with protruding lips, vagina straight covering more than half of the corresponding body diameter. Spermatheca weakly developed, rounded. Tail conoid, distinctly annulated, gradually tapering to a heart or V-shaped terminus. Phasmids near or slightly posterior to mid-tail.Not found.Tylenchorhynchus capitatus Quinisulcius capitatus  was originally described in the rhizosphere of pear from the USA by Allen in 1955. Afterward, this species has been found in various geographic and agricultural locations  was only described in Quinisulcius, males were not described for 10 species ; Taber Rural Municipality, Alberta, Canada.G. brevidens; MW023248, MW023249 for Q. capitatus), 28S , and the ITS region of the rRNA  of both species were obtained.The sequences of partial 18S . However, it is noted that another population of G. brevidens (AY284597) from the Netherlands distantly arranged from the G. brevidens clade. Additionally, sequences of Nagelus obscurus , from the Netherlands and Belgium do not cluster together. Therefore, these data suggest that a misidentification is probable and requires a detailed re-evaluation based on integrative taxonomy for ascertaining their species status. In subclade (II), the Q. capitatus grouped with Tylenchorhynchus microphasmis (AY593903), T. maximus, and two unidentified Tylenchorhynchus spp. Since the Quinisulcius sequences deposited in the GenBank were the first sequences deposited for this genus, we anticipate that inclusion of more Quinisulcius sequences will rearrange the position of Q. capitatus. We noted that the sequences of T. leviterminalis  arranged distantly from the T. leviterminalis clade. T. leviterminalis may consist of a species complex; therefore, a detailed re-evaluation based on integrative taxonomy is required to determine the exact status of these populations.Phylogenetic relationships among the isolates were determined separately for each dataset using Bayesian inference (BI) . The 18SQ. capitatus within the species of Tylenchorhynchus in Clade (I). However, due to a lack of Quinisulcius sequences, phylogenetically close species cannot be determined. Clade (II) indicates that the Canadian population of G. brevidens grouped with G. brevidens populations from China , Iran . It is noted that in this tree, Q. capitatus does not cluster within Tylenchorhynchus species but independently appeared as a different, well-supported clade (III) (PP = 1.00).The 28S tree presents two well-supported (PP = 1.00) main clades  Brzeski, 1991 and G. longus  Tarjan, 1973 G. brevidens being the third species discovered in this province. Geocenamus brevidens is a native American species described by Currently, the genus  species , with ei species . Among taminatus  Brzeski,G. brevidens were previously examined by several researchers, who demonstrated that this species requires relatively cooler soils and low temperature for achieving an optimal reproduction rate. Moreover, the damage potential of G. brevidens increased in clay sand and sandy loam soils , and molecular characters. In our 28S phylogenetic trees, G. brevidens sequences formed two separated subclades. Our data of G. brevidens are coincident with the original descriptions as well as with other populations that have been described using integrative taxonomy (Geocenamus rather than to G. brevidens. We agree with G. brevidens that may clarify this situation. Moreover, in our phylogenetic trees, Q. capitatus grouped with Tylenchorhynchus species; the closely related species cannot be determined due to the lack of Quinisulcius sequences in GenBank. Based on integrative taxonomical characterization, we propose our G. brevidens and Q. capitatus as standard and reference populations for each species until topotype specimens become available and molecularly characterized.In this work, populations of taxonomy . ConsequG. brevidens and Q. capitatus from Alberta, Canada. Moreover, our light micrographs and sequence-based information will enable prompt identification of these species. Last, it will be important for future research to conclusively determine whether these stunt nematodes should be incorporated into nematode management programs.The current knowledge of the occurrence and distribution of non-target nematode species in Alberta is very scarce. Here, we provide the descriptions of two stunt nematodes based on an integrative taxonomical approach. Data on geographical distribution and host plant associations of these species are also provided and discussed. The present study will help to update the taxonomic records of


Fusobacterium nucleatum (F. nucleatum) is recognised as a key microorganism in subgingival dental plaque, and is linked to periodontitis as well as colorectal cancer and systemic diseases. Five subspecies of F. nucleatum have been identified: animalis, fusiforme, nucleatum, polymorphum, and vincentii. Differential integration of subspecies into multispecies biofilm models has been reported, however, biofilm forming ability of individual F. nucleatum subspecies is largely unknown. The aim of this study was to determine the single-subspecies biofilm forming abilities of F. nucleatum ATCC type strains. Static single subspecies F. nucleatum biofilms were grown anaerobically for 3 days on untreated or surface-modified  plastic and glass coverslips. Biofilm mass was quantified using crystal violet (CV) staining. Biofilm architecture and thickness were analysed by scanning electron microscopy and confocal laser scanning microscopy. Bioinformatic analysis was performed to identify orthologues of known adhesion proteins in F. nucleatum subspecies. Surface type and treatment significantly influenced single-subspecies biofilm formation. Biofilm formation was overall highest on poly-L-lysine coated surfaces and sandblasted glass surfaces. Biofilm thickness and stability, as well as architecture, varied amongst the subspecies. Interestingly, F. nucleatum ssp. polymorphum did not form a detectable, continuous layer of biofilm on any of the tested substrates. Consistent with limited biofilm forming ability in vitro, F. nucleatum ssp. polymorphum showed the least conservation of the adhesion proteins CmpA and Fap2 in silico. Here, we show that biofilm formation by F. nucleatum in vitro is subspecies- and substrate-specific. Additionally, F. nucleatum ssp. polymorphum does not appear to form stable single-subspecies continuous layers of biofilm in vitro. Understanding the differences in F. nucleatum single-subspecies biofilm formation may shed light on multi-species biofilm formation mechanisms and may reveal new virulence factors as novel therapeutic targets for prevention and treatment of F. nucleatum-mediated infections and diseases.Development of dysbiosis in complex multispecies bacterial biofilms forming on teeth, known as dental plaque, is one of the factors causing periodontitis.  The oral environment consists of a multitude of bacterial species living on both soft and hard tissues in complex multispecies communities known as biofilms . Dental Streptococcus, Neisseria, Prevotella, Haemophilus, and Rothia genera, among other less abundant genera, creating a distinct signature of a healthy, symbiotic state [Health-related supra- and sub-gingival bacterial communities are often composed of Gramme-positive and Gram-negative early colonisers including ic state –7. Howevic state –12.Fusobacterium nucleatum, an anaerobic commensal member of dental biofilms [F. nucleatum have been identified to date: animalis, fusiforme, nucleatum, polymorphum, and vincentii [fusiforme and vincentii as one subspecies vincentii [biofilms , is presbiofilms  but is ebiofilms . It has biofilms . It is abiofilms , 16, as biofilms . Five suincentii . Some auincentii .nucleatum (ATCC 25586), polymorphum (ATCC 10953) and vincentii (ATCC 49256) were shown to prevent superoxide generation induced by N-formylmethionyl-leucyl-phenylalanine in neutrophil-like HL-60 cells. Additionally, subspecies polymorphum increased the rate of apoptosis in HL-60 cells when compared to the other subspecies [F. nucleatum subspecies were also studied in in vitro multi-species biofilm models. Differences in the incorporation of subspecies into the biofilm were described, where bacterial numbers of F. nucleatum ssp. vincentii and ssp. polymorphum were significantly lower in comparison with ssp. nucleatum [Despite the close genomic relatedness of all subspecies, differences in pathogenicity have been recognised, such as host immune response modulation: subspecies bspecies . F. nuclucleatum .F. nucleatum adhesion proteins have been identified, which mediate coaggregation and biofilm formation with various oral microbes as well as salivary proteins: outer membrane protein RadD with a putative accessory protein Aid1 [F. nucleatum in silico, such as YadA-like adhesin originally found in Yersinia species [ein Aid1 , autotraein Aid1 , porin Fein Aid1 , outer mein Aid1 , and adhein Aid1 . Homolog species .F. nucleatum subspecies is poorly understood. Moreover, few authors have used type strains as reference strains to the clinical isolates in studies of F. nucleatum, and many have not reported the subspecies used, making a comparison of these results difficult. While it is clinically relevant to study F. nucleatum in multi-species biofilms, information obtained from single-subspecies F. nucleatum biofilms without the presence of additional binding partners is necessary for better understanding of biofilm-related immunogenic and pathogenic properties of F. nucleatum subspecies and virulence factor expression.Surprisingly, despite its structural importance in dental plaque and its role in pathogenic biofilms in colorectal cancer and other diseases, biofilm formation by single F. nucleatum subspecies along with analysis of differences in adhesins known to mediate biofilm formation and aggregation. A further aim was to utilise a simplified in vitro single-subspecies biofilm model which could be repeated by other researchers, using widely available substrates and substrate coatings, to better understand biological properties of each subspecies. We hypothesised that there is no difference in biofilm formation and architecture among F. nucleatum subspecies.Therefore, this study aimed to investigate single-subspecies biofilm formation abilities by F. nucleatum were obtained from the Periodontal Research Group culture collection  and were originally purchased from the American Tissue Culture Collection (ATCC): F. nucleatum ssp. animalis ATCC 51191 (FNA), F. nucleatum ssp. fusiforme ATCC 51190 (FNF), F. nucleatum ssp. nucleatum ATCC 25586 (FNN25), F. nucleatum ssp. polymorphum ATCC 10593 (FNP), and F. nucleatum ssp. vincentii ATCC 49256 (FNV). Additionally, a genetically tractable strain F. nucleatum ssp. nucleatum ATCC 23726 (FNN23) was kindly provided by Dr. Daniel Slade . The identity of all subspecies was confirmed by 16S rRNA sequencing.The following type strains of 2, 10% CO2, and 10% H2; Don Whitley DG250 Anaerobic Workstation, Don Whitley Scientific, Bingley, UK) on Schaedler anaerobe agar plates . Liquid cultures were grown in Schaedler anaerobe broth .All subspecies were grown at 37°C in an anaerobic chamber  and plastic  coverslips placed in a 24-well-plate. Artificial saliva (AS) was used as a coating agent to mimic human saliva and formation of salivary pellicle to promote bacterial adhesion [In order to evaluate biofilm formation of adhesion . AS was adhesion , gelatinadhesion , and poladhesion .in vivo conditions. Fibronectin diluted in PBS (5 μg/cm2) was applied and incubated for 45 min at room temperature. Gelatin solution (0.1% in PBS) was incubated for 20 min at 37°C. Poly-L-lysine coating was performed for 10 min at room temperature. These surface coatings were left to dry in a laminar flow hood as per the manufacturers' instructions without washing. Additionally, a set of glass coverslips was sandblasted to create a roughened surface. Sandblasting was completed using a Basic quattro sandblasting unit , in which each coverslip was treated for 5 s with aluminium oxide .Substrate coating was performed as follows: AS was added to coverslips and incubated at 37°C for 1 h . Next, AF. nucleatum cultures were washed once with PBS and the optical density of each culture was adjusted to OD600 = 1 in SAB, corresponding to 1.62 × 109 CFU/ml for each subspecies. For each single-subspecies biofilm, 400 μl of bacterial suspension was added to each well containing studied substrates and biofilms were incubated static for 72 h, under anaerobic conditions. SAB was replaced after 24 and 48 h and biofilms were monitored for contamination daily.To initiate biofilm growth, planktonic ®, Tecan; software SparkControl, v. 2.3, Tecan). To account for differences between glass and plastic coverslip surface area, absorbance readings per cm2 were calculated using the formula below. The surface area of a glass coverslip was 113.04 mm2, the area of a Thermanox coverslip was 132.67 mm2.Biofilm biomass was quantified by crystal violet (CV) staining. After 72 h of incubation, biofilms were carefully washed once with 100 μl PBS and air-dried for 2 h at 37°C, then stained with a CV solution  at room temperature for 30 min. After staining, biofilms were gently washed with 200 μl PBS and air dried at 37°C for 2 h. Ethanol  was used to destain the biofilms for 1 h on a plate shaker. Ethanol solution from each well was diluted in Milli-Q water 1:10 in a 96-well plate and the absorbance was measured at 600 nm  according to the manufacturer's instructions. Briefly, 3 μl of SYTO™ 9 were added to 1 ml of sterile PBS and biofilms were stained with 200 μl of the diluted stain for 20 min at room temperature in the dark.Biofilms for CLSM analysis were grown in 24-well black plates with clear base  either with no surface treatment, or coated with fibronectin, gelatin or poly-L-lysine as stated above. In order to avoid excessive detachment, biofilms were first fixed with 4% paraformaldehyde for 10 min, then washed in PBS and stained using green fluorescent, cell permeant nucleic acid stain SYTO™ 9 , with a 40X oil immersion objective at 488/500 nm. Maximum thickness of the biofilms was estimated by obtaining z-stack horizontal images at 1.3 μm intervals. Biofilms were grown in triplicate in one experiment and images were acquired in the centre of each well using Zeiss Zen 2011 software.In order to examine single-subspecies biofilm architecture using SEM, biofilms were grown on poly-L-lysine coated plastic (Thermanox™) coverslips in 24-well plates. Biofilms were fixed using 2.5% glutaraldehyde  in 0.1 M sodium cacodylate buffer  for 10 min at room temperature. Following fixation, biofilms were dehydrated with increasing ethanol concentrations (20–100%) and incubated for 10 min at each step.Finally, drying agent hexamethyldisilizane  was applied and left to evaporate overnight. Coverslips with biofilms were mounted onto aluminium specimen stubs , sputter coated with two layers of gold and visualised using a scanning electron microscope (Zeiss EVO MA10). A visible layer of biofilm was chosen after visual evaluation of each specimen under low magnification (50X) and recorded at 1000X and 5000X magnification.F. nucleatum ssp. animalis ATCC 51191 (GCA_000220825), F. nucleatum ssp. nucleatum ATCC 23726 (GCA_000178895), F. nucleatum ssp. nucleatum ATCC 25586 (GCA_000007325), F. nucleatum ssp. polymorphum ATCC 10953 (GCA_000153625), F. nucleatum ssp. vincentii ATCC 49256 (GCA_000182945) were retrieved from EnsemblBacteria (Release 52) [F. nucleatum ssp. fusiforme data were not available on this portal, possibly due to their close genetic relatedness with F. nucleatum ssp. vincentii.Bacterial genomes of GCA_0001845 were rF. nucleatum, ssp. nucleatum ATCC 25586 proteins as queries, with a cutoff e-value of 1e-10. Protein domains were annotated using InterPro [Orthologues were identified using blastp (v. 2.12.0)  with defInterPro  with a cInterPro  with theInterPro , JTT subpost-hoc test. Results were considered statistically significant if p < 0.05. Graphs were created using GraphPad Prism, version 9.3.0.Statistical analysis was performed using GraphPad Prism . Biomass quantification data were analysed using Shapiro-Wilk normality test and shown to conform to normal distribution. Samples were compared within subspecies and either untreated Thermanox™ surface or untreated glass surface were considered as controls. Values were analysed by one-way ANOVA, followed by Dunnett's All subspecies formed biofilms detectable with CV, with the exception of FNP, which did not form a continuous biofilm on any of the tested surfaces. When examined visually during the incubation period, this subspecies was found to remain planktonic in the biofilm supernatant.p = 0.04 and p = 0.02, respectively), the amount of biofilm quantified was consistently low . FNF adhered significantly better to uncoated Thermanox coverslips when compared to uncoated glass (p = 0.002), however the other subspecies showed no difference in biofilm formation between glass and plastic surfaces. FNA and FNF were the best biofilm formers on plastic surfaces.Differences in biofilm mass were seen amongst the subspecies and on the different surfaces : GeneralBiofilm thickness was also examined using CLSM and mean values estimated from three-dimensional biofilm images . SimilarLarge standard deviations were associated with some subspecies-surface combinations  and were seen in both CV and CLSM experiments. These reflect a high degree of visually observed biofilm detachment during handling, indicating low adhesive strength of these biofilms.Thermanox coverslips coated with poly-L-lysine were chosen as the surface for biofilm analysis by SEM based on CLSM results, which showed higher biofilm thickness on this type of surface. Overall, high magnification 1000X,  revealedF. nucleatum subspecies with regard to thickness and formation of aggregates and water channels. Cell-to-cell cohesion did not seem to differ among subspecies.Cell-to-cell cohesion was observed in more detail under 5000X magnification . In all F. nucleatum ATCC subspecies genomes publicly available on EnsemblBacteria (Release 52) [To investigate possible differences in adhesion proteins amongst the subspecies, which might explain the observed lack of biofilm formation in FNP, analysis of the bacterial genomes was carried out. Sequence alignments of Aid1, CmpA, FadA, Fap2, FomA, RadD, and YadA from FNN25 identified orthologous proteins in the ease 52) . As expeOrthologues of all the considered proteins were found in FNP. Interestingly, CmpA and Fap2 orthologues were identified in all subspecies; however, FNP orthologues were less conserved with the lowest identity percentage among the four analysed subspecies . AnnotatF. nucleatum as a commensal microorganism, opportunistic pathogen, and emerging oncobacterium in the oral cavity and at extraoral sites has received considerable attention in recent years [nt years . Individnt years –36; whilnt years , 34; intnt years .in vitro studies that differences in pathogenicity among F. nucleatum subspecies exist, such as differences in coaggregation with other oral bacteria and biofilm formation in multispecies biofilm models [F. nucleatum ATCC type strains has not been addressed in the literature to date, and data presented in this study showed for the first time that not all F. nucleatum subspecies have the ability to form stable single-subspecies biofilms. In our study, FNP did not form biofilms but only small bacterial aggregates. In support of this finding, similar results were obtained by Karched et al., who showed poor single-subspecies biofilm formation by FNP, whilst a strong autoaggregating ability was shown in this subspecies [Differential involvement of subspecies in health and disease might suggest differences in their virulence properties. It is increasingly evident from m models . To the bspecies .F. nucleatum subspecies has not been addressed in the literature to date. The present study showed considerable biomass variation likely caused by unstable biofilm formation. Under physiological conditions in the oral cavity, F. nucleatum grows as a component of complex multi-species biofilms in the presence of multiple binding partners, especially early colonisers [polymorphum formed stable biofilms with the early coloniser Actinomyces naeslundii, however, it did not form single-subspecies biofilms in saliva [polymorphum [Biofilm stability of lonisers . Periasan saliva . This fuymorphum , 41. Invin vivo conditions and improve adhesion of bacteria to substrates [F. nucleatum single-species biofilm formation on AS coated surfaces has not been reported in the literature to date, however Tavares et al. showed stable single-species biofilm formation by FNF on plastic surfaces coated with human saliva [F. nucleatum single-species can help to understand whether and how specific salivary properties may influence the selection and growth of certain subspecies in the oral cavity.In our study, formation of stable biofilms was also substrate-specific. First, surfaces were coated with AS, which is used to mimic bstrates . The advbstrates . AS signn saliva . Using hF. nucleatum was shown to adhere to fibronectin [polymorphum ATCC 10953 was found to adhere to fibronectin-coated gingival epithelial cells and fibronectin-coated coverslips as single cells, whilst biofilm formation was not investigated. This supports our finding that single FNP cells adhere to fibronectin coated surfaces, however, studies analysing FNP biofilm formation on fibronectin coated surfaces are missing.Secondly, surfaces coated with fibronectin, a glycoprotein present in physiological fluids such as plasma and saliva , were coronectin  and moreEscherichia coli (E. coli) was improved by gelatin, but was dependent on specific buffering conditions and bacterial strains [F. nucleatum biofilms. Additionally, surfaces coated with poly-L-lysine, a cationic coating agent promoting bacterial adhesion by electrostatic interactions as mostly shown in E. coli studies [F. nucleatum.Thirdly, gelatin, a derivative of collagen found in the extracellular matrix of tissues, is a commonly used polymer for coating tissue culture vessels . Benn et strains . In our  studies , 47, ledIn vivo, periodontal biofilms predominantly adhere to enamel, dentine [Apart from surface coatings, surface roughness was also found to promote biofilm formation on multiple types of surfaces by providing a larger surface area for adhesion of bacteria and also protecting adherent bacteria from detachment by shear forces . In this dentine  and ceme dentine . In futu dentine  or atomi dentine , 54 coulStaphylococcus aureus and Pseudomonas aeruginosa, showing strain-specific differences such as large mushroom-like structures, aggregates and differences in thickness [Assessment of the architecture of single-subspecies biofilms showed observable differences among subspecies. Similar observations were made in a previous study investigating hickness . PossiblOf note, biofilm structure also differed between microscopy techniques in our study. For example, FNA biofilms analysed with CLSM appeared as mono-layered structures, while FNA biofilms imaged with SEM were shown to be multi-layered. This may have been due to the fixation procedures affecting the degree of biofilm detachment. Fixation preserves cell morphology and the native structure of biofilms , howeverF. nucleatum biofilms has been observed in a number of studies, suggesting that F. nucleatum forms biofilms according to the definition by Costerton et al. [F. nucleatum ssp. nucleatum (ATCC 25586) [F. nucleatum ssp. fusiforme cells in single-species biofilms [F. nucleatum subspecies has not been studied in a single-species biofilm context.In general, biofilm sample preparation for imaging is often a destructive process, involving multiple washing steps, which may affect biofilm structure and introduce artefacts . Presumen et al. . Ali MohC 25586) . SubsequC 25586) . Tavaresbiofilms . HoweverF. nucleatum subspecies. FomA binds to human protein statherin found in saliva [Yersinia species and was identified in F. nucleatum [In order to address the limited ability of FNP to form stable biofilms, bioinformatic analysis of known adhesion proteins was performed and showed that a number of orthologues found in this subspecies have very low identity when compared to FNN25, a biofilm forming strain often used as a control in biofilm models . The anan saliva . The Yaducleatum , adheresucleatum .in silico analysis found the autotransporter domain in Fap2 in all subspecies. Hence, one may speculate that these adhesion proteins and perhaps the autotransporter domain are important for cell-cell adhesion in single-subspecies biofilms. CmpA and Fap2 had a very low identity in FNP, and this subspecies was the most distant in the phylogenetic tree. This might indicate an inability to form stable single-subspecies biofilms. It is, however, important to note that these bioinformatic results have to be considered with caution and no final conclusions can be drawn due to the small scale of the analysis. Only a selected set of adhesion proteins reported in the literature was analysed and it is likely that other putative adhesion proteins involved in biofilm formation remain to be discovered.Based on our analysis, all proteins were found in the FNP genome, however four of these  were not well-conserved. Only two proteins, CmpA and Fap2 were detected in all subspecies and were found to share a common autotransporter domain in FNN25. In addition, our With the method employed here, no autotransporter domains for CmpA were detected in subspecies FNA and FNN23. Additionally, despite a high identity of the YadA-like protein in FNP, a general lack of enhanced adhesion of FNP to fibronectin and gelatin in our study might suggest that YadA in FNP does not play a key role in adhesion to these proteins. Mutagenesis studies could be performed in the genetically modifiable strain FNN23 in order to validate these findings. Mutants lacking each of the above-mentioned adhesion proteins could be studied regarding their adhesion, autoaggregation and biofilm forming ability.F. nucleatum, however this might not reflect true virulence of this anaerobic microorganism, as virulence genes have been shown to be upregulated in biofilms [F. nucleatum single-subspecies biofilm characterisation data might help researchers to perform more biologically relevant F. nucleatum pathogenicity studies using single-subspecies biofilms. Our experiments showed that in general both glass and plastic surfaces supported biofilm growth and thus can be utilised for studies of F. nucleatum subspecies biofilms when transparent substrates are required. Additionally, surface coatings do not seem to be necessary when quantity of the biofilm is not a priority.Many authors have utilised planktonic cultures in pathogenicity studies of biofilms , 68. AvaF. nucleatum, represented by widely available ATCC type strains, to build a knowledge base, which can later be utilised to study virulence of specific clinical isolates. As biofilm formation is one of the virulence properties of bacteria, understanding the differences in pathogenicity of individual F. nucleatum subspecies may reveal new virulence factors as novel therapeutic targets for prevention and treatment of F. nucleatum-mediated infections and diseases.Whilst we appreciate that behaviour of laboratory type strains might differ significantly from clinical isolates obtained from patients, we believe that it is important to first characterise virulence properties of subspecies of The original contributions presented in the study are included in the article/MM carried out experiments and data analysis and wrote the manuscript. DB performed bioinformatic analysis and wrote the corresponding parts of the manuscript. MG, SK, IC, and JH were involved in planning experiments, supervised the project, and contributed to the interpretation of the results. JH provided support with statistical analysis and figure preparation. All authors provided critical feedback on the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by a grant provided by Birmingham Community Healthcare NHS Foundation Trust. Publication of this study was possible thanks to funds provided by the University of Birmingham, Birmingham, UK.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Leishmaniinae: Leishmania martiniquensis (Thailand), Leishmania orientalis (Thailand), Leishmania enriettii (Brazil), Leishmania sp. Ghana, Leishmania sp. Namibia and Porcisia hertigi (Panama). De novo assembly was performed using Nanopore long reads to construct chromosome backbone scaffolds. We then corrected erroneous base calling by mapping short Illumina paired-end reads onto the initial assembly. Data has been deposited at NCBI as follows: raw sequencing output in the Sequence Read Archive, finished genomes in GenBank, and ancillary data in BioSample and BioProject. Derived data such as quality scoring, SAM files, genome annotations and repeat sequence lists have been deposited in Lancaster University’s electronic data archive with DOIs provided for each item. Our coding workflow has been deposited in GitHub and Zenodo repositories. This data constitutes a resource for the comparative genomics of parasites and for further applications in general and clinical parasitology.We provide the raw and processed data produced during the genome sequencing of isolates from six species of parasites from the sub-family  Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.15134085 Leishmania and 18 different species are known to infect humans1. 98 sandfly species are suspected or confirmed vectors of Leishmania2. There are three major types of leishmaniasis: visceral, also known as kala-azar, is fatal if left untreated in over 95% of cases; cutaneous, the most common form, causes skin lesions leaving life-long scars and serious disability or stigma; mucocutaneous, leads to partial or total destruction of mucous membranes of the nose, mouth and throat3. Over one billion people live in endemic areas and are at risk of leishmaniasis. It is estimated that each year, globally, new cases of cutaneous leishmaniasis occur at an incidence of 700,000 to 1.2 million or more in over 100 countries4. Additionally, up to 300,000 visceral leishmaniasis cases cause more than 200,000 deaths annually5.Leishmaniasis is a neglected tropical disease. It is considered to be a disease of poverty, primarily affecting low and middle-income countries (LMICs). Leishmaniasis is caused by parasites of the genus Leishmania is divided into four subgenera: L. Leishmania, L. Viannia, L. Sauroleishmania and the newest subgenus L. Mundinia, the latter now accommodating several species from the L. enriettii complex and others, from five continents12. In 1994, the Leishmania Genome Network was initiated13 and announced, ten years later, the assembly of the Leishmania major Friedlin strain as the first Leishmania reference genome14. Since then, a total of 58 genomes have become available publicly, assembled at a variety of levels of completeness ranging from contigs to chromosome level. Prior to our project, only two L. Mundinia subgenus genomes have been sequenced and assembled: Leishmania enriettii, strain LEM3045 (GCA_000410755) and Leishmania sp. MAR, strain LEM2494 (GCA_000410755). The genus Porcisia is a sister genus of Leishmania within the sub-family Leishmaniinae. Prior to the release of our genome, there were no genome sequences for genus Porcisia. Subsequently, the partial genome of P. deanei was released and published15.The genus L. Mundinia species – those of L. martiniquensis, L. orientalis, L. enriettii, L. sp. Ghana and L. sp. Namibia - and one genome in the genus Porcisia – that of P. hertigi, formerly known as L. hertigi16 - using Illumina and Nanopore sequencing. The two isolates from Ghana and Namibia are from new species that have not yet been formally named. The World Health Organization (WHO) codes for the six isolates are: L. martiniquensis MHOM/TH/2012/LSCM1;LV760; L. orientalis MHOM/TH/2014/LSCM4;LV768; L. enriettii MCAV/BR/2001/CUR178;LV673; L. sp. Ghana MHOM/GH/2012/GH5;LV757; L. sp. Namibia MPRO/NA/1975/252;LV425; and P. hertigi MCOE/PA/1965/C119;LV43. Nanopore long reads were used for the initial scaffolding assemblies, followed by mapping of the Illumina short reads onto these scaffolds, thus increasing quality of the assembled sequence while preserving whole chromosome integrity. Final polishing, reordering and reorienting of chromosomes, along with masking and classifying of repeat regions, was guided by the most closely related reference genome for each species. Finished genome annotation was both evidence-based and ab initio.We assembled and annotated the genomes of five Figure 28. Illumina HiSeq 4000 and MiSeq sequencing was contracted to BGI Genomics and Aberystwyth University. Nanopore sequencing was performed in-house using MinION FLO-MIN106 flow cells with SQK-LSK109 ligation sequencing protocol. Throughout the text we provide literature citations to software where available. Links to both published and unpublished software used are provided in Table 30.From the parasite cryobank at Lancaster University, we selected six samples of the species listed above without publicly available reference genomes. Table De novo assemblies were performed with Nanopore MinION long reads using Flye31. Due to the low quality scores in Nanopore long reads, we mapped high quality Illumina short reads onto the assemblies and created corrected consensus sequences using minimap232 and SAMtools33. The consensus sequence was scanned for any contamination or any sequence of vector origin by BLAST+34 on the UniVec database35. Finally, a polishing step was done to minimise gaps using Pilon36.37 release-47 genomes. The output for each genome was then visualized using wordcloud to suggest the closest relative among TriTrypDB genomes38. Then, synteny was plotted for each genome by aligning each of its chromosomes with the corresponding chromosomes of its wordcloud-predicted closest relative, using MUMmer39 .For all chromosomes of each polished genome, we then ran BLAST + (parameters: -max_target_seqs. 1 -max_hsps 1) against all TriTrypDB41. Then, we generated a stratified genome-wide repeat plot for each assembly38  an evidence-based annotation round using EST, mRNA-seq and protein homology evidence from TriTrypDB release-47 along with our repeat-masking output, 2) an ab initio round using AUGUSTUS43, with the pre-trained L. tarentolae as the model organism. After each round, Annotation Edit Distance (AED) scores were calculated and plotted  and quantified using Qubit® dsDNA HS Assay Kits (ThermoFisher Scientific) prior to sequencing. Concentrations ranged between 68.2 and 120 ng/µL. For consistency, we used the same extracted DNA for all three sequencing platforms . Furthermore, we assessed the gDNA high molecular weight using N50 estimates of MinION long reads which were ranged between 12.07 and 22.92 kilobases.We scanned all assemblies for any contamination or any sequence of vector origin by first building a UniVec Database and then using BLAST+ . All contaminants were found either at the beginning or at the end of contigs and then deleted. No contaminants affected assembly integrity.123 to output all sequence quality scores in one interactive report110.We used FastQC to check the sequence quality of Illumina short reads sequences and pycoQC to check the Nanopore long reads sequence quality. We used MultiQC124 as a benchmark for the presence of expected universal single-copy orthologues. Other checks focussed on the correct order and orientation of the chromosomes, for instance MUMmer alignment to find synteny between assemblies and other Leishmania genomes. Yet further checks focussed on the accuracy and precision of annotation, for instance using Annotation Edit Distance score (AED) in MAKER2 (Fig. Since the analysis took many steps to finish, quality checks were introduced between each step. Some checks were focused on completeness, for instance using BUSCOER2 Fig. . We chec


Passiflora quadrangularis L. belongs to the family Passifloraceae which bears larger fruit with edible juicy mesocarp and pulp known as a good source of phytochemicals. Cultivation and plant management practices are known to influence the phytochemical compositions of agricultural produce. This study aimed to examine the influence of the cultivation practices on the antioxidant activities and secondary metabolites of the organically and conventionally grown P. quadrangularis. Findings revealed organically treated P. quadrangularis plants showed enhancement in their antioxidant properties and secondary metabolites profiles. Among the plant parts, leaves of P. quadrangularis grown organically possessed higher antioxidant activities compared to the conventional in all assays evaluated. The antioxidant activities in the edible parts of the P. quadrangularis fruit have also been enhanced through organic cultivation with significantly higher total phenolic content and DPPH in mesocarp, and the pulp showed higher total flavonoid content, DPPH and FRAP. This observation is supported by a higher level of vitamins and secondary metabolites in the samples. The secondary metabolites profile showed mesocarps were phenolic rich, the pulps were flavonoids rich while leaves showed good composition of phenolics, flavonoids and terpenoids with outstanding antioxidant activities. The common secondary metabolites for organically produced P. quadrangularis in different plant parts include 2-isopropyl-3-methoxycinnamic acid (mesocarp and pulp), myricetin isomers (pulp and leaves), and malvidin-3-O-arabinoside isomers (pulp and leaves). This study confirmed that organic cultivated P. quadrangularis possessed higher antioxidant activities contributed by its vitamins and secondary metabolites. Statistical filters of p < 0.05, max fold change > 2 and minimum coefficient of variation < 30 were applied. Top candidates from library matches were assigned tentatively to represent the features. The abundance of features elevated in samples from organic cultivation with potential matched identity belong to classes of phenolics, flavonoids, terpenoids and alkaloids were presented by hierarchical clustering technique and visualized in heatmap using Metaboanalyst software . Correlation analysis (CA) was carried out using XLSTAT software version 2016  to obtain the relationship between antioxidant activities and the putatively identified metabolites in samples of organic and conventional cultivations.Progenesis QI 2.0 enables the library matching of the potential markers based on the accurate mass of precursor and also MS/MS fragments obtained from MSP. quadrangularis fruits cultivated under organic and conventional practices. There were no significant difference (p > 0.05) between pH and TTA of organic and conventional pulp and mesocarp of P. quadrangularis. This is contradicting to the findings by Janzantti et al.  and [M-H] from MS/MS library matching. A total of 197 putatively identified secondary metabolites  from organic cultivation were conducted.The MSabolites  Tables wabolites  and edibabolites  of P. qu content , 54, 55. content . also haoperties , 52, 56.operties , 57. FurPassiflora sp. namely isoquercitin, myricetin, isovitexin 2’’-O-(6‴-(E)-P-Coumaroyl), Glucoside 4’-O-Glucoside, 3,6,3’,4’-tetrahydroxyflavone are among the prominent metabolite markers [P. quadrangularis (vitexin-2”-O-xyloside) reported by Costa et al. [P. quadrangularis were not elevated from organic cultivation in leaves sample of this study.In this study, 149 putatively identified secondary metabolites were detected in leaves extract  had show markers , 58. Thea et al. . has shoa et al. . in the P. quadrangularis has not been reported. Flavonoid derivatives, phenolics and terpenoids were the major classes of the secondary metabolites in edible parts Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Endometrial cancer is among the most common malignant tumors threatening the health of women. Recently, immunity and long noncoding RNA (lncRNA) have been widely examined in oncology and shown to play important roles in oncology. Here, we searched for immune-related lncRNAs as prognostic biomarkers to predict the outcome of patients with endometrial cancer.  RNA sequencing data for 575 endometrial cancer samples and immune-related genes were downloaded from The Cancer Genome Atlas (TCGA) database and gene set enrichment analysis (GSEA) gene sets, respectively. Immune-related lncRNAs showing a coexpression relationship with immune-related genes were obtained, and Cox regression analysis was performed to construct the prognostic model. Survival, independent prognostic, and clinical correlation analyses were performed to evaluate the prognostic model. Immune infiltration of endometrial cancer samples was also evaluated. Functional annotation of 12 immune-related lncRNAs was performed using GSEA software. Prognostic nomogram and survival analysis for independent prognostic risk factors were performed to evaluate the prognostic model and calculate the survival time based on the prognostic model.  Twelve immune-related lncRNAs  were obtained, and a prognostic model was constructed. The survival rate in the high-risk group was significantly lower than that in the low-risk group. Patient age, pathological grade, the International Federation of Gynecology and Obstetrics (FIGO) stage, and risk status were the risk factors. The 12 immune-related lncRNAs correlated with patient age, pathological grade, and FIGO stage. Principal component analysis and functional annotation showed that the high-risk and low-risk groups separated better, and the immune status of the high-risk and low-risk groups differed. Nomogram and receiver operating characteristic (ROC) curves effectively predicted the prognosis of endometrial cancer. Additionally, age, pathological grade, FIGO stage, and risk status were all related to patient survival.  We identified 12 immune-related lncRNAs affecting the prognosis of endometrial cancer, which may be useful as therapeutic targets and molecular biomarkers. Endometrial cancer is one of the most common malignant tumors in women. However, the mechanisms underlying endometrial cancer occurrence and development are unknown. Recent studies indicated that hypertension, diabetes, obesity, and estrogen replacement therapy are risk factors for endometrial cancer –4. The mLong noncoding RNAs (lncRNAs) are more than 200 nucleotides in length and were recently shown to dynamically regulate gene expression at multiple levels –18. LncRRecently, tumor immunotherapy has gained attention. Immune cells and molecules are complex components in the tumor microenvironment and can promote or inhibit malignant tumor progression. The immune response in the tumor microenvironment has an important effect on tumor proliferation, invasion, and metastasis –29. ManyIn this study, we explored the transcriptome data of endometrial cancer samples in the TCGA database. Immune-related lncRNAs coexpressed with immune-related genes were identified, and a prognostic nomogram was constructed to predict the overall survival rate. These lncRNAs show potential as therapeutic targets and molecular biomarkers of endometrial cancer.https://portal.gdc.cancer.gov/), with 23 normal samples and 552 endometrial cancer samples. Clinical information on endometrial cancer samples was downloaded from UCSC Xena (https://xenabrowser.net). We downloaded the gene sets IMMUNE_RESPONSE (systematic name: M13664), IMMUNE_SYSTEM_PROCESS (systematic name: M19817), GO_NEGATIVE_REGULATION_OF_ADAPTIVE_IMMUNE_RESPONSE (systematic name: M10422), GO_NEGATIVE_REGULATION_OF_IMMUNE_RESPONSE (systematic name: M15641), and GO_T_CELL_ACTIVATION_INVOLVED_IN_IMMUNE_RESPONSE (systematic name: M10714) from the gene set enrichment analysis (GSEA) database (https://software.broadinstitute.org/gsea/index.jsp). Immune-related genes were obtained from the IMMUNE_RESPONSE (systematic name: M13664) and IMMUNE_SYSTEM_PROCESS (systematic name: M19817) gene sets. Functional annotation was performed using the gene sets IMMUNE_RESPONSE (systematic name: M13664), GO_NEGATIVE_REGULATION_OF_ADAPTIVE_IMMUNE_RESPONSE (systematic name: M10422), GO_NEGATIVE_REGULATION_OF_IMMUNE_RESPONSE (systematic name: M15641), and GO_T_CELL_ACTIVATION_INVOLVED_IN_IMMUNE_RESPONSE (systematic name: M10714).Fragments per kilobase per million RNA sequencing data of 575 samples were downloaded from TCGA database .By sorting and analyzing the transcriptome data of the endometrial cancer samples from TCGA database, the expression matrices of mRNA and lncRNA of endometrial cancer samples were obtained. Immune-related genes were obtained from the IMMUNE_RESPONSE (systematic name: M13664) and IMMUNE_SYSTEM_PROCESS (systematic name: M19817) gene sets. The immune-related gene expression matrix was obtained by collecting and analyzing the mRNA expression matrix and immune-related genes together using the limma package in p value filter = 0.01) and multivariate Cox regression analyses were performed using the survival package in R software. The coexpression network was plotted using Cytoscape software (Cytoscape v. 3.7.2). The survival and survminer packages of R software were used to draw the survival curves of high- and low-risk groups. Risk curves were drawn using the pheatmap package in R software. Univariate- and multivariate-independent prognostic analyses were performed using the survival package in R software, and a multi-index receiver operating characteristic (ROC) curve was plotted using survivalROC package in R software to evaluate the accuracy of the constructed model. The ggpubr package in R software was used for clinical correlation analysis.By collecting and analyzing the clinical data, univariate .Principal component analysis was performed using the limma and scatterplot3d packages in R software, 70% of the tumor samples were placed into the training group and 30% into the validation group. The rms, foreign, and survival packages in R software were used to perform multivariate Cox regression analysis of the training group and calculate the C-index of the training and validation groups. A nomogram was constructed using the rms, foreign, and survival packages in R software. The survival and timeROC packages in R software were used to construct multi-index ROC curves for the training and validation groups. The survival package in R software was used to perform Kaplan-Meier survival analysis for the training group.Using the foreign, survival, and caret packages in R software (R-3.6.1), strawberry-Perl-5.30.0.1. p values <0.05 were considered to indicate statistically significant results.The statistical analysis was performed using To identify immune-related lncRNAs associated with prognosis, 332 immune-related genes were selected from the GSEA data sets. Through data collection and analysis, we identified 137 immune-related genes and 363 immune-related lncRNAs showing a coexpression relationship with these immune-related genes. Next, univariate Cox regression analysis of all immune-related lncRNAs was performed, and a Forest plot was obtained . As showp < 0.05).To compare the survival rates of different risk statuses based on grouping results, survival analysis was performed, and survival curves were obtained . As showTo analyze the impact of the patients' age, pathological grade, and FIGO stage on prognosis, we performed univariate and multivariate independent prognostic analyses of clinical data Figures . The resTo compare tumor microenvironment differences for different risk statuses using the ESTIMATE algorithm, the ESTIMATE, immune, and stromal scores of all samples were calculated. The results showed that the ESTIMATE scores ranged from −3166.978 to 3990.147, immune scores ranged from −1359.509 to 3614.677, and stromal scores ranged from −2224.623 to 860.431. Next, we drew box plots of these scores for different risk statuses Figures . The avePrincipal component analyses of the expressions of all immune-related lncRNAs and 12 immune-related lncRNAs associated with prognosis were performed to determine whether there were differences in the distribution between the high- and low-risk groups Figures . As showTo further evaluate the effect of the model constructed based on the 12 immune-related lncRNAs on prognosis, we used the patient's risk status based on these lncRNAs as an independent risk factor affecting prognosis and divided all samples into training (70%) and validation (30%) groups. Multivariate Cox regression analysis using training samples was performed to analyze the effects of patient's age, pathological grade, FIGO stage, and risk status on prognosis . The agep values, hazard ratios, and 95% confidence intervals of the clinical characteristics are shown.Age ≥65 years was compared to age <65 years; Grade 2 and Grade 3 were compared to Grade 1; Stage II, Stage III, and Stage IV were compared to Stage I; and high-risk group was compared to low-risk group. Regression coefficients, p < 0.05).To analyze the effects of age, pathological grade, FIGO stage, and risk status on overall survival, each risk factor was subjected to Kaplan-Meier survival analysis, and survival curves were drawn Figures . As showγ inhibits tumor progression [ALOX15, CISD1, CS, GCLC, GPX4, SLC7A11, EMC2, G6PD, and ACSF2) that could predict the prognosis of breast cancer [κB signaling pathway in hepatocellular carcinoma cells, lncRNA miR503HG inhibits tumor metastasis [Recent studies showed that the immune system has a dual effect on tumor progression. The immune components in the tumor microenvironment can both promote or inhibit the progression of malignant tumors . Infiltrgression . Recentlgression –44. Intet cancer . LncRNAst cancer . In gastt cancer . In livetastasis .κB activity, thereby facilitating immune escape [LncRNAs also affect tumor progression by regulating immune components in the tumor microenvironment. For example, in lung and breast cancer, lncRNA NKILA can upregulate the sensitivity of tumor-specific cytotoxic T lymphocytes and type 1 helper T cells to activation-induced cell death by inhibiting NF-e escape . In coloe escape .However, it is relatively unknown whether lncRNAs modulate endometrial cancer progression via the regulation of immune components in the tumor microenvironment. In the present work, the expression matrix of immune-related lncRNAs was analyzed by collecting immune gene sets from the GSEA database and information on endometrial cancer samples from TCGA database. Univariate and multifactorial Cox regression analyses were performed, and 12 immune-related lncRNAs were identified as having an important influence on endometrial cancer: ELN-AS1, AC103563.7, PCAT19, AF131215.5, LINC01871, AC084117.1, NRAV, SCARNA9, AL049539.1, POC1B-AS1, AC108134.4, and AC019080.5.Among the 12 immune-related lncRNAs, PCAT19 level is downregulated in non-small cell lung carcinoma and regulates the development of prostate cancer –53. By mBy sorting and analyzing the transcriptome information of endometrial cancer samples from TCGA database, we identified 12 immune-related lncRNAs. These molecules may play important regulatory roles in the occurrence and development of endometrial cancer and represent potential therapeutic targets. However, their specific roles and mechanisms require further experimental validation.


Bacteroides uniformis CBA7346, isolated from the gut of healthy Koreans, on mice with high-fat diet (HFD)-induced NAFLD. Administration of B. uniformis CBA7346 reduced body and liver weight gain, serum alanine aminotransferase and aspartate aminotransferase levels, liver steatosis, and liver triglyceride levels in mice on an HFD; the strain also decreased homeostatic model assessment for insulin resistance values, as well as serum cholesterol, triglyceride, lipopolysaccharide, leptin, and adiponectin levels in mice on an HFD. Moreover, B. uniformis CBA7346 controlled fatty liver disease by attenuating steatosis and inflammation and regulating de novo lipogenesis-related proteins in mice on an HFD. Taken together, these findings suggest that B. uniformis CBA7346 ameliorates HFD-induced NAFLD by reducing insulin resistance and regulating de novo lipogenesis in obese mice.Dietary habits and gut microbiota play an essential role in non-alcoholic fatty liver disease (NAFLD) and related factors such as insulin resistance and de novo lipogenesis. In this study, we investigated the protective effects of  Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, characterized by irregular lipid accumulation in hepatocytes even without alcohol abuse . The gloThe pathogenesis of NAFLD has not yet been clearly elucidated, and the main hypothesis that explains the progression of NAFLD is the “multiple-hit hypothesis”. A variety of factors including dietary habits such as a high-fat diet (HFD), and genetic and environmental factors, can lead to obesity accompanied by insulin resistance and adipocyte proliferation . InsulinLactobacillus and Bifidobacterium can ease NAFLD symptoms in animal models ..17].Liver tissues were frozen with optimum cutting temperature compound , sectioned (4 μm), and stained with ORO for evaluation of lipid accumulation in the liver. All slide samples were scanned using a Pannoramic 250 Flash III slide scanner . The ORO-stained area was calculated using ImageJ software .Fecal samples were collected on the non-administration day at 12 weeks, and the microbiota was analyzed by sequencing the V3–V4 region of 16S rRNA using a MiSeq system  at Macrogen , as per the manufacturer’s instructions. Pairs of reads from the DNA fragments were assembled using FLASH , operatip < 0.05, and all data were expressed as the mean ± standard error of the mean. Statistical analysis was performed using IBM SPSS Statistics for Windows, version 20 .To assess the relative significance among the groups, Duncan’s multiple range test was performed at p < 0.05) compared to the NC group, at 12 weeks  compared to the HC group at the end of 12 weeks. Moreover, the HU group exhibited a significantly lower (p < 0.05) liver/body weight ratio compared to the HC group  (p < 0.05)  than the HC group.Serum insulin levels were significantly higher in the HC group than in the NC group ( < 0.05) A. In add < 0.05) B. B. unip < 0.05), whereas the HU group showed remarkably reduced levels  compared to the HC group. These results suggest that B. uniformis CBA7346 alters HFD-induced changes in biochemical and hormonal parameters.HFD-induced changes in biochemical and hormonal parameters were evaluated using serum cholesterol, triglyceride, leptin, and adiponectin levels . No signB. uniformis CBA7346 reduced HFD-induced steatosis and hepatocyte ballooning (p < 0.05) compared to the HC group  than in the HC group. Furthermore, the HU group showed significantly less reduction in hepatic triglyceride levels (p < 0.05) compared to the HC group  compared to the HC group (except for SCD1 protein expression). In addition, the p-AMPKα/AMPKα ratio was significantly higher in the HU group than in the HC group (p < 0.05). These results suggest that B. uniformis CBA7346 alters HFD-induced changes in hepatic lipid metabolism.To evaluate whether the administration of measured . B. unifB. uniformis CBA7346 on the intestinal microbiota of mice on an HFD  through the gut-liver axis, leading to hepatocyte death and lipid accumulation [B. uniformis CBA7346, showed decreased LPS release in the serum confirming that B. uniformis CBA7346 also prevented the passage of LPS into the blood.HFDs have been associated with lipid accumulation, insulin resistance, and metabolic diseases ; to indueriments . Insulineriments , and sereriments . ALT andmulation . Mice onB. uniformis CBA7346 suppressed extensive hepatic steatosis, lobular inflammation, and hepatocellular ballooning in mice on an HFD. In addition, B. uniformis CBA7346 administration while on an HFD affected the de novo lipogenesis pathway in the liver. SREBP1 activated by insulin, and ChREBP activated by glucose regulates hepatic lipogenesis [B. uniformis CBA7346 efficiently attenuated the accumulation of triglycerides by downregulating this protein. Furthermore, mice on an HFD, when treated with B. uniformis CBA7346, showed increased AMPKα activation levels. AMPKα regulates hepatic lipid metabolism by modulating transcription factors such as SREBP-1 and ChREBP [B. uniformis CBA7346 can inhibit hepatic lipid synthesis by regulating lipid metabolism-related proteins in obese mice.Hepatic steatosis plays an essential role in the progression of NAFLD, and a prolonged HFD sequentially leads to steatosis and steatohepatitis, progressing to bridging fibrosis ,27. Our ogenesis ,28. In togenesis . As exped ChREBP . Likewisd ChREBP , was sigB. uniformis CBA7346 reduced the serum levels of lipid profiles in mice with HFD-induced NAFLD. Leptin and adiponectin—important hormones in obesity management—were also suppressed in B. uniformis CBA7346 treated mice on an HFD. These factors could also possibly regulate the expression of lipid homeostasis-related proteins in the liver [Dyslipidemias, i.e., abnormal levels of lipid profiles such as total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides, are also induced by HFDs in animals . In our he liver .B. uniformis CBA7346 did not significantly influence microbial composition changes such as the changes in the F/B ratios caused by the HFD. Another study reported that daily administration of 5 × 108 CFU B. uniformis ameliorated intestinal dysbiosis in mice with HFD-induced NAFLD [6 CFU B. uniformis CBA7346 only thrice a week. Therefore, further studies are needed to identify the dose of B. uniformis CBA7346 that produces the most efficient results in mice with HFD-induced NAFLD with regard to NAFLD attenuating effects.Many studies have shown that the gut-liver axis plays an important role in NAFLD development ,9; we foed NAFLD , but we B. uniformis CBA7346 isolated from the gut of healthy Koreans, on mice with HFD-induced NAFLD. Administration of B. uniformis CBA7346 inhibited body and liver weight gain, reduced insulin resistance, and influenced serum metabolic biomarkers related to hepatic injury and steatosis in mice on an HFD. The administration of B. uniformis CBA734 also healed NAFLD by attenuating steatosis, inflammation, and ballooning, and regulating de novo lipogenesis-related proteins induced by an HFD in mouse liver tissues. We conclude that B. uniformis CBA7346 may offer protection against HFD-induced NAFLD by regulating the release of LPS and lipid metabolism-related proteins and controlling insulin resistance (B. uniformis strain used in this animal study.This study demonstrates, for the first time, the effects of administrating sistance . However