Luticolamicrocephala M. Rybak, Peszek & Kochman-Kędziora, sp. nov., Luticolaasymmetrica M. Rybak, Kochman-Kędziora & Peszek, sp. nov., Luticolaterrestris Kochman-Kędziora, M. Rybak & Peszek, sp. nov. and Microcostatusmeridionalis Peszek, M. Rybak & Kochman-Kędziora, sp. nov. Algal community composition had low species richness (9–15 taxa per sample) and samples were dominated by: Humidophilacontenta, Nitzschiabrevissima and Eunotiaaff.pseudominor. The three new Luticola species formed numerous populations, exceeding 10% of the share, whereas Microcostatusmeridionalis reached 5.4%. Basic morphological data of associated diatom flora together with detailed micrographs are also provided.The knowledge about terrestrial diatom assemblages in southern Africa is rather limited, despite a long history of diatom research in this area. Terrestrial habitats are places of characteristic diatom floras, dominated by species resistant to desiccation which are able to thrive in conditions of limited water availability. The presented work expands the knowledge about these unique habitats. During the study on terrestrial moss-inhabiting diatoms from Western Cape Province (South Africa), four taxa with a unique set of valve features have been found and described herein as new species, based on light and scanning electron microscopy. These new species are:  Research on African diatoms has a long tradition, with the first works dating back to the 19th century i.e. . The dynMuelleriataylorii Van de Vijver & Cocquyt from Drakensburg, Freestate Province, South Africa.Despite a long history of diatom research in southern Africa, the knowledge about terrestrial diatom assemblages is rather limited . AlthougLuticola and genus Microcostatus (Luticola D.G. Mann (Navicula to accommodate species included in Naviculaesect.Punctatae with Luticolamutica (Kütz.) D.G. Mann as generitype. So far, over 200 species have been documented worldwide , whereas summers (December–February) are warm and dry . The WesFor this study, the moss samples were collected in September 2018 from three different study sites in Western Cape Province, South Africa. Two sites were located on the edge of the forest by the Prince Alfred’s Pass. The third site was situated in the Jonkershoek Nature Reserve, where the sample was collected from moss growing on the rock (Table LM) Carl Zeiss Axio Imager A2, equipped with a 100× Plan Apochromatic objective with differential interference contrast (DIC) for oil immersion. Diatom images were captured with a Zeiss AxioCam ICc5 camera. For the observations in a scanning electron microscope (SEM), the samples were applied to a polycarbonate membrane filter with a 3 µm mesh, attached to aluminium stubs and sputter-coated with 20 nm of gold, using a turbo-pumped Quorum Q 150T ES coater. Diatoms were analysed using a Hitachi SU8010 SEM. The storage locations of holotype and isotype slides (Diatom Collections) for each of the newly-described species are indicated in the descriptions. Diatom terminology and identification was based on the following references: In the laboratory, the moss samples were digested in sulphochromic mixture – a mixture of concentrated sulphuric acid and potassium dichromate in order to obtain clean diatom frustules. To remove the sulphochromic mixture, the material was centrifuged at 2500 rpm with distilled water. The cleaned diatom suspension was dried on microscope cover-slips and mounted in the synthetic Naphrax Brunel Microscopes Ltd., Chippenham, UK (refractive index 1.73). Diatoms were identified and counted under a light microscope  at North-West University, Potchefstroom, South Africa.Slide no. 20-093 stored at the South African National Diatom Collection (SZCZ) hosted by the University of Szczecin.Slide no. 27525 and unmounted material with the same number at the Szczecin Diatom Collection  at North-West University, Potchefstroom, South Africa.Slide no. 20-091 stored at the South African National Diatom Collection (SZCZ) hosted by the University of Szczecin.Slide no. 27523 and unmounted material with same number stored at the Szczecin Diatom Collection  at North-West University, Potchefstroom South Africa.Slide no. 20-092 stored at the South African National Diatom Collection (SZCZ) hosted by the University of Szczecin.Slide no. 27524 and unmounted material with same number at the Szczecin Diatom Collection  at North-West University, Potchefstroom, South Africa.Slide no. 20-093 stored at the South African National Diatom Collection (SZCZ) hosted by the University of Szczecin.Slide no. 27525 and unmounted material with same number at the Szczecin Diatom Collection .The name refers to the area from where the new species is described  Kale, Levkov & Karthick . Additionally, L.jogensis has 5–6 areolae per striae, whereas striae in L.microcephala sp. nov. are composed of 3 to 4 areolae. The second of the Indian species L.gandhii differs from L.microcephala sp. nov. in having rostrate, not capitate apices. The most pronounced difference amongst all three species is the number of rows of areolae on the valve mantle. Luticolamicrocephala sp. nov. has only one row of elongated areolae, becoming more rounded in the central part of the valve and near the apices. On the contrary, L.jogensis has 2 or 3 rows .Amongst other species described from South Africa, Luticolaasymmetrica sp. nov. shows morphological similarity to five Luticola species reported from the African continent: L.imbricatiformis Levkov, Metzeltin & Pavlov , L.falknerorum Metzeltin & Lange-Bertalot  and more areolae per striae (5–6 versus 3–5 in Luticolaasymmetrica sp. nov.). Both species can also be separated, based on external raphe structure. Raphe branches of L.falknerorum are bordered by silica ridges and have short proximal raphe endings  and L.micra Levkov, Metzeltin & Pavlov  from the group A and L.incana Levkov, Metzeltin & Pavlov from the group B. Luticolatenuis and Luticolaterrestris sp. nov. are observed from terrestrial habitats. Both species overlap dimensions; however, Luticolaterrestris sp. nov. has higher striae density (20–23 in 10 µm versus 18–20 in L.tenuis). Moreover, L.tenuis has more linear valve outline and slightly deflected proximal raphe endings  and the lack of ghost areolae in the central area. Additionally, L.micra has elongated stigma . Moreover, L.incana does not show a ghost areolae in the central area and has short, slightly deflected distal raphe endings  show some degree of similarity to L.terrestris sp. nov. Naviculaguluensis can be easily distinguished, based on striae density (15 in 10 µm versus 20–23 in 10 µm) and shape of raphe endings. Naviculaguluensis has shortly deflected distal raphe endings  Lange-Bertalot  J.R. Johansen & Sray  can be observed in LM. Microcostatusmeridionalis sp. nov can be separated, based on its cuneate valve ends, whereas M.werumii has slightly rostrate valve ends. Additionally, proximal raphe endings are different in both species – in M.werumii, they are are poorly separated and located close to each other , but can be separated, based on the striae pattern and present of central area in. M.meridionalis .In the studied material, some species were scarce and their exact identification was not entirely possible. In the literature data, there is a lack of information about valve dimensions of rom Asia . For thiudominor  and 9.9–M.meridionalis, which has fully covered areolae in the central part of the valve. Accompanying Pinnularia and Hantzschia taxa also show the presence of external areolae occlusions. This type of structure is also present on the surface of Eunotiacf.pseudominor, whereas the genus Eunotia does not create hymen or other occlusions . This low species richness is quite typical for terrestrial environments, such as soils, rock crevices or clumps of terrestrial bryophytes. Especially in the Southern Hemisphere, terrestrial diatom assemblages are still poorly known, both in taxonomic and ecological aspects. The present study showed that these environments are often “hot spots” for the occurrence of potentially new and rare taxa.


From time immemorial, humans have exploited plants as a source of food and medicines. The World Health Organization (WHO) has recorded 21,000 plants with medicinal value out of 300,000 species available worldwide. The promising modern “multi-omics” platforms and tools have been proven as functional platforms able to endow us with comprehensive knowledge of the proteome, genome, transcriptome, and metabolome of medicinal plant systems so as to reveal the novel connected genetic (gene) pathways, proteins, regulator sequences and secondary metabolite (molecule) biosynthetic pathways of various drug and protein molecules from a variety of plants with therapeutic significance. This review paper endeavors to abridge the contemporary advancements in research areas of multi-omics and the information involved in decoding its prospective relevance to the utilization of plants with medicinal value in the present global scenario. The crosstalk of medicinal plants with genomics, transcriptomics, proteomics, and metabolomics approaches will be discussed. Papaver somniferum L. was the first reported in the early 1800s [The welfare of humankind depends on 12% of Earth’s approximately 300,000 . Payne ely 1800s . Countrily 1800s . The Worly 1800s . The WHOly 1800s . The WHOly 1800s . The Intly 1800s ,22. Arouly 1800s ,24. Globly 1800s . The WHOly 1800s . Medicinly 1800s . The Indly 1800s . Consequly 1800s . India cly 1800s . Herbacely 1800s . There aly 1800s  and 13 mly 1800s .Artemisia annua L. with novel hybrids with high-yielding abilities so as to gain a robust cropping system, and secondly in a re-engineered microbial host by the reconstitution of the biosynthetic pathway of artemisinin, which was obtained after the sequencing of its genome [Vitis vinifera L. multi-omics  identified 238 genes and 2012 metabolites that are upregulated by UV-C irradiation. This supports the concept that stilbene biosynthesis encourages transcription factor-mediated regulation [Papaver somniferum L. have been identified mostly by omics approaches [Papaver somniferum L. [Modern day omics approaches, which include genomics, transcriptomics, proteomics and metabolomics, are becoming extremely significant for the identification and characterization of vital gene-protein-metabolite networks, new drug metabolites, complete genomes, transcriptomes, and proteomes of medicinal plants, and the responses of human cells to drugs or whole ethno-botanical plants for the medicinal use and large-scale production of plant-derived medicines ,35,36,37s genome . The cros genome . Vitis vgulation . Genes oproaches ,43. The ferum L. . In an eferum L. , phytochferum L. . The potferum L. . The freferum L. . Our preOryza sativa L., barley [http://metnetdb.org/mpmr_public/ accessed on 17 November 2020) is accessible for the development of a hypothesis regarding the role of genes [Glycyrrhiza uralensis Fisch. ex DC. (licorice), two cytochrome P450 genes of the glycyrrhizin biosynthetic pathway were detected [High-throughput omics platforms such as genomics, transcriptomics, proteomics and metabolomics  generate, barley  and popp, barley . Advanceof genes . The resof genes . In Glycdetected , and thehttp://www.genoscope.cns.fr/spip/ accessed on 17 November 2020) [Phalaenopsis equestris (Schauer) Rchb.f [Brassica napus L. [Capsicum annuum L. [Momordica charantia L. [Coffea canephora Pierre ex A. Froehner [Salvia miltiorrhiza Bunge [Ziziphus jujuba Mill. [Glycyrrhiza uralensis Fisch. Ex DC. [Dendrobium officinale Kimura et Migo [Azadirachta indica A. Juss., 1830 [Catharanthus roseus (L.) G. Don chloroplast and genome [Pogostemon cablin (Blanco) Benth. [Salvia miltiorrhiza Bunge (Danshen) and Catharanthus roseus (L.) G. Don synthesizes triterpenes, indole alkaloids and diterpene quinone. The draft genome sequence of Catharanthus roseus (L.) G. Don offers verification of the partial gene clustering for alkaloid (vinblastine and vincristine) biosynthesis. With bacterial artificial chromosome sequencing, Kellner et al. [Ziziphus jujuba Mill. is rich in vitamin C and sugar, and has a variety of medicinally essential flavonoids, phenolics and alkaloids. Azadirachta indica A. Juss. is medicinally effective as an antitumorigenic, antidiabetic and antimalarial. The genome of neem sequenced with the next generation sequencing approach is loaded with AT sequences and modest repetitive DNA, and has 20,000 genes [Salvia miltiorrhiza Burge (Danshen) and the characterization of its putative genes involved in the terpenoid biosynthetic pathway was studied by Ma et al. [Salvia miltiorrhiza Burge (Danshen) [Salvia miltiorrhiza Burge (Danshen), 40 terpenoid biosynthetic pathway genes have been discovered. Among these, 27 genes were new, which comprise 19 families (10 with single and 9 with multigene). In the terpenoid biosynthetic pathway of S. miltiorrhiza Burge (Danshen), HDR, DXS, HMGR and GGPPS enzymes are transcribed by multigene families that have diverse expression profiles and subcellular positions [Capsicum annuum pepper variety Zunla-1 with wild progenitor Capsicum annuum var. glabriusculum (Chiltepin) [Solanum tuberosum L., Capsicum, Atropa and tomato give information about the evolution of various members of the Solanaceae family. Innumerable anticancerous plant-based chemical molecules, such as camptothecin and podophyllotoxin derivatives [Panax ginseng cp genome gives an insight into its evolution and polymorphism [Panax ginseng promotes health and is useful in clinical therapy. The genome sequencing of Dendrobium officinale Kimura et Migo, which is a medicinal plant, and the orchid Cypripedium macranthos Sw. provides an insight into the content and order of their genes and latent RNA editing sites [Ocimum sanctum L. and Ocimum basilicum L. genome sequencing and annotation reveals higher expressions of genes of the phenylpropanoid/terpenoid biosynthetic pathway, cytochrome P450s and transcription factors. This has provided a new approach for the mining of biosynthetic pathways of important medicinal metabolites in related species [Ocimum tenuiflorum L. disclosed that the amino acid mutations at the gene loci of biosynthesis give it incomparable pharmaceutical traits. Ocimum tenuiflorum L. generates specialized metabolites with anticancer potential, such as ursolic acid, oleanolic acid, luteolin, taxol, eugenol, apigenin and sitosterol, as a defense mechanism. The genes responsible for the expression of these metabolites with anticancer potential can be identified and used for the development of targeted drugs [Papaver somniferum L.), the genes of benzylisoquinoline alkaloid (BIA) biosynthesis have been identified by virus-induced gene silencing (VIGS) technology. This has also helped us to discover the genes of the morphine biosynthetic pathway involved in reactions of O-demethylation with metabolite thebaine to produce codeine and then the conversion of the metabolite codeine into morphine [Viscum coloratum (Kom.) Nakai, with anticancerous properties, 19 new polymorphic microsatellite markers were developed, so that the ecological conservation and population genetics of this plant can be studied [Sinopodophyllum hexandrum (Royle) Ying showed its genetic diversity and structure of population, and provided data for the studies of evolution and conservation [The DNA sequences of a genome integrate vital information of the origin, development and epigenomic regulation of a plant. This may act as the foundation of decoding genomic and chemical diversity at minute levels . The higer 2020) ,61. Phal) Rchb.f , Brassicnapus L. , Capsicunnuum L. ,65, Momoantia L. , Coffea Froehner , Salvia za Bunge , Ziziphuba Mill. ,70, Glyc. Ex DC. , Dendrob et Migo , Azadiras., 1830 , and Catd genome ,74 and t) Benth.  have bee) Benth. ,76. Salvr et al.  showed s00 genes . The gena et al. . The cp Danshen)  verifiesositions . The domiltepin) . The genivatives , can be morphism . Panax gng sites . Ocimum  species . The cp ed drugs . In opiumorphine ,43. The morphine , which imorphine . Microsa studied . The stu studied . ISSR anervation . The genervation . To concPolygonum cuspidatum Sieb. et Zucc. [Rhodiola algida (Crassulaceae) [Taxus mairei [Salvia sclarea L. (Lamiaceae) [The transcriptomes of hundreds of therapeutic plants for instance, Oenothera (Onagraceae) , Fabaceaet Zucc. , Rhodiolulaceae) , Taxus ms mairei , Salvia miaceae) , etc., smiaceae) ,95,96,97miaceae) . Whole tPodophyllum hexandrum Royle, candidate genes were selected and combinatorially expressed in Nicotiana benthamiana Domin. This way, six enzymes from the podophyllotoxin biosynthetic pathway to etoposide aglycone were identified [Cannabis sativa L.) Glandular trichomes of the female flower of cannabis are the primary sites of cannabinoid biosynthesis, suggesting their unnoticed role in the generation of chemical diversity [Chlorophytum borivilianum Santapau & R. R. Fern. has revealed antitumerogenic and anticancerous potential [Catharanthus roseus (L.) G. Don generates diverse iridoid-based monoterpene indole alkaloids along with anti-cancerous vinblastine [Curcuma longa L. (rhizome) transcriptomics showed transcripts associated with the terpenoid biosynthetic pathways and other biosynthetic pathways of various anticancerous phytochemicals, such as vinblastine, curcumin and taxol. This information paved the way for the biosynthetic pathways of a variety of terpenoids in Curcuma longa L., along with its transcriptomic database [Ophiorrhiza pumila Champ. ex Benth., accrues a monoterpenoid indole alkaloid known as camptothecin, which is an anticancerous metabolite. The deep transcriptome analysis of Ophiorrhiza pumila Champ. ex Benth. yielded a 2GB sequence from which novel genes of plant secondary metabolic biosynthetic pathways were predicted [Withania somnifera (L.) Dunal synthesizes bioactive secondary metabolites known as withanolides. The leaf and root tissue chemo-profiling of Withania somnifera (L.) Dunal chemovars showed variations in the composition and traits of withanolides. The genes of chemotypes and the tissue-specific biosynthesis of withanolide of distinct chemotypes was characterized [Papaver somniferum L. [Salvia miltiorrhiza Burge (Danshen), Cui et al. [Salvia miltiorrhiza Burge (Danshen), a group of novel genes related to terpenoid-derived tanshinone and salvianolic acid secondary metabolite biosynthesis was identified [Lupinus angustifolius L. chemo-varieties for quinolizidine alkaloids, the gene that encodes the enzyme lysine decarboxylase involved in the first step of the alkaloid biosynthetic pathway, was undertaken, and the catalytic potential of the lysine decarboxylase was explained by site-directed mutagenesis and protein modeling [Camptotheca acuminata Decne. [Camptotheca acuminata Decne., Rauvolfia serpentina (L.) Benth. ex Kurz and Catharanthus roseus (L.) G. Don for the respective biosynthesis of camptothecin, ajmaline and vinblastine [Vaccinium macrocarpon Aiton (Cranberry) fruits at different developmental stages was annotated from public domains such as NCBI, KEGG, GO and NT, and the genes CHS, F3H, CHI, F3 ′ H, and LDOX of the bioactive flavonoid biosynthetic pathway were identified [UDP-glucose flavonoid 3-O-glucosyl transferase (UFGT) enzyme of the flavonoid biosynthetic pathway, which characterizes more different types of flavonoids that are accessible in the cranberry transcriptome. Further, the ABC transporters and glutathione S-transferases (GST), WD40, WRKY and bHLH regulatory transcription factors, involved in flavonoid biosynthesis were also found in this transcriptome [Rubus coreanus Miq. 1867 (rich in anthocyanins) were assessed by metabolomics and transcriptomics [Magnolia sprengeri Pampanini, and Nuovo Giorn with red and white flowers, revealed some key enzymes of the flavonoid biosynthetic pathway, such as phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, F3′H, F3H and CHS. The families of MYB, bHLH and WD40 transcription factors also regulate the anthocyanidin metabolic pathway. With FPKM investigation, eight of these genes for transcription factors associated with the transcript abundance of genes, metabolic processes, and the color of flowers showed an eightfold boost in expression level in Magnolia sprengeri Pampanini, and Nuovo Giorn red-flowered strain, compared to the white-flowered strain [Chlorophytum borivilianum Santapau & R.R.Fern., which is an endangered species, an adaptogen, antiaging agent and promoter of general health, the transcriptome analysis enabled insights into the molecular mechanism of flavonoid glycosylation [From transcriptome data of entified . Podophyentified . Simultaiversity ,101. Chlotential  due to cotential . The trablastine . From thblastine  and cytoblastine . Iridoidblastine ,107. Curdatabase . Phylotrdatabase . Rubiaceredicted . Withanicterized . The difcterized ,111. Theferum L. . Advancei et al.  found vaentified . In the modeling . The putmodeling . Then, ta Decne. . RNA-seqblastine ,117. In blastine . The RNAcriptome . The metiptomics . From thiptomics ,122,123.d strain . In Chlosylation . Transcrsylation . To concTripterygium wilfordii Hook. f. has been extensively and effectively utilized for the treatment of several human syndromes, for instance rheumatoid (RH) arthritis and skin psoriasis. The anticancerous value and intrinsic mechanisms of action of Tripterygium wilfordii Hook. F. have been examined, and at the proteomic level the effect of a bioactive metabolite diterpenoid epoxide triptolide in curing colon cancer has been demonstrated [Andrographis paniculata (Burm. F) also contains diterpene compounds that have medicinal applicability against various human disorders, such as cancer and hepatitis, and viral and pathogenic bacteria [Periploca graeca L., and can help fight cancers in the lungs and colon both in the laboratory and under natural conditions via the beta-catenin/TCF signaling pathway, by means of inducing apoptosis. With tandem mass spectrometry (TMS) and 2D gel electrophoresis, the outcome of periplocin’s action on the cell line A549 of lung cancer was studied. Western blot analysis validated and investigated the proteins subjected to modifications and protein–protein interactions [Curcuma longa L. has antioxidant, antineoplastic, anti-angiogenic and anticancerous value. With proteomics, the activity of curcumin in diverse cancerous cell lines was confirmed. The proteomic investigation differentiated twelve proteins with differential expression patterns that enhance functions such as transcription, glycolysis, RNA translation, the splicing of mRNA and lipid metabolism, the synthesis of proteins, protein folding and the degradation of proteins, amino acid synthesis, and the motility of cells in the MCF-7 cell line of human breast cancer [Coptis chinensis Franch., and its anti-proliferative properties lead to the arrest of the cell cycle at the G0 stage of mitosis and the apoptosis of cells [Garcinia hanburyi Hook. f. resin. It has revealed promising antitumor activity in clinical trials and hinders the growth of a range of cancer cells through multiple signaling pathways [Garcinia oblongifolia Champ. ex Benth contains the bioactive metabolite 1, 3, 6, 7-tetrahydroxyxanthone that restricts cell proliferation in hepatocellular cancerous cells via the upregulation of p16 and 14-3-3σ [The field of proteomics is a potent platform to investigate comprehensively the proteins regulated by the drugs and explore signaling pathways of cell perturbations. Proteomics has a range of functions in medicinal plant research. Proteomics illustrates the structures, functions and modifications of proteins, and the protein–protein interactions taking place under in vitro and in vivo conditions . The autnstrated . The metnstrated . Androgrbacteria . Proteombacteria . Periploractions . Curcumit cancer . The proof cells . Gambogipathways ,139,140. 14-3-3σ , and 1,  14-3-3σ , which p 14-3-3σ . In anot 14-3-3σ .Catharanthus roseus (L.) G. Don, the systematic analysis of the proteome was undertaken via 2D polyacrylamide gel electrophoresis. Mass spectrometry identified proteins such as strictosidine synthase and tryptophan synthase, involved in alkaloid biosynthetic pathway [Cannabis sativa L. with different levels of cannabinoids were performed by two-dimensional gel electrophoresis (2D-gel), followed by mass spectrometry. The counting of resolved gel spots on 2D-gel indicated the presence of at least 800 proteins in leaves and flowers. Less than 100 proteins expressed in the flowers were characterized by mass spectrometry [Artemisia annua L., and quantitative targeted sub-proteomic analysis of two chemotypes of high (HAP) and low (LAP) artemisinin content enabled identification of 13403 proteins on the basis of the genome sequence annotation database, and 182 proteins on the basis of mass spectrometry, respectively [Artemisia annua L. and its role in the production of artemisinin [Withania sominifera (L.) Dunal) has major secondary metabolites of withanolides. When resolved on the 2D-gels, Indian ginseng root tissue showed 56 unique spots, whereas 22 proteins were identified by MALDI-TOF/TOF [In  pathway . Differetrometry . The firtrometry . Global ectively . MS-baseemisinin . Kim et emisinin  reviewed-TOF/TOF .To conclude, the proteomics of medicinal plants provided information on the structure, function and post-translational modifications of proteins, protein–protein interactions, protein targets of plant-based bioactive drugs, protein–drug interactions at the molecular level, and the signaling pathways of cell perturbations. This provides insights into the mechanisms of plant-based medicines in tumor cells and a complex understanding of the inheritable traits and physiological status of plant. Proteins undergo post-transcriptional and post-translational modifications. Thus, proteomics data do not always complement transcriptomics data. Therefore, future efforts require integrated omics approaches to explore the biology of any medicinal plant .cells-1Metabolomics is mainly significant in the plant kingdom, because of the huge quantity of metabolites (primary and secondary) produced by plants . MetabolAloe vera (L.) Burm. f., an anticancer and antitumorigenic plant. The metabolome influences the genome of hepatocellular carcinoma cells by escalating the gene expressions of p53 and Bcl-2 [Panax ginseng C. A. Mey., Panax notoginseng (Burkill) F. H. Chen and Panax japonicus (T. Nees) C.A.Mey. by the UPLC-QTOF-MS technique identified secondary metabolites such as chikusetsu saponin Ⅳa and ginsenosides  [Persicaria minor (Huds.) Opiz syn. Polygonum minus Huds, 48 compounds with via GC × GCTOF MS, 42 compounds via GC-MS, 37 volatile compounds via GC-MS investigation, and 85 flavonoids via LC-TOF were successfully identified [Pulcaria crispa (Forssk.) Benth. ex Oliv. shows proficient in- vitro anticancerous action [Rubus coreanus Miq. [Metabolomics studies undertaken with the analytical technique of nuclear magnetic resonance spectroscopy are useful for compiling metadata in two diverse dimensions for the detection of any alterations . The metnd Bcl-2 . The metRg2, R0) . In Persentified ,166. Oveentified ,168. Thes action . Mass spnus Miq. . SeveralModern day omics-based technology allows extensive and nano-scale assessments of biological samples for the probing of compounds with medicinal properties. Traditional medicinal plants are very prolific in the discovery of novel plant-based drugs. Medicinal plants have unexploited potential for use in novel molecular target discovery, which adds to the process of drug development. It is imperative to manipulate and investigate not only at the genome level with high-throughput sequencing and recombinant DNA technology, but also at the levels of the proteome with mass spectrometry, the transcriptome with RNA Seq and the metabolome with nuclear magnetic resonance spectroscopy and/or LC-MS. In modern day biology, big data is derived from omics technologies, which allow the discovery of unidentified metabolic pathways/enzymes, metabolites, genes, gene networks, and protein–protein interactions, because of which interdisciplinary research on medicinal plant genomics accompanied by high-throughput sequencing platforms for DNA and RNA, metabolomics and proteomics is entirely essential. These studies will continue to disclose novel secondary metabolites that are biologically active and thus make the immense biological diversity of plants applicable for use in for novel drug discovery. The systems biology tool will help in identifying networks by which pharmacological substances of individual medicinal herbs or synergistic action networks of herbal prescriptions can be resolved and their target signaling pathway networks can be studied. However, the databases, computational models and infrastructure required to pool the disciplines are in their infancy. Systems biology will prove to be a revolutionary research area in plant-based drug discovery and diseases in future.


Neuroendocrine tumors (NETs) are neoplasms with variable malignant potential that may arise from neuroendocrine cells. The gastrointestinal tract is a common site for primary NETs and annually 1.3 for every 100,000 persons are newly detected . AlthougHere, we report the case of a 64-year-old man with jaundice who was diagnosed with small-cell NEC in the distal common bile duct (CBD).A 64-year-old man was referred from a local clinic with complaint of painless jaundice. The patient had past medical histories of diabetes mellitus, hypertension, chronic obstructive pulmonary disease, and gout. He had no surgical or family history of cancer.At admission, laboratory test results that present obstructive jaundice were as follows: total bilirubin 17.4 mg/dL, alkaline phosphatase 372 IU/L, carcinoembryonic antigen (CEA) 3.0 ng/mL, and carbohydrate antigen 19–9 235 U/mL. Magnetic response cholangiopancreatography revealed a focal wall thickening in the distal CBD and combined dilatation of the upstream bile duct . PositroThe patient underwent pylorus-preserving pancreaticoduodenectomy (PPPD) under a presumptive diagnosis of distal CBD cancer. In the resected specimen, 2-cm tumor tissue was located in the distal CBD . On micrNECs in the biliary system are very rare , 7, 8. Tet al. [To identify the characteristics of tumors and the clinical features, we reviewed the literature, in which 21 cases including our case were reported about the SCC from 2000 . All 21 et al.  reportedDespite the development of the imaging studies, the preoperative diagnosis of NEC is still difficult. Especially, cholangiocarcinoma presents very similar characteristics and morphology compared with NEC with various modalities including ultrasound, computed tomography, or magnetic resonance imaging. In the present study, the patient was also diagnosed with distal CBD cancer in preoperative imaging studies. Although a preoperative tissue confirmation through ERCP could be helpful, the risks of false-negative results always exist. Moreover, preoperative biopsy does not tell us whether the tumor is SCC or LCC. Measurement of serum chromogranin A is helpful to diagnose NET . ChromogAlthough WHO made the formal classification of NECs in extra-hepatic bile ducts, still little is known about the effective treatment strategy for this tumor due to its rare incidence and difficult preoperative diagnosis. Compared with the poor prognosis of conventional cholangiocarcinoma, NECs in extra-hepatic bile ducts have various clinical courses and prognoses with or without recurrences. So far, previous studies have reported quite different results about the prognosis of this tumor. Therefore, further studies with large cohorts from multiple institutions are needed to identify the clinical characteristics, to predict the true prognosis, and to establish the optimal management guidelines for this rare tumor.Gastroenterology Report online.H.I.S., J.R.K., and Y.P. conceived the study and participated in its design and coordination. J.R.S. and H.I.S. drafted the manuscript. All authors read and approved the final manuscript.None.goaa051_Supplementary_DataClick here for additional data file.


Psammolestes, a genus endemic of the SDTF and naturally infected with Trypanosoma cruzi (agent of Chagas disease), using a combination of phylogenetic, population genetics and niche model methods, and evaluated the reliability of the three morphospecies currently recognized.The evolutionary history of biodiversity in South America has been poorly studied in the seasonal dry tropical forest (SDTF). Species diversification in this ecosystem may have a twofold explanation. First, intermittent connections in the middle and late Pleistocene promoted species dispersal and/or genetic connectivity between lineages isolated in disjunct patches of forest. Second, allopatric speciation proceeded immediately after the formation and colonization of the SDTF in the Neogene. Here we studied the diversification of P. coreodes and P. tertius in a monophyletic clade sister to P. arthuri. Species delimitation tests recovered these lineages as different species despite the shared genetic variation observed between P. coreodes and P. tertius in five genes. Also, genetic variation of the genus clustered in three groups that were consistent with the three morphospecies. Our demographic model predicted a scenario of divergence in absence of gene flow, suggesting that mixed haplotypes may be the result of shared ancestral variation since the divergence of the subtropical-temperate species P. coreodes and P. tertius. In contrast, the tropical species P. arthuri was highly differentiated from the other two in all tests of genetic structure, and consistently, the Monmonier’s algorithm identified a clear geographical barrier that separates this species from P. coreodes and P. tertius.Our multilocus analyses recovered Psammolestes that diverged in absence of gene flow in the late Miocene. This result supports a scenario of species formation driven by geographical isolation rather than by divergence in the face of gene flow associated with climatic oscillations in the Pleistocene. Also, we identified the Amazon basin as a climatic barrier that separates tropical from subtropical-temperate species, thus promoting allopatric speciation after long range dispersion. Finally, each species of Psammolestes occupies different climatic niches suggesting that niche conservatism is not crucial for species differentiation. These findings influence the current vector surveillance programs of Chagas disease in the region.We found three genetically structured lineages within The online version contains supplementary material available at 10.1186/s12862-022-01987-x. The Andes uplift and the formation of the Amazon Basin promoted species diversification via vicariance and/or dispersal which may be associated with climatic oscillations. Many examples from multiple organisms show the effect of such geological events in species differentiation –6, but oPhyllopezus was not influenced by Pleistocene climatic oscillations, but show a high phylogenetic structure associated with Miocene geomorphology [Phacellodomus and arthropods such as Nephila or Drosophila gouveai seems to be a consequence of Pleistocene climatic variation [Species diversification in SDTF may be the result of these dry forest patches being intermittently connected during cold and dry periods in the middle and late Pleistocene, thus promoting species dispersal and/or genetic connectivity between isolated lineages  occurs in SDTF in apparent association with nests of Furnariidae birds [P. arthuri , P. tertius  and P. coreodes , whose ecology and behavior remain largely unknown [Psammolestes arthuri occurs across the eastern plains of Colombian and Venezuela, P. tertius is found in coastal regions near the Cerrado, Caatinga and the Mata Atlantica in Brazil, and P. coreodes distributes across the Chaco in Argentina, Paraguay, Bolivia, and Brazil [P. arthuri is the most easily recognizable species based on a smooth and shiny cuticle in the thorax and the head, lack of cervical constriction, long hairs restricted to the apex of the second and third segments of the stylet, an anterolateral pronotal margin distinctly extended, and male genitalia with basal plate struts completely fused [P. tertius and P. coreodes are recognized based on male genitalia morphology, anteocular distance, and post-ocular distance. Specifically, P. tertius has basal plate struts broadly S-shaped, while those of P. coreodes are hook shaped. Also, the anteocular distance in P. tertius is at least 2× higher than its post-ocular distance, while that of P. coreodes is always less than 2× [P. tertius and P. coreodes [The genus ae birds –33. This unknown . PsammolPinto, 19, P. tertd Brazil . These sd Brazil –37, but d Brazil . For exaly fused . In addi than 2× , 38. AddPsammolestes were initially grouped into the tribe Psammolestini and separated from Rhodniini [Psammolestes and Rhodnius were kept as separate genera as the femur and head of Psammolestes are wider and shorter than those of Rhodnius [Rhodnius is paraphyletic compared to Psammolestes [Psammolestes, and found P. arthuri sister to P. tertius and this clade sister to P. coreodes [. These findings suggest that Psammolestes is a monophyletic clade within the prolixus group [Species of hodniini , 39, buthodniini . NonetheRhodnius . These tmolestes , 40–44. coreodes . These fus group .Psammolestes remains to be tested. This is especially relevant as P. arthuri is a tropical species but P. tertius and P. coreodes have temperate distributions.Additionally, multiple studies have revealed a major role of niche conservatism in the diversification of the subfamily Triatominae –49. For Psammolestes and investigate the role of the niche in maintaining these species. Our hypothesis was that the Amazon basin acts as a dispersion barrier that separates tropical and subtropical-temperate species thus suggesting a major role of geomorphology events in the divergence of Psammolestes. This scenario predicts that: (i) P. coreodes and P. tertius are most closely related to each other than they are to P. arthuri, and (ii) species differentiation proceeds despite niche conservatism. The understanding of the biotic and abiotic processes that shape vector species diversity of tropical diseases, as well as, the factors involved in their speciation process are essential for the settlement of adequate strategies for disease transmission control [In this study, we used phylogenetic, population genetics analyses, and niche modeling to test the existence of discrete lineages within  control .P. coreodes and P. tertius as sister monophyletic clades, while 28S, CISP, LSM, TRNA and UPCA topologies  P. arthuri, and (ii) P. coreodes + P. tertius  from temperate species (P. coreodes and P. tertius). This geographic break was recovered in all genes, suggesting that the tropical P. arthuri diverged from the other two temperate species in allopatry  and nucleotide diversity (π) values were similar among the three ci Table . The throns Fig. . In agreies Fig.  and 18, ata Fig. , 20. Thilgorithm  supportsOur results suggest that the demographic model of ‘divergence without gene flow’ fitted our data better than other models with unidirectional or bidirectional gene flow. However, this scenario shows some uncertainty (wAIC = 0.30) as AIC values were not considerable different between models . This model showed different non-overlapping suitable areas for each species of tes Fig. . Overalluri Fig. . Consisted Table .Fig. 4EnP. coreodes, P. tertius and P. arthuri [P. coreodes and P. tertius are genetically more similar than they are to P. arthuri. The species distribution analyses suggest that these species are restricted to tropical savannas and have a low probability of occurrence in humid areas. These findings support a role for the Amazon basin as an absolute barrier for the dispersal of species of Psammolestes.We recovered three well supported lineages that are concordant with the previously described morphospecies and experimental crosses:  arthuri . Both phP. tertius and P. arthuri were recovered as sister species (bootstrap support = 66%), and this clade sister to P. coreodes (bootstrap support = 87%) [P. tertius and P. coreodes, sister to P. arthuri. This result is consistent both with the geographic and genetic distance between these taxa. Despite of the contentious systematics of the genus [Psammolestes thus supporting the original species description based on morphological traits [Our phylogenetic reconstruction contrasts with a previous study where t = 87%) . Howeverhe genus , 40–44, l traits , 35, 38.Psammolestes is not explained by recent dispersal events across corridors in the forested Amazon basin nor by the Pleistocene arc hypothesis [Psammolestes is the result of extinction in the Amazon basin. Interestingly, the diversification times of Psammolestes do not mirror those of Phacellodomus rufifrons (Furnariidae), a bird whose nests are commonly invaded by these kissing bugs [Psammolestes. Nevertheless, future studies are needed to understand the evolutionary importance of this peculiar association with Furnariidae birds, which seems to be exclusive to these Triatominae species.Our mtDNA divergence times estimation, the strong genetic structure we observed, and the absence of gene flow between species suggest that the diversification of pothesis . In contpothesis –55. Howeing bugs  and whosing bugs . TherefoPsammolestes occur in the SDTF, they have divergent niches shaped by different climatic predictors, indicating that niche conservatism does not play a role in the diversification of these triatomines. This finding agrees with previous studies that documented nonoverlapping niches for P. coreodes and P. tertius [P. tertius and P. coreodes [Our niche modeling results suggest that, although all species of  tertius , 32. Succoreodes . HoweverPsammolestes has three genetically structured species that also differ in their climate niches and morphology. They diverged in allopatry without gene flow, and their differentiation involved long distance dispersal event(s) across the Amazon basin . Further investigation is needed to elucidate the behavior and ecology of each species as well as the reproductive barriers maintaining their integrity. These findings are relevant in terms of understanding the transmission dynamics of Chagas disease and future improvement of vector control strategies in endemic countries.In summary, Psammolestes species, from 12 localities in Venezuela, Colombia, and Brazil . Outgroup selection was based on previous phylogenetic reconstructions, where Psammolestes was shown to be sister taxa to some of the prolixus group species (Rhodnius seems to be paraphyletic with respect to Psammolestes), including R. prolixus [We collected a total of 92 individuals of the three zil Fig. . We alsoprolixus . The samGenómica, evolución y biogeografía de especies del género Rhodnius: vectores de la enfermedad de Chagas” act number 007/2016.This study was submitted and approved by the ethics committee of Universidad del Rosario entitled “® Blood & Tissue kit, with modifications in the original protocol suggested by the manufacturer for extractions in insects [Psammolestes: Four new nuclear loci, tRNA Guanine (37) -N (1) methyltransferase (TRNA), Putative juvenile hormone inducible protein (PJH), Probable cytosolic iron sulfur protein assembly protein Ciao 1 (CISP), Lipoyl synthase, mitochondrial (LSM), along with the previously reported Uncharacterized Protein for Cell Adhesion (UPCA) [https://digitalinsights.qiagen.com). Sequence alignment per locus was performed using MAFFT [We extracted DNA from leg tissue, using the DNeasy insects . We ampln (UPCA) , 58 and n (UPCA)  and Cyton (UPCA) , 59  using maximum likelihood (ML) inference in IQ-Tree 2 [We reconstructed phylogenetic relationships among the three Q-Tree 2 . We seleQ-Tree 2  based onQ-Tree 2 , aBayes Q-Tree 2  and SH-aQ-Tree 2  with 10,Q-Tree 2 .Psammolestes species tree using multilocus coalescence species approach in BEAST2 v.2.6.6 with genes included in this study [We also estimated the is study , 71. We is study  and usedis study . The Logis study  tool wasis study , obtainiis study .Finally, we estimated divergence times using the mitochondrial locus CYTB in BEASTv.2.6.6 . We onlyPsammolestes lineages with two delimitation methods: The Bayesian Phylogenetics and Phylogeography method ), shallow population sizes ), deep divergence times ) and shallow divergence times ). Each analysis used 100,000 iterations per run, sampling every 2 iterations, and using 10% of the iterations in the chain as burn-in.We established the number of od  and theod . For thod , number of segregating sites (S), population substitution rate (θ), and nucleotide diversity (π) to characterize the genetic variability of each Psammolestes species in DNASP v6.12.03 [Psammolestes with a relative measure (FST) and two absolute ones . To evaluate deviations from panmixia, we implemented the Hudson permutation test [2 , Tajimand Rozas ) and Fu nd Rozas ), in ordnd Rozas  for eachnd Rozas .Psammolestes testing for isolation by distance implementing a Mantel test in the R package vegan [ST) and the geographical distances calculated in the package geosphere [adegenet [We explored the geographical diversification of ge vegan  and a lieosphere . Additioeosphere  in the Radegenet  using a http://taylor0.biology.ucla.edu/structureHarvester/; Evanno et al. [distruct [.Lastly, STRUCTURE v2.3.4  was implo et al. ). The reo et al.  and plotdistruct .Psammolestes species using four algorithms: Artificial Neural Networks  environmental conditions are not suitable for these taxa [Psammolestes species based on the combination of the four models produced by the previously mentioned algorithms. Two metrics were used to choose the model that best predicts the distribution of the taxa: The True Skill Statistic (TSS) and the area under the curve (AUC) of the receiver-operating characteristic (ROC) [Models were constructed using BIOMOD2 package  for eachks , Generaks , and Maks . We obt DataTri . As we dese taxa , 103. Anese taxa . Five enese taxa  because ese taxa . Algoritic (ROC) . Variablic (ROC) .Psammolestes species using R package humboldt [Schoener’s D statistic was calculated. This statistic goes from 0 to 1, meaning no overlap and full overlap respectively [We estimated the environmental niche equivalence between all pairs of humboldt . To do tectively . D statiectively . This waectively . We inteectively .Additional file 1. CYTB Phylogenetic reconstruction and Barrier test. (A) Phylogenetic reconstruction with the ML algorithm based on the mitochondrial marker CYTB (B) Barrier test algorithm based on molecular and geographical arrays. Bootstrap values on the internal nodes are shown in the following order: SH-aLRT/aBayes/ultrafast bootstrap support. Only nodes with bootstrap values higher than 60 are shown.Additional file 2. PJH Phylogenetic reconstruction and Barrier test (A) Phylogenetic reconstruction with the ML algorithm based on the nuclear marker PJH (B) Barrier test algorithm based on molecular and geographical arrays. Bootstrap values on the internal nodes are shown in the following order: SH-aLRT/aBayes/ultrafast bootstrap support. Only nodes with bootstrap values higher than 60 are shown.Additional file 3. 28S Phylogenetic reconstruction and Barrier test (A) Phylogenetic reconstruction with the ML algorithm based on the nuclear marker 28S (B) Barrier test algorithm based on molecular and geographical arrays (B). Bootstrap values on the internal nodes are shown in the following order: SH-aLRT/aBayes/ultrafast bootstrap support. Only nodes with bootstrap values higher than 60 are shown.Additional file 4. CISP Phylogenetic reconstruction and Barrier test (A) Phylogenetic reconstruction with the ML algorithm based on the nuclear marker CISP (B) Barrier test algorithm based on molecular and geographical arrays. Bootstrap values on the internal nodes are shown in the following order: SH-aLRT/aBayes/ultrafast bootstrap support. Only nodes with bootstrap values higher than 60 are shown.Additional file 5. LSM Phylogenetic reconstruction and Barrier test. (A) Phylogenetic reconstruction wit the ML algorithm based on the nuclear marker LSM (B) Barrier test algorithm based on molecular and geographical arrays. Bootstrap values on the internal nodes are shown in the following order: SH-aLRT/aBayes/ultrafast bootstrap support. Only nodes with bootstrap values higher than 60 are shown.Additional file 6. TRNA Phylogenetic reconstruction and Barrier test. (A) Phylogenetic reconstruction with the ML algorithm based on the nuclear marker TRNA (B) Barrier test algorithm based on molecular and geographical arrays. Bootstrap values on the internal nodes are shown in the following order: SH-aLRT/aBayes/ultrafast bootstrap support. Only nodes with bootstrap values higher than 60 are shown.Additional file 7. UPCA Phylogenetic reconstruction and Barrier test. (A) Phylogenetic reconstruction with the ML algorithm based on the nuclear marker UPCA (B) Barrier test algorithm based on molecular and geographical arrays. Bootstrap values on the internal nodes are shown in the following order: SH-aLRT/aBayes/ultrafast bootstrap support. Only nodes with bootstrap values higher than 60 are shown.Additional file 8. Bayesian inference of species tree based on multilocus data (A) Maximum clade credibility tree based on Bayesian inference of the seven genes used in this study. The values observed represent posterior probabilities. (B) Bayesian species tree from multilocus data.Additional file 9. Bayesian inference phylogenetics tree for the locus CYTB obtained in *BEAST. Horizontal purple bars illustrate the 95% HPD for the nodes’ divergence time. Branch with a posterior probability above 0.95 are showAdditional file 10. Posterior probabilities on nodes, calculated by the mPTP algorithm.Additional file 11. Heatmaps calculated for three different statistics: A) Fst, B) Dxy and C) Da for three species based on the molecular data obtained from the nuclear marker 28S.Additional file 12. Heatmaps calculated for three different statistics: A) Fst, B) Dxy and C) Da for three species based on the molecular data obtained from the nuclear marker CISP.Additional file 13. Heatmaps calculated for three different statistics: A) Fst, B) Dxy and C) Da for three species based on the molecular data obtained from the mitochondrial marker CYTB.Additional file 14. Heatmaps calculated for three different statistics: A) Fst, B) Dxy and C) Da for three species based on the molecular data obtained from the nuclear marker LSM.Additional file 15. Heatmaps calculated for three different statistics: A) Fst, B) Dxy and C) Da for three species based on the molecular data obtained from the nuclear marker PJH.Additional file 16. Heatmaps calculated for three different statistics: A) Fst, B) Dxy and C) Da for three species based on the molecular data obtained from the nuclear marker TRNA.Additional file 17. Heatmaps calculated for three different statistics: A) Fst, B) Dxy and C) Da for three species based on the molecular data obtained from the nuclear marker UPCA.Additional file 18. Demographic models created with Phylogeographic Inference Using Approximate Likelihoods (PHRAPL) to test the evolution of Psammolestes. (A) Divergence with no migration (B) Divergence with bidirectional migration between P. coreodes and P. tertius. (C) Divergence with bidirectional migration between P. tertius and P. arthuri. (D) divergence with bidirectional migration between P. tertius with P. coreodes, and P. tertius with P. arthuri. (E) Divergence with bidirectional migration between P. coreodes and P. arthuri. (F) Divergence with bidirectional migration between P. tertius with P. coreodes, and P. coreodes with P. arthuri. (G) Divergence with bidirectional migration between P. tertius with P. arthuri, and P. arthuri with P. coreodes (H) Divergence with bidirectional migration between the three Psammolestes species. Starting from this point, all of the demographic models include bidirectional migration between P. arthuri and the MRCA (most recent common ancestor) of P. tertius and P. coreodes. (I) Divergence with bidirectional migration between P. arthuri and the MRCA of P. tertius and P. coreodes. (J) Divergence with bidirectional migration between P. coreodes and P. tertius. (K) Divergence with bidirectional migration between P. tertius and P. arthuri. (L) divergence with bidirectional migration between P. tertius with P. coreodes and P. arthuri. (M) Divergence with bidirectional migration between P. coreodes and P. arthuri. (N) divergence with bidirectional migration between P. coreodes with P. tertius and P. arthuri. (O) Divergence with bidirectional migration between P. arthuri with P. coreodes and P. tertius. (P) Divergence with bidirectional migration between the three Psammolestes species. Support values for the demographic scenarios are shown under each figure.Additional file 19. Linear correlations of Isolation by distance (IBD) test. (A) 28S (B) CISP (C) CYTB (D) LSM (E) TRNA (F) UPCA (G) PJH.Additional file 20. Mantel’s test for isolation by distance (IBD) and linear correlation results. The result of the Mantel’s test is shown in the two first columns of the table, and the results of the Pearson’s correlation test correspond to the third column. The last two columns show the results of the linear correlation tested between geographical and genetic distances.Additional file 21. Fit of the demographic models tested in.Additional file 22. Individuals of Psammolestes species collected in this study.Additional file 23. Genes included in this study, the primers used to obtain their corresponding sequence, and the length of each one of them. “*” symbolizes a new marker used for the delimitation of the Psammolestes species.Additional file 24. GenBank accession numbers of seven loci analyzed in this study and samples origin included in this study.


Creptotrema was investigated using morphological and molecular tools, including data for Creptotrema creptotrema (type-species). Three new species, parasites of Brazilian fishes, are described: Creptotrema conconae n. sp. , Creptotrema schubarti n. sp.  and Creptotrema megacetabularis n. sp. ). The diagnosis of the genus was amended to include new features. The new species differ from each other mainly in terms of body shape, relative sucker size, and testes position. DNA sequences were obtained from Creptotrema spp. from Brazil, including 28S, ITS and COI. Genetic divergences among the new species and C. creptotrema varied from 2.1 to 5.2% (21–49 bp) for 28S, and 6.6 to 16.4% (21–45 bp) for COI. Phylogenetic analysis (28S) placed the newly generated DNA sequences of Creptotrema in a clade (Creptotrema clade sensu stricto) composed of C. creptotrema, the new species described herein, and all species previously described as Auriculostoma, revealing that Auriculostoma is best interpreted as a synonym of Creptotrema based on the principle of priority of zoological nomenclature. Creptotrema funduli, the single sequence of the genus previously available, was not grouped within the Creptotrema clade sensu stricto, suggesting the need for reevaluation of the taxonomic status of this species. Our results showed that Creptotrema represents a monophyletic genus of trematodes widely distributed across the Americas, which currently comprises 19 valid species of parasites of teleosts and anurans.Diversity of  Allocreadiidae Looss, 1902 includes relatively small digeneans that appear to be restricted to freshwater systems distributed across the Americas, Asia, and Europe , 24. TheThe delimitation of genera in Allocreadiidae is usually defined by the possession of a combination of four non-unique morphological characters: the number of muscular “papillae” in the oral sucker; posterior extent of the intestinal ceca; posterior extent of the uterus, and anterior extent of the vitelline follicles. This approach has led to several controversies, resulting in polyphyletic groups and controversial genera and subgenera .Creptotrema Travassos, Artigas & Pereira, 1928 was described to accommodate the type-species Creptotrema creptotrema Travassos, Artigas & Pereira, 1928 as allocreadiids presenting one pair of muscular ventrolateral papillae associated with the oral sucker and a uterus that does not reach the posterior region of the testes. Species of this genus parasitize freshwater teleosts belonging to the family Anostomidae in Brazil. There are currently seven valid species in this genus . Photomicrographs of the vouchers of C. funduli (CMNPA1989–0566) deposited in the Canadian Museum of Nature, C. lynchi (UNSM20251) deposited at the University of Nebraska State Museum, and Auriculostoma guacurarii Montes, Barneche, Croci, Balcazar, Almirón, Martorelli & Pérez-Ponce de León, 2021 deposited in the Museo de Ciencias Naturales de La Plata  were also examined.Vouchers of C. creptotrema [CHIOC 39702]; C. conconae n. sp. [CHIOC 39596 a–g]; C. schubarti n. sp. [CHIOC 39599] and C. megacetabularis n. sp. [CHIOC 39703]).To confirm morphological identification, each parasite specimen was mounted on a slide with 96% ethanol, covered with a coverslip, and photographed. Conspecific specimen vouchers  were moC. creptotrema, and specimens of the new species described herein: one specimen of Creptotrema conconae n. sp., one specimen of Creptotrema schubarti n. sp., and four specimens of Creptotrema megacetabularis n. sp.), using the DNeasy Blood & Tissue Kit , following the manufacturer’s protocol. Fragments of 28S rDNA, ITS rDNA, and COI mtDNA were amplified using the primers and cycling conditions presented in Genomic DNA was extracted from the species described herein , Dicrocoelium Dujardin, 1845 (Dicrocoeliidae), Degeneria Campbell, 1977, and Phyllodistomum Braun, 1899 (both Gorgoderidae), used as the outgroup. The second contained the newly generated COI mtDNA sequences of C. creptotrema and the three new Creptotrema spp., along with 16 published sequences of the Allocreadiidae retrieved from GenBank and Phyllodistomum parasiluri Yamaguti, 1934, and Dicrocoelium spp. as the outgroup  model and a bootstrap procedure with 1000 replicates in the MEGA7 program , 32.Allocreadiidae Looss, 1902Creptotrema Travassos, Artigas & Pereira, 1928Type species, host, and locality:Creptotrema creptotrema Travassos, Artigas & Pereira, 1928 from Megaleporinus obtusidens  (= Leporinus elongatus), Cachoeira de Emas , Pirassununga municipality, São Paulo State, Brazil.Other species:Creptotrema funduli Mueller, 1934 (species inquirenda); Creptotrema macrorchis  comb. n.; Creptotrema platense  comb. n.; Creptotrema stenopteri  comb. n.; Creptotrema lynchi Brooks, 1976; Creptotrema paraense Vicente, Santos & Souza, 1978; Creptotrema pati Lunaschi, 1985; Creptotrema astyanace  comb. n.; Creptotrema lamothei Curran, 2008; Creptotrema sucumbiosa Curran, 2008; Creptotrema diagonale  comb. n.; Creptotrema foliaceum  comb. n.; Creptotrema totonacapanense  comb. n.; Creptotrema lobata  comb. n.; Creptotrema tica  comb. n.; Creptotrema guacurarii comb. n. ; Creptotrema conconae n. sp. (present study); Creptotrema schubarti n. sp. (present study); Creptotrema megacetabularis n. sp. (present study).Amended diagnosis: Body small, round to elongate or lanceolate, dorsoventrally flattened. Slight constriction at prepharyngeal level (neck region) present or absent. Tegument smooth. Remnants of eyespots present  or absent. Oral sucker subterminal, rounded or funnel-shaped with variable morphology . Small dome-like papillae over the margin of oral sucker (around 19–21) and ventral sucker (around 6), with a variable arrangement. Ventral sucker round, pre-equatorial to equatorial, with similar size or larger than oral sucker. Prepharynx short or absent. Pharynx subspherical. Esophagus short to moderately long. Intestinal ceca blind, bifurcating at anterior margin of ventral sucker; ceca end immediately or well posteriorly beyond testes. Genital pore median, at level of intestinal bifurcation or slightly anterior or posterior, equidistant between ventral sucker and cecal bifurcation. Seminal vesicle bipartite; pars prostatica and cirrus present. Cirrus unarmed, eversible. Cirrus-sac well-developed, long, usually forming several loops, sometimes overlapping ventral sucker or reaching the ovary posteriorly. Testes in posterior half of body, oval or elongate, entire or slightly lobed, tandem, symmetrical or oblique, intra or extracecal. Ovary posterolateral to ventral sucker, slightly pre-equatorial to equatorial, pretesticular, entire to lobed, submedian. Seminal receptacle posterior to ovary. Laurer’s canal with dorsolateral opening. Vitelline follicles lateral, exclusively extracecal or extra- and intracecal, with large or small follicles extending from level of oral sucker to posterior end of body, completely separated into two fields or confluent (scarcely or not) in post-testicular region. Uterus pretesticular, between anterior testis and genital pore, but loops may extend to the post-testicular region; intra- or extracecal. Eggs operculate, unembryonated, without spines or filaments; small knob opposite to operculum present or absent. Excretory pore terminal at posterior region of body; excretory vesicle I-shaped to claviform, reaching to anterior testis. Intestinal parasites of freshwater fishes and anurans in the Americas.Remarks: Travassos et al.  μm long versus 1254 [1084–1439] μm long in C. guacurarii comb. n.), and the body shape is usually oval (versus well elongated in C. guacurarii comb. n.). The testes of Creptotrema schubarti n. sp. are symmetrical or slightly oblique, intercecal, but may overlap the cecal region, while in C. guacurarii comb. n., they are clearly oblique and strictly occupy the intercecal region. In Creptotrema schubarti n. sp., the uterus extends between genital pore and may exceed the testes region, and the loops may extend into the extracecal area; in C. guacurarii comb. n., the uterus extends between the genital pore and mid-level of testes, with loops extending into pre- and inter-testicular regions, intercecal. The post-testicular space length is longer in C. guacurarii comb. n., representing 27 (25–30) percent of body length, while in the new species this space represents approximately 15% of the body length. Both species presented few eggs; however, they were smaller in the new species (55  and OK075291 [completely developed adult]), one of C. conconae n. sp. (GenBank accession number OK075288), one of C. schubarti n. sp. (GenBank accession number OK075293), and one of C. megacetabularis n. sp. (GenBank accession number OK075289).We successfully obtained eight partial sequences of the 28S rDNA: two of ss indicated no saturation in either the transitions or transversions; the critical index of substitution saturation (Iss.c) values were above the Iss values.The 28S rDNA final alignment was 1038 bp long. The IAcrolichanus auriculatus  Ward, 1917, parasites of sturgeons (Acipenseriformes) from the Holarctic region , and the other included all the other sequences of allocreadiids from Neotropical and Holarctic regions . The clade composed of allocreadiids from the Neotropical region comprised sequences of the genera Creptotrematina, Wallinia, Creptotrema , Paracreptotrema, Paracreptotrematoides, and Pseudoparacreptotrema; the North American Megalogonia ictaluri Surber, 1928 appears as a sister taxon of this clade. Creptotrema funduli from Mississippi (USA) is basal to the clade composed of allocreadiids from the Neotropical region and M. ictaluri.The 28S rDNA ML and BI phylogenetic analyses presented identical topologies, with most clades being well supported. Both analyses also recovered the Allocreadiidae as monophyletic. In the final 28S rDNA phylogenetic reconstruction , two maiCreptotrema spp. formed a clade referred to as Creptotrema sensu stricto , with Wallinia spp. recovered as its sister group. The Creptotrema sensu stricto clade included the sequences of the type-species C. creptotrema, the sequences of the new species described herein, and all sequences previously deposited in the GenBank as Auriculostoma spp.. Creptotrema funduli, the only sequence of the genus previously deposited in GenBank, was not positioned within the Creptotrema sensu stricto clade, suggesting that this species might not belong to Creptotrema and should be reevaluated  . Creptotrema megacetabularis n. sp. (from siluriforms belonging to Auchenipteridae) appeared as a sister taxon in this group. Creptotrema conconae n. sp. (the only species that parasitizes heptapterids) appeared as a sister clade of this group, clustering together with C. astyanace comb. n. and C. lobata comb. n.Among the Crepidostomum spp. were distributed in three different clades, one of them well-supported  and comprised of sequences of Crepidostomum metoecus  (type-species), Crepidostomum farionis  Lühe, 1909 and Crepidostomum oschmarini Zhokhov & Pugacheva, 1998, parasites of salmoniform (C. metoecus and C. farionis) and cypriniform (C. oschmarini) fishes, and referred to herein as the clade Crepidostomum sensu stricto. Another clade  was positioned as a sister group of Margotrema spp. from Mexico and was composed of Crepidostomum auritum  and Crepidostomum cornutum  Stafford, 1904, both parasites of perciform fishes from Mississippi, USA. The last clade  included Crepidostomum affine Tkach, Curran, Bell & Overstreet, 2013 and Crepidostomum illinoiense Faust, 1918, both parasites of hiodontiform fishes from the USA ( the USA .C. creptotrema (type-species) varied from 2.1 to 5.2% (21–49 bp); among Creptotrema spp. (except C. funduli) and species previously described as Auriculostoma, it varied from 0.4% to 5.0% (6–49 bp); from Creptotrema spp. (including the new sequences and Auriculostoma spp. reassigned herein) and Wallinia spp. (its sister clade), it varied between 2.9% and 6.9% (39–63 bp). There was no intraspecific genetic divergence within the sequences of C. creptotrema and C. megacetabularis n. sp. that presented differences between their morphology. For the other species of this genus, only one sequence per species was available; therefore, intraspecific comparisons could not be inferred. See Supplementary Table S1 for information on the genetic divergence values using the 28S rDNA gene.For the partial 28S rDNA, the interspecific genetic divergences found among the sequences of the new species and ss indicated no saturation in either the transitions or transversions, and the critical index of substitution saturation (Iss.c) values were above the Iss values.The final COI mtDNA alignment was 352 bp long. The ICreptotrema spp. sequences grouped together in a well-supported monophyletic clade  and were positioned as a sister group of Wallinia chavarriae Choudhury, Daverdin & Brooks, 2002 . Creptotrema schubarti n. sp. appeared to be the most closely related species to C. creptotrema , although poorly supported in the BI analyses  (see Supplementary Table S2). No intraspecific genetic divergence was observed between the three C. creptotrema sequences; for the other species of this genus, only one specimen was sequenced.For the mtCOI gene, the genetic divergences between all e.g., Crepidostomum, Paracreptotrema, Megalogonia, and herein demonstrated for species of Auriculostoma and Creptotrema), their relationships, and host-parasite associations [Information regarding the species diversity of allocreadiids from Neotropical freshwater fishes has increased recently as a result of integrative taxonomic approaches involving morphological and phylogenetic analyses . Howeverciations , 25, 49.Creptotrema deposited in museums and the phylogenetic positioning among isolates provided in this study allowed us to formally describe three new species and to amend the diagnosis of Creptotrema. We observed intraspecific morphological variation among specimens of C. creptotrema collected in this study, C. conconae n. sp., and C. megacetabularis n. sp. However, the absence of intraspecific genetic divergence between the sequences of the different morphotypes of each species , reinforces the hypothesis that these morphological differences represent distinct stages of ontogenetic development of these species.The accurate morphological analyses of type-specimens of Creptotrema schubarti n. sp. and C. guacurarii comb. n. , the set of morphological differences presented herein supports the erection of the new species. These are the only sequences of the Creptotrema clade sensu stricto generated from parasites of fishes belonging to the genus Characidium from Brazil and Argentina, respectively, which may have contributed to the low divergence observed. The 28S rDNA represents a highly conserved fragment and is not an ideal molecular marker for the delimitation of species or for making inferences about interspecific divergences. Unfortunately, to date, there are no other deposited sequences of C. guacurarii comb. n. from other molecular markers useful for species delimitation, such as the mtCOI gene, which would enable a more precise analysis of molecular divergences between them. However, a comprehensive and meticulous analysis of the morphological features of both species provided important evidence for their delimitation.Although we have found low interspecific genetic divergences between the partial 28S rDNA sequences of Auriculostoma as a synonym of Creptotrema, and supports the monophyly of the clade Creptotrema sensu stricto. Based on our morphological analyses, it is possible to affirm that the morphology of the oral sucker and muscular lobes (“auricles”) is highly variable among Creptotrema spp. , and represents a shared feature among other “papillose” allocreadiids as a result of convergent evolution. Therefore, this character should not be considered sufficient to delimitate and distinguish species of Creptotrema or genera of “papillose” allocreadiids, since this character arose or was secondarily lost several times during the evolutionary history of the group [The molecular evidence demonstrated herein identified he group . We recoResults section), the sequence of C. funduli was not grouped within the Creptotrema sensu stricto clade. Considering that this sequence was not sourced from the type-host or type-locality [Creptotrema and may represent a new genus yet to be properly described, corroborating the paraphyly of the genus recently proposed by Pérez-Ponce de León et al. [C. funduli is proposed herein as species inquirenda; future studies must be conducted to resolve its taxonomic status.According to our 28S rDNA phylogenetic inference  on the dorsal surface of the oral sucker. Considering this morphological feature and the fact that C. mulleri was described from specimens collected in salmonids (trout) from Turkey, its supposed relationship with Creptotrema spp. was not accepted in this study.farionis . Manter farionis  examinedCreptotrema is now one of the most widely distributed genera of trematodes across the Americas, represented by 19 valid species of parasites of freshwater teleosts and anurans , and South America seems to be an important center of diversification for these allocreadiids. Excluding the nominal species C. funduli, which requires a taxonomic and systematic review, it is possible that the Neotropical species of Creptotrema (Creptotrema sensu stricto clade) comprises a separate monophyletic group as suggested by Choudhury et al. [Creptotrema spp. are preferentially freshwater fishes belonging to multiple orders , excluding C. lynchi as originally described in toads, there seems to be a close relationship between this lineage of digeneans and characiforms, in which 10 of the 19 valid species have been reported , indicating the need for supplementary studies regarding their morphology and new molecular data to investigate whether they may belong to the same genus or if they may be erected in a new genus.niforms  and molecular studies . Our results describe new species, molecular data, and important information, increasing our understanding of the relationships among allocreadiids, especially Creptotrema, contributing substantially to clarifying the phylogeny of this genus.Considering that South America is exceedingly rich in freshwater fish fauna , less tThe authors declare that they do not have any conflict of interest.https://www.parasite-journal.org/10.1051/parasite/2021065/olmThe supplementary material of this article is available at Table S1. Nucleotide divergence (p-distance expressed in %) estimated for the 28S rDNA among Creptotrema spp. and selected digeneansTable S2. Nucleotide divergence (p-distance expressed in %) estimated for the mitochondrial cytochrome c oxidase I (COI mtDNA) among Creptotrema spp. and selected digeneansThe authors would like to thank FAPESP (The São Paulo Research Foundation), CAPES (Coordination for the Improvement of Higher Education Personnel), CNPq , and Pro-Rectory of Research (PROPe – UNESP) for financial and scientific support, and the post-graduate scholarships granted to L.F. (CAPES/PNPD 17/2016); A.A. (FAPESP 2018/25554-2); A.C.Z. (FAPESP 2016/07829-9); P.O.F.Y. (FAPESP 2013/25786-7); and M.B.E (CNPq 140873/2017-1); R.J.S. is supported by FAPESP (2016/50377-1) and CNPq .


Biofilms are intricate bacterial assemblages that attach to diverse surfaces using an extracellular polymeric substance that protects them from the host immune system and conventional antibiotics. Biofilms cause chronic infections that result in millions of deaths around the world every year. Since the antibiotic tolerance mechanism in biofilm is different than that of the planktonic cells due to its multicellular structure, the currently available antibiotics are inadequate to treat biofilm-associated infections which have led to an immense need to find newer treatment options. Over the years, various novel antibiofilm compounds able to fight biofilms have been discovered. In this review, we have focused on the recent and intensively researched therapeutic techniques and antibiofilm agents used for biofilm treatment and grouped them according to their type and mode of action. We also discuss some therapeutic approaches that have the potential for future advancement. Biofilms are multicellular clusters of microbes that adhere to various surfaces using an extracellular polymeric substance (EPS) made up of proteins, polysaccharides, or extracellular DNA (eDNA) . The EPSStaphylococcus epidermidis and Staphylococcus aureus), followed by Pseudomonas aeruginosa . Th. Th28]. S. aureus medical device associated biofilm development at concentrations safe in humans  pyrimidines (compounds 19 a–c) and found that it was able to inhibit Gram-positive bacteria including s luteus . Remarkand death ,84.E. coli. In a screening of six plant and animal derivatives of indole, indole-3-carboxaldehyde and 3-indolylacetonitrile were found to be potential biofilm inhibitors against P. aeruginosa and E. coli O157: H7. These compounds decrease biofilms by reducing curli production without affecting microbial growth . . Citrus Penicillium spp isolated from the sponge Axinella corrugata which inhibited biofilm formation by S. epidermidis [Streptomyces gandocaensis that were the inhibitors of Acinetobacter baumannii biofilms [Since marine organisms are a rich source of novel bioactive metabolites, studies on marine fungal and bacterial secondary metabolites have been gradually growing for the development of novel therapeutic agents . For exadermidis . Similarbiofilms . Thus, sSeaweed secondary metabolites such as phlorotannin possess antibacterial properties. Several in vivo studies and clinical trials exist on the health benefits of Phlorotanin. However, these studies were not based on the antibiofilm properties of phlorotannin ,97. The Antimicrobial peptides (AMPs) are a type of innate defense mechanism in different eukaryotes which was first discovered by Kiss and Michl in the 1960s. AMPs are cationic and hydrophobic residues containing molecules that can interact with various microorganisms such as bacteria, fungus, protozoa, and some enveloped viruses ,99,100. S. aureus adhesion to the polystyrene surfaces and altered the cell as well as polystyrene surface hydrophobicity. These AMPs also changed the biofilm-related gene expressions in planktonic cells [P. aeruginosa biofilm formation. It was likely achieved by inhibiting the transcription of Las and Rhl QS systems [P. aeruginosa by upregulating the expression of genes needed for type IV pili biosynthesis and function [L. monocytogenes and Gram-negative pathogens Burkholderia cenocepacia and P. aeruginosa. Peptide 1037 directly reduced biofilm production by decreasing swarming and swimming motilities, inducing twitching motility, and downregulation of several genes related to biofilm production [AMPs such as nisin and bovicin HC5 reduced the ic cells . On the  systems ,106. LL-function . LikewisE. coli, A. baumannii, P. aeruginosa, Klebsiella pneumoniae, MRSA, S. Typhimurium, and Burkholderia cenocepacia [P. aeruginosa to a greater amount than 1018 [Fuente Nunez et al. found an effective anti-biofilm peptide 1018 which could bind and degrade (p)ppGpp, a crucial signal required for the formation of biofilm . Peptideocepacia . D-enanthan 1018 . E. coli DNA gyrase by trapping an enzyme-DNA cleavable complex [Apidaecin, pyrrhocoricin, and drosocin are 18–20 amino acid, proline-rich residues that were initially obtained from insects. These AMPs attack a target microbial protein in a stereospecific manner. They interact with the bacterial heat-shock protein DnaK by inhibiting chaperone-assisted protein folding and limiting the DnaK ATPase activity ,109,110. complex . PR-39,  complex .P. aeruginosa PAO1 inside the biofilms and lead to a discharge of β-galactosidase. However, this impact on biofilm cells was slower when compared to the planktonic cells [P. aeruginosa biofilm [S. aureus biofilm cells and caused the ATP flux from the cells [Some antimicrobial peptides polarize and permeabilize the cell membrane and finally cause membrane disruptions. Esculentin (Esc (1–21) is the AMP derived from the frog skin. It could breach the cytoplasmic membrane of Gram-negative bacteria for example ic cells . Similaric cells . Another biofilm . Bacterihe cells .Streptococcus mutans that resulted in decreased biofilm formation on polystyrene and saliva-coated hydroxyapatite [S. epidermidis by inhibiting PIA [icaA and icaD, and at the same time upregulated icaR genes of S. epidermidis thus decreasing PIA production resulting in biofilm inhibition [Several AMPs also target biofilm EPS components. AMPs such as P1 could destroy biofilm EPS formed by yapatite . Anotherting PIA . Similarhibition . As all hibition .A few antibiofilm peptides have even been used in treating biofilms in animals and agriculture. Since antibiofilm peptides have a complex mechanism of action, improving our understanding of these mechanisms in biologically relevant situations appears to be important for determining structure–function associations and finally optimizing synthetic antibiofilm peptides for improved antibiofilm potential .B. subtilis [E. coli, UPEC strain) by using mass spectrometry-based targeted and untargeted metabolomic methods, together with cytological imaging, that enabled in identifying the targeted metabolites and related metabolic pathways involved in biofilm development [S. aureus , and DNase I is a popular antibiofilm strategy . The damruginosa . Polyami. aureus ,48,126. . aureus . Similar in vivo . A simil. aureus . Thus, ES. aureus [Bacteriophage therapy has been used for over 50 years to treat bacterial infections. Recent experimental and clinical studies have demonstrated a remarkable impact on treating both wound biofilm infections as well as device-related infections . The exi. aureus . It is p. aureus . One of . aureus .luxS gene and fimbriae-associated gene (fimH) using the CRISPRi technology for regulating biofilm-associated infections [ompA (outer membrane protein A) gene knockout in a dominant mosquito microbiota Cedecea neteri [ompA mutant displayed a reduction in biofilm formation capability, significantly decreased infection in adults, and was also found to be less prevalent in adults [Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9 system) is a novel technology that has been applied for genomic editing in some prokaryotes and eukaryotes . Most refections ,133. Sima neteri . The ompn adults . Thus, CS. aureus [E. coli IHF can bind to DNABII in many bacterial species and destabilize biofilms by releasing individual bacterium. DNA II, when combined with antibiotic therapy, showed a synergistic effect in treating biofilms of numerous bacterial species such as oral bacteria [P. aeruginosa [S. aureus [Haemophilus influenzae (NTHi) showed early NTHi eradication and prevention of disease [agr signal disruption could be effective against S. aureus SSTI [Antibiotic-resistant species also evolve to colonize and evade host immunity. The approach of targeting biofilms using vaccines poses numerous difficulties in targeting bacterial biofilms since vaccines are specific to a particular microorganism and show large variability in the expression of vaccine-targeted epitopes . Neverth. aureus . The DNA. aureus . Studiesbacteria , UPEC [1bacteria , P. aeruruginosa , and S. . aureus ,147 in a. aureus . The nex disease . Alternaeus SSTI ,136. Beteus SSTI .Pathogenic biofilms formed on implantable medical devices (IMDs) or human tissues have caused a huge risk in global healthcare. Functionalized biomaterials could be a novel approach to combat and eradicate pre-existing biofilms. Adherence of bacteria is the initial stage of IMD-associated infections that enables bacteria to colonize in the implants. The inhibition of biofilms could be achieved by coating the implants with biomaterials with antifouling and antibacterial properties. Biofilm eradication could be achieved by using nanoparticle (NP)-coated drugs to disperse biofilms .NPs are regarded as a substitute for antibiotics for combating multidrug-resistant and biofilm-associated infections . The bioS. mutans virulence . The study also proposed that CaF2-NPs could also suppress the enzymatic activities related to cell adhesion, glucan synthesis, acid production and tolerance, and quorum sensing that result in biofilm inhibition [Naturally forming and engineered NPs could differ largely in their physicochemical properties including size, shape, and charge . For thehibition P. aeruginosa PAO1, which suggests NPs-delivered natural compounds can be efficiently used in biofilm-related infection [NPs such as AuNPs (gold NPs) were also developed in combination with hordenine. Hordenine-AuNPs displayed greater antibiofilm properties on nfection . Most renfection . CARs utnfection . Nitric oxide-releasing nanoparticles (NO NPs) employ various simultaneous antimicrobial mechanisms, so the chances that microbes will develop resistance to NO NPs is low . SchaireS. mutans biofilms-related infections [S. aureus biofilm bacteria in vivo than that of PTT or PDT alone [Photodynamic therapy (PDT) has been utilized for treating several types of viral, fungal, bacterial, protozoan, or even parasitic infections in recent years. It is known that PDT has effectively decreased the clinically important drug-resistant Gram-negative and Gram-positive bacteria . PDT confections . Li et aDT alone . The detection and extraction of newer compounds are now more convenient with the accessibility of innovative techniques . These nCancer cells and bacterial cells share some similar traits, such as virulence high replication rates, modes of dispersal within the host, and rapid development of drug resistance . Cancer Synthetic biology (SB) is a promising interdisciplinary research field that could be used to design and construct newer fabricated devices, artificial metabolic pathways, and organisms, or as well as reform current natural and biological systems, targeting critical problems in health, materials, energy, and the environment . Using s


Hepatocellular carcinoma (HCC) is one of the most burdened tumors worldwide, with a complex and multifactorial pathogenesis. Current treatment approaches involve different molecular targets. Phytochemicals have shown considerable promise in the prevention and treatment of HCC. We investigated the efficacy of two natural components, 1,8 cineole (Cin) and ellagic acid (EA), against diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) induced HCC in rats. DEN/2-AAF showed deterioration of hepatic cells with an impaired functional capacity of the liver. In addition, the levels of tumor markers including alpha-fetoprotein, arginase-1, alpha-L-fucosidase, and ferritin were significantly increased, whereas the hepatic miR-122 level was significantly decreased in induced-HCC rats. Interestingly, treatment with Cin (100mg/kg) and EA (60mg/kg) powerfully restored these biochemical alterations. Moreover, Cin and EA treatment exhibited significant downregulation in transforming growth factor beta-1 (TGF-β1), Fascin-1 (FSCN1), vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), and epithelial-mesenchymal transition (EMT) key marker, vimentin, along with a restoration of histopathological findings compared to HCC group. Such effects were comparable to Doxorubicin (DOX) (2mg/kg); however, a little additive effect was evident through combining these phytochemicals with DOX. Altogether, this study highlighted 1,8 cineole and ellagic acid for the first time as promising phytochemicals for the treatment of hepatocarcinogenesis via regulating multiple targets. Hepatocellular carcinoma (HCC) is the fourth main cause of cancer-related mortality, creating a large global cancer burden , 2. UnfoEllagic acid (EA) is a natural phenolic constituent present in berries, walnuts, grapes, pomegranates, black currents, and dried fruits . EA is rWe aimed to assess the potential anticancer effect of EA and Cin alone and when combined with the standard chemotherapeutic agent, Doxorubicin, in an in-vivo model of rat HCC. Furthermore, we initially shed light on their possible effects on EMT and its regulators.Diethylnitrosamine (DEN) (#bN0756), ellagic acid (#bE2250), doxorubicin hydrochloride (#bD1515), 1,8 cineole (#bC80601), acetylaminofluorene (2-AAF) (#bA7015) were purchased from Sigma-Aldrich Co.Group 1  rats were fed with a standard diet, i.p. injected once weekly with saline, and served as a negative control. Group 2 (Induced-HCC) rats with DEN/2-AAF as described below and served as untreated HCC animals. Groups 3–5  were DEN/2-AAF induced HCC rats treated with daily administration of ellagic acid (60 mg/kg p.o.) Reviewers' comments:https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  16 Aug 2021Additional Editor Comments:• Well performed and well written manuscript. But language is the major issue and i suggest to send the manuscript for language editing company for correction.Response: We appreciated the editor's comment. Accordingly, we sent the manuscript to a language editing company and the proof was attached.Journal Requirements:1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfResponse: We respect the journal requirements, and the corrections have been done.2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Response: We appreciated the reviewer's comment. We revised the whole references list and added four references to be a final of 60. All the changes have been done to meet the journal requirement.The added references are listed below:1. Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Pineros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941-53. Epub 2018/10/24. doi: 10.1002/ijc.31937. PubMed PMID: 303503102. Sobreira Dantas Nobrega de Figueiredo FR, Monteiro AB, Alencar de Menezes IR, Sales VDS, Peticia do Nascimento E, Kelly de Souza Rodrigues C, et al. Effects of the Hyptis martiusii Benth. leaf essential oil and 1,8-cineole  on the central nervous system of mice. Food Chem Toxicol. 2019;133:110802. Epub 2019/09/08. doi: 10.1016/j.fct.2019.110802. PubMed PMID: 31493462.3. Abdel-Hamid NM, Abass SA, Mohamed AA, Muneam Hamid D. Herbal management of hepatocellular carcinoma through cutting the pathways of the common risk factors. Biomed Pharmacother. 2018;107:1246-58. Epub 2018/09/28. doi: 10.1016/j.biopha.2018.08.104. PubMed PMID: 302573394. Choudhari AS, Mandave PC, Deshpande M, Ranjekar P, Prakash O. Phytochemicals in Cancer Treatment: From Preclinical Studies to Clinical Practice. Front Pharmacol. 2019;10:1614. Epub 2020/03/03. doi: 10.3389/fphar.2019.01614. PubMed PMID: 321166653. In your Methods section, please provide additional information on the animal research and ensure you have included details on : (1) how often the animal health was monitored (2) whether any animals died during induction of hepatocarcinogenesis, and if so, how many were found dead? (3) specific criteria of how animal health was monitored.Response: We are thankful for the reviewer's accuracy and in order to respond to the reviewer comment, we added the specific criteria of animal health and the number of dead animals during induction of hepatocarcinogenesis in the method section.(1) The general health conditions of animals concerning food consumption, water intake, and body weight were observed during all the experimental time besides signs of distress including respiratory changes, body temperature, or even unusual behavior were also observed. We did not notice a significant variation in the previous parameters between the experimental groups except for the DEN/2-AAF group that showed a modest weight loss compared with the others .(2) Throughout the initiation phase of DEN treatment (first eight weeks), only one rat was dead. After starting 2-AAF administration (promotion phase), another two rats from the DEN/2-AAF induced HCC group died .https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at plosone@plos.org if you have any questions.In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at Response: We understand the journal requirement and accordingly we uploaded a separate PDF file with the raw data of the original uncropped blot images. 5. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.Response: We respect the reviewer's comment. So, the ethics statement is only now in the method section.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 11 Oct 20211,8 cineole and ellagic acid inhibit hepatocarcinogenesis via upregulation ofMiR-122 and suppression of TGF-β1, FSCN1, vimentin , VEGF, and MMP-9PONE-D-21-18911R1Dear Dr. Rashna M. AllamWe’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Vikas Kumar Academic EditorPLOS ONE