Synodontis  from the Nile River and Lake Turkana revealed the presence of both S. schall-like and S. frontosus-like morphotypes, with a phenotypic gradient between them. We concluded phylogenetic and population genetic analyses based on two mitochondrial and one nuclear marker including 131 coxI (565 bp), 96 cytb (973 bp) and 19 RAG2 (896 bp) sequences from the Nile-Turkana population, plus additional GenBank data of Synodontis spp. Whilst nuclear data were inconclusive, mitochondrial sequences suggested that both morphotypes and intermediate forms are conspecific. The results imply probable synonymy of S. frontosus with S. schall. Conversely, a strong biogeographical signal was revealed among widely distributed and supposedly conspecific S. schall-like catfish of the Nilo-Sudanian ichthyological province. Synodontis schall sensu stricto (=Eastern clade), as defined by type locality in the Nile, is apparently restricted to the eastern part of the Nilo-Sudanian ichthyological province . Synodontis schall Western clade  most probably represents a cryptic taxon, unrecognized thus far due to the absence of distinctive morphological differences.Cryptic genetic diversity and erroneous morphological species determination represent frequent problems in biodiversity research. Here, examination of 138 specimens of  Traditionally, cryptic species/diversity in fishes is mostly discovered in highly variable taxa, especially in the species rich lineages of ray-finned fishes, such as the neotropical Characidae5 or Cichlidae7. Cryptic diversity was also found - albeit less frequently - in other lineages, e.g. spiny eels8 and Neotropical11 and African catfish13, including the family Mochokidae16. Generally, the main contributing factor to the undiscovered cryptic diversity of some species is the lack of clear distinguishing morphological features in combination with the absence of relevant molecular data3. This not only underestimates the species richness but also limits our understanding of species geographical distribution.Determining and identifying independent lineages and species is critical not only for taxonomy, but also for understanding the processes leading to diversification and the origin of population structure. Due to the extensive sampling, discovering cryptic species within the scope of population genetic studies is not a rare phenomenonSynodontis specimens. Two of three Synodontis spp. in our sample, Synodontis nigrita Valenciennes, 1840 and Synodontis serratus Rüppell, 1829 (both absent in Lake Turkana) could be readily morphologically determined. The third species Synodontis schall  showed deviation from both, the data in the literature, as well as the differential diagnosis and neotype proposed recently by Musschoot & Lalèyè17. Among Synodontis spp. occurring in the Nile and Turkana basins, S. schall might be confused with the morphologically very similar Synodontis frontosus Vaillant, 1895. According to the literature, S. schall is common and abundant in both the Nile River and Lake Turkana21. Although the two species might co-occur in both basins, S. frontosus is considered absent in Lake Turkana itself, reportedly being restricted to its main source, the Omo River22. Diagnostic features of both species are similar and partly overlapping, therefore, distinguishing between the two in the field is difficult.Within the scope of research on fish parasites in the Nile River and Lake Turkana, we examined a total of 138 S. schall is important as the species has been used as a model in parasitological research23. Therefore, we decided to base the taxonomical assignment of our Nile-Turkana S. schall specimens on a multidisciplinary approach, particularly on molecular identification with an assessment of selected morphological traits. Herein, we assess the identity of S. schall-like catfish from the Nile and Turkana basins by means of phylogenetic and haplotype analyses of a representative set of Synodontis spp. coxI, cytb and RAG2 sequences, revealing a previously unrecognized biogeographical pattern. To the best of our knowledge, this is the first confrontation of morphological features traditionally used for determination of S. schall and similar Nilo-Sudanian catfishes with molecular data to aid proper distinction of these catfishes of high significance from both an ichthyological and regional fisheries perspective.Resolving the species status of 24. With 131 nominal species currently recognized, Synodontis Cuvier, 1817 is the most speciose mochokid genus widely distributed in all African ichthyological provinces except for Maghreb and South Africa25. Despite numerous studies based on genetic markers28 morphology remains the main classification tool for identification of Synodontis spp.29. Several molecular phylogenetic studies were published during the last 13 years which elucidated general biogeographic patterns and relationships within the genus31. Synodontis schall is traditionally considered widespread throughout the Nilo-Sudanian ichthyological province from the Atlantic to Indian Ocean17. Its widespread distribution, abundance and easy harvesting make it one of the most important subsistence fish species in Sub-Saharan Africa north of equator and along the whole Nile valley34. Importantly, this ecologically plastic species28 is less likely to be affected by climatic changes and anthropogenic drainage alterations, making it a prospective source for fisheries in the future35. Despite its significance for fisheries and a long taxonomical history, morphology-based distinction between S. schall and similar species such as S. frontosus, remains unclear.The Mochokidae  is an endemic African family of freshwater catfish containing 211 species in nine generaS. schall n = 120, S. nigrita n = 8, S. serratus n = 10, Total: 138; see Table Sampled fish were obtained in seven different localities in the Nile and Turkana basins , cytochrome b (cytb) and recombination activating gene 2 (RAG2), respectively  were taken from Ivanova et al.37. PCR reactions contained 12.5 μl of PPP Master mix, 10.5 μl of ultra pure water, 5 pM of each PCR primer and 0.5 μl of isolated DNA (100 to 170 ng). Published primer sequences (5′-GAC-TTGAAGAACCACCGTTG-3′ forward and 5′-TTTAGAATTCTGG CTTTGGGAG-3′ reverse) to amplify part of cytb (~1000 bp) according to Pinton et al.27. For RAG2 (~1100 bp) amplification primers (forward 5′-TGY TAT CTC CCA CCT CTG CGY TAC C-3′ and reverse 5′-TCA TCC TCC TCA TCK TCC TCW TTG TA-3′) and reaction conditions followed those provided by Sullivan et al.38. Amplification reactions for cytb and RAG2 contained the same PCR reagents and amount of DNA as mentioned above. All products were verified on a 1% agarose gel and purified using enzymes exonuclease I (Exo I) and shrimp alkaline phosphatase (SAP)39. Purified fragments were directly sequenced by Macrogen Inc.  using the same primers used for PCR amplification.DNA was isolated using the phenol:chloroform:isoamyl alcohol (25:24:1) method described in Sambrook & Russell40. In addition to our original sequences  to interpret our results in a broader phylogenetic context  in PHYML v3.045. BI analyses were performed under the same conditions for all datasets - using 5 million MCMC replications, with four chains and three independent runs. The following nucleotide substitution models were used: GTR + I + G for coxI data, GTR + G for cytb, HKY + I + G for RAG2 and GTR + I + G for concatenated mt data. ML analyses were run using bootstrap analyses with 1000 replications and models mentioned above. Convergence for BI runs was estimated by TRACER v1.746, ESS values were assessed and found to exceed 200 for all parameters. Trees computed for cytb were rooted using Auchenoglanis occidentalis  and Mochokus niloticus (KY483655) as an outgroup. The remaining trees  were rooted using GenBank sequences of Microsynodontis sp. (DQ886604 for cytb and HF565741 for RAG2). All trees were deposited in TreeBASE database (treebase.org) and are accessed under the following link http://purl.org/phylo/treebase/phylows/study/TB2:S23311.Alignments were constructed in MAFFT v. 6S. schall sequences and included sequences of a closely related taxon (S. serratus) and a distantly related congener (S. nigrita) were included for comparison. Due to the limited availability of the nuclear data, genetic distances were computed for mitochondrial data only.Pairwise genetic distances were computed to assess the divergence of S. schall clades revealed by phylogenetic analyses using PopART v1.747 with settings for TCS networks48.To examine the level of population variability in the dataset, statistical parsimony networks based on pairwise differences were constructed for individual Fst values, genetic distances and differentiation were calculated using DNASP v549. Due to the limited number of GenBank sequences from the western part of S. schall geographic range, population statistics were performed only for S. schall samples obtained for this study (Nile and Turkana); data were split according to their resemblance to the S. schall-like and S. frontosus-like morphotypes (see Table Statistics of Haplotype diversity (Hd), S. schall specimens ranged from 142 to 300 mm . Weight ranged from 75 to 783 g . Synodontis schall from the Nile and Turkana showed remarkable variation in body shape, proportions, as well as coloration and other qualitative diagnostic features        and S. frontosus (33–48) morphotypes  Fig.  to S. frly) Fig. . Gradualct) Fig.  to S. frns) Fig. . Remarkant) Fig.  to S. frne) Fig. . MandibuS. schall/S. frontosus/intermediate morphotypes was as follows: El-Molo Bay 35/16/7, Kalokol 19/11/0, Todonyang 32/31/14, implying somewhat higher relative incidence of S. frontosus-like morphotype in the freshwater part of Turkana   vs. snout more broadly rounded with wider interorbital region and orbits impinging on the lateral outline of the head in dorsal view (as in S. frontosus) (sensu Hopson22). The two states are well demonstrated by the two specimens showing S. schall and S. frontosus morphotype in Fig. In addition, several other features corresponding either to coxI (565 bp), and two smaller sets comprising 96 cytb (973 bp) and 19 RAG2 (896 bp) sequences were generated. Data representing individual populations obtained from the Nile River and Lake Turkana were used for phylogenetic analyses together with Synodontis spp. sequences retrieved from GenBank. The newly generated sequence data were deposited in GenBank under accession numbers coxI: KY483652-KY483782, cytb: KY483783-KY483878, RAG2: MF136700-MF136718, MF150161- MF150164  and one sequence of S. caudovittatus (HF565865), all from the Nile, also clustered within the Eastern clade in all analyses. In contrast, GenBank sequences labeled S. schall , Synodontis ouemeensis HF565953, all from western part of Nilo-Sudanian and Eburneo-Ghanian provinces, together with sequences labeled Synodontis aff. bastiani and Synodontis aff. haugi HF565950, HF565954-56 (pet trade), created a separate group with high nodal support. Due to the clear separation of this group from the “S. schall Eastern clade” and taxonomic assignment of most its sequences, we named the clade “S. schall Western clade”.In cytb included sequences representing 52 Synodontis spp. , as well as S. schall sequences  and EU781915 from the Nile and/or Chad basin. This clade clearly corresponds with the S. schall Eastern clade, hence its name in the tree. The rest of S. schall sequences  from Senegambia, Niger and Chad basin created a group corresponding to the S. schall Western clade, closely related to, but clearly distinct from the S. schall Eastern clade. Interestingly, cytb sequences of S. ouemeensis (HF566058), S. aff. bastiani (HF566004) and S. aff. haugi (HF566027) clustered with the S. schall Western clade data. This result is similar to coxI analysis, however; with a stronger branch support and placing sequences of S. aff. bastiani and S. aff. haugi within the Western clade. In conclusion, analyses of both mitochondrial markers revealed very similar patterns with strong biogeographic signals, indicating phylogenetic distinctiveness and geographic affinity of two clades, i.e. S. schall Eastern and S. schall Western clade  was less informative compared to the clear separation of Eastern and Western clades in all analyses performed for both mitochondrial genes. A total of 19 RAG2 sequences of Synodontis spp. were analyzed , S. caudovittatus (HF565755), S. violacea (HF565838) and S. aff. bastiani (HF565751) from Eastern Nilo-Sudan province, as well as S. schall (HF565817-19) from Western Nilo-Sudan province. Although sequences of S. serratus from our sample and from GenBank clustered together with high support, the S. serratus branch was placed within the S. schall clade, probably as a result of low sequence variability in the whole dataset (100-94.5%).The phylogenetic analysis of the nuclear gene  and non-significant for cytb (P > 0.05). Similarly, a difference was seen in haplotype diversities for coxI, but not in cytb  was considered a basic feature distinguishing S. schall from S. frontosus. The genetic uniformity of the Nile-Turkana sample on one hand, and its remarkable morphological variability lead us to assume that diagnostic features of S. frontosus merely describe part of the intraspecific variability of S. schall. In other words, explicit evidence of remarkable intraspecific variability provided herein renders the two nominal species from East Africa, S. schall and S. frontosus, most probably synonymous, hence requiring taxonomical reassessment. Additionally, from a strictly phylogenetic point of view, the clustering of Synodontis sequences assigned to several species  implies that these data might represent a single species S. schall Western clade, especially considering uncertain status of many Synodontis nominal species.In the Nile and Turkana basins, typical morphotypes of both S. schall Eastern clade, as represented in our sample to S. schall sensu stricto: (i) the most prevalent morphotype matching S. schall, (ii) phylogenetic affinity (see below), (iii) geographic origin matching with the type locality in the Nile and (iv) with respect to probable synonymy, the specific name S. schall has priority over S. frontosus. The interpretation is further indirectly supported by the supposed absence of S. frontosus in Lake Turkana itself, being presumably limited to Omo River (as highlighted by Hopson 1982 and other authors). Although in our Turkana sample S. frontosus morphotype was not restricted to the freshwater Omo delta, it was most prevalent here, whereas the S. schall morphotype was the most prevalent in the Turkana saline and medium salinity parts. Differences in the prevalence of morphotypes between the localities may also coincide with the moderate (intraspecific) level of genetic differentiation found in the population genetic analysis of coxI  of mitochondrial DNA introgressed from one species to another. Unfortunately, we were not able to corroborate the mitochondrial results by an independent marker. The phylogeny based on the nuclear RAG2 gene was not informative, due to the lack of resolution in the respective part of the tree. Lack of representative data from the Western Nilo-Sudanian province, together with the generally low variability of the gene did not allow clear distinction of mitochondrial lineages in the nuclear dataset. Although generally rare, complete fixation of introgressed mitochondrial DNA was found for example in African cichlids53 and coral reef fish54. Analyses of more variable nuclear loci  would be required to completely exclude such a possibility in S. schall-like catfishes. However, considering the weak differentiation of morphological traits defining S. schall and S. frontosus, as opposed to the high level of cytb diversity required to be introgressed between the two species across all sampled localities, we consider the “introgression and fixation” alternative to be much less probable than conspecificity.Mitochondrial data revealed no significant differentiation between specimens representing S. schall and S. frontosus, i.e. Nile River in Assouan and in Khartoum, respectively, is necessary for sound taxonomical measures . Unfortunately, studies of other authors do not aid further resolution, because even recent taxonomic studies38 on Nilo-Sudanian Synodontis spp. lack molecular phylogenetic analyses, and vice versa, all available phylogenetic studies lack clues for the determination of sampled specimens, and/or the exact origin of the samples28.Concurrent morphological and molecular sampling throughout the Nile basin, including terra typica of both S. schall-like catfish throughout the Nilo-Sudanian province. The results of the phylogenetic reconstruction, haplotype analyses and genetic distance recognized different S. schall clades. Phylogenetic analyses of both mitochondrial markers unequivocally revealed separate S. schall Eastern and Western clades, each comprising a mixture of samples assigned by different authors to S. schall, S. frontosus or other taxa. In addition to that, the genetic similarities between S. schall Eastern and S. schall Western clades sequences showed more than 5% divergence for both mitochondrial genes, a phenomenon generally associated with taxa recognized as distinct species55. Regardless of GenBank taxonomic sequence assignment, the two well supported clades, S. schall Eastern and Western clade, exclusively comprise either samples from eastern part of the Nilo-Sudanian ichthyological province , or those from its western part , respectively. The taxonomically incongruent, but geographically non-random phylogenetic pattern reveals a strong biogeographical signal among the supposedly homogenous, conspecific and widely distributed S. schall-like catfish.The combined Nile-Turkana and GenBank dataset comprises all major limnic systems of the Nilo-Sudanian province, i.e. rivers Gambia, Senegal, Volta, Niger, Nile (including Lake Albert), endorheic basins of Chad and Turkana, as well as Eburneo-Ghanian (sub) province. As such, the presented dataset allows us to draw conclusions regarding diversity of Synodontis schall was long considered morphologically uniform and widely distributed throughout the Nilo-Sudanian ichtyological province from the Atlantic to Indian Ocean21. In contrast, our results imply the presence of two relatively distant phylogenetic lineages hidden under this name. Synodontis schall sensu stricto, as defined by location of type locality in the Nile, corresponds to the Eastern clade in our analyses, and is restricted to the province’s eastern part, i.e. Nile, Turkana and Chad basins, and possibly elsewhere in north-east Africa  probably represent a cryptic species more closely related to congeners from the same region than to their eastern siblings. Interestingly, there seems to be an overlap in distribution ranges of the “Eastern clade” and the “Western clade” in the Chad basin, probably as a result of independent and temporally distinct colonization events.ica Fig. . On the Hydrocynus56. The cryptic diversity, i.e. presence of three lineages among Hydrocynus vittatus populations from three separate drainages , was revealed based on a combination of morphological and molecular (cytb) data. Similarly, as in S. schall-like catfishes in this study, the diversity of H. vittatus has long been overlooked due to the absence of definitive morphological features and molecular evidence. Another possible example of overlooked diversity among African fish, but revealed morphologically in this case, might be two cyprinid species, Labeo niloticus and L. senegalensis. The two Labeo spp. show a distribution pattern almost identical to that of S. schall-like catfishes are also distinguished by subtle, sometimes overlapping morphological features. Morphological studies including samples from western  and eastern (L. niloticus) parts of Nilo-Sudan ichthyological province revealed apparent hidden diversity correlated with geographical origin of specimens57. However, molecular data are not yet available for L. senegalensis and L. niloticus and their species status remains to be confirmed. The multiple examples of overlooked diversity in H. vittatus and Labeo spp., as well as S. schall-like catfish herein, suggest that the phenomenon of hidden diversity among long known and geographically widely distributed African fishes might be more common than previously thought.The phenomenon of cryptic, geographically determined diversity in African fish described herein is not a new discovery. A similar pattern was described for African tigerfish genus Synodontis schall , as it has been defined and recognized for more than two centuries, apparently includes two distinct phylogenetic units (Eastern and Western clades), i.e. two distinct species are hidden within this name. The Eastern clade represents S. schall sensu stricto sensu stricto as defined by the type locality in the Nile, while the Western clade from the western part of Nilo-Sudanian province probably represents a cryptic species, which remained unrecognized due to a lack of differentiating morphological traits. A candidate name Synodontis gambiensis Günther, 1864 (Terra typica: Gambia), currently considered a junior synonym of S. schall, is available for eventual taxonomical amendment of the Western clade. In contrast, another long-recognized nominal species, S. frontosus Vaillant, 1895, might be merely a junior synonym of S. schall sensu stricto. An interesting, rather unusual taxonomical case has emerged from this study, since concurrent taxonomical deflation  and taxonomical split  might be inevitable in the future for the two long recognized congeners.Supplementary infoSupplementary Tables S1, S2, S3, S4


With the advent of novel molecular platforms for high-throughput/next-generation sequencing, the communities of commensal and pathogenic microorganisms that inhabit the human body have been defined in depth. In the last decade, the role of microbiota-host interactions in driving human cancer plasticity and malignant progression has been well documented. Germ-free preclinical models provided an invaluable tool to demonstrate that the human microbiota can confer susceptibility to various types of cancer and can also modulate the host response to therapeutic treatments. Of interest, besides the detrimental effects of dysbiosis on cancer etiopathogenesis, specific microorganisms have been shown to exert protective activities against cancer growth. This has strong clinical implications, as restoration of the physiologic microbiota is being rapidly implemented as a novel anticancer therapeutic strategy. Here, we reviewed past and recent literature depicting the role of microbiota-host interactions in modulating key molecular mechanisms that drive human cancer plasticity and lead to malignant progression. We analyzed microbiota-host interactions occurring in the gut as well as in other anatomic sites, such as oral and nasal cavities, lungs, breast, esophagus, stomach, reproductive tract, and skin. We revealed a common ground of biological alterations and pathways modulated by a dysbiotic microbiota and potentially involved in the control of cancer progression. The molecular mechanisms most frequently affected by the pathogenic microorganisms to induce malignant progression involve epithelial-mesenchymal transition- (EMT-) dependent barrier alterations and tumor-promoting inflammation. This evidence may pave the way to better stratify high-risk cancer patients based on unique microenvironmental/microbial signatures and to develop novel, personalized, biological therapies. The collective genome content of the human microbiota  is known as the human microbiome, although the words “microbiome” and “microbiota” are often used interchangeably . The totose to 1 . Despiteose to 1 . Howeverose to 1 . The metose to 1 –8.vs “gene mutation AND cancer” (Helicobacter pylori (H. pylori) is the etiologic agent of gastritis and gastric ulcers, which can be precursors of gastric adenocarcinoma [Major milestones have been reached in the last decade by the Human Microbiome Project (HMP). This is a two-phase research initiative aimed at identifying and characterizing the whole human microbiota through the extensive use of metagenomics and whole genome sequencing , and at elucidating the role of microbes in human diseases through the use of multiple “omics” technologies  , 10. In  cancer” . Thus, tarcinoma .12 bacteria/gm stool) [Here, we reviewed the most important evidence obtained in the understanding of the microbiota-host interaction mechanisms that drive human cancer plasticity and lead to malignant progression. Most studies aimed at elucidating the effects of microbiota-host interactions on human cancer pathogenesis have been focused on colorectal cancer (CRC), for the obvious reason that the greatest number and diversity of microbes in the human body inhabits the large intestine (10m stool) . HoweverPorphyromonas gingivalis , can induce dysbiosis [Fusobacterium nucleatum (F. nucleatum), can become opportunistically pathogenic and lead to dysregulated immune response and increased risk to develop periodontal diseases and OSCC [Streptococcus sp., Peptostreptococcus sp., and Prevotella sp., have been identified to correlate strongly with OSCC [P. gingivalis is able to stimulate the expression of the cancer stem cell markers CD133 and CD44 [P. gingivalis promote migratory and invasive properties [P. gingivalis mutant strains lacking the fimbrial protein FimA were attenuated in their ability to activate Zeb1 expression, demonstrating a role for the FimA adhesin in triggering EMT [Oral cancer, primarily oral squamous cell carcinoma (OSCC) arising from the oral mucosa, is caused by both genetic and environmental factors, such as tobacco and alcohol consumption, betel quid chewing, and human papillomavirus infections . Howeverysbiosis . In thisand OSCC , 17. Othith OSCC . P. gingand CD44 . It has operties , 20. Theoperties , 20. Morα-toxin/hemolysin A from Staphylococcus aureus (S. aureus) induces improper cytoskeletal remodeling due to destabilization of cell-cell contacts and focal adhesions [S. aureus plays a key role in the pathogenesis of nasal polyposis, through secreted products, such as the protein A that induces mast cell degranulation and the staphylococcal enterotoxins (SAE) that induce release of proinflammatory cytokines [β and vimentin as compared with the healthy nasal mucosa [S. aureus could play an important role as driver of this epithelial plasticity.The olfactory epithelium microbial community is mainly dominated by 4 phyla: Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria . Signifidhesions . This redhesions . S. aureytokines . Nasal pl mucosa . Thus, tPrevotella, Veillonella, and Streptococcus. There is large overlap in the community composition between the lung microbiota and that of the oral cavity as compared with other body sites [Kras mutations and p53 deletion was utilized to compare germ-free and specific pathogen-free conditions, revealing that germ-free mice are significantly protected against LUAD [β (IL-1β) and IL-23, and γδ T cells to promote inflammation and tumor cell proliferation through IL-17 [The normal lung was considered as a microbe-free organ for a long time. Over the past decade, novel culture-independent techniques for microbial identification have challenged this dogma and revealed that the lung is constantly exposed to a variety of air-borne microbes through inhalation . The mosdy sites . The numdy sites . The moldy sites . It has dy sites . As an enst LUAD . The lungh IL-17 .vs paired normal adjacent tissues [Enterobacteriaceae and Staphylococcus were recently found at higher abundance in breast cancer patients than in healthy controls [Escherichia coli (E. coli) and Staphylococcus epidermidis (S. epidermidis) from normal adjacent tissue of breast cancer patients were found to induce DNA double-strand breaks (DSBs), which are the most detrimental type of DNA damage [Listeria fleischmannii is associated with genes involved in EMT, and Haemophilus influenzae is correlated with genes involved in the control of G2-M checkpoint, E2F signaling, and mitotic spindle assembly [Studies performed on human samples have demonstrated that different bacterial profiles can be detected in the normal adjacent breast tissue from breast cancer patients and in normal tissues from healthy controls  Figure . This is tissues   and butyrate . A numbeern diet . This apt cancer . Third, t cancer . In addit cancer . LCA levt cancer . The rolt cancer .Streptococcus viridans as the most frequent microorganism present in the normal esophagus and the oropharynx [Streptococcus, Prevotella, and Veillonella were the most represented bacterial populations [Streptococcus); the type II was largely correlated with GERD and BE and composed by Gram-negative anaerobes/microaerophiles  [Streptococcus and Prevotella dominate the upper gastrointestinal tract and the ratio of these two species can be associated with two known EAC risk factors in BE  [κB pathways. Activation of NF-κB leads to production of IL-1β or TNF-α by inflammatory cells. This state of chronic inflammation is thought to play a crucial role in the progression from benign to malignant disease [β, IL-8, and NF-κB was found to occur in the transition from normal esophageal epithelium to BE and EAC [N-nitroso compounds in acidic environments [β- and IL-6-dependent inflammation, leading to BE and EAC [F. nucleatum, which could migrate from the oral cavity and colonize the esophagus; the mechanisms involve production and activation of various chemokines, such as CCL20 [Lactobacillus fermentum [Treponema denticola and Streptococcus anginosus, which are minor bacteria in the saliva, were found to preferentially infect the normal mucosa of the esophagus as well as the esophageal cancer tissues [Bulleidia, Lautropia, Catonella, Peptococcus, Moryella, Corynebacterium, and Cardiobacterium and increased levels of colonization by Prevotella, Streptococcus, and Porphyromonas in ESCC vs normal tissues [Streptococci invade their target cells through a binding between the hyaluronic acid capsular polysaccharide on the bacterial surface and the hyaluronic acid-binding protein CD44 on the epithelial cells. The signaling pathway induced by Streptococci upon adhesion to CD44 involves cytoskeletal reorganization via Rac1 and ezrin, as well as loss of intercellular junctions by changed distribution of the junctional proteins ZO-1 and E-cadherin [The interest on the esophageal microbiota remains limited as compared with other human tissues, primarily because the esophagus was considered as a channel connecting the oral cavity with the stomach. The first studies on esophageal microbiota demonstrated that the bacterial species found in the esophagus came from other districts, such as the oropharynx by swallowing or the stomach by reflux –49. Gastopharynx , 54, 55.opharynx . Six majulations . Distal ulations . Two typnomyces) . Gall an length) . Gram-ne disease , 59–61.  and EAC . Gram-neronments . Bile ac and EAC . The proas CCL20 . Recentlermentum . Epithel tissues . The cor tissues , 69. Thicadherin , 71. Thucadherin , 73, couH. pylori, the stomach was considered as a sterile organ, because of its thick mucus layer, the acidic gastric juice, and the peristaltic movement. H. pylori survives under the acidic conditions in the stomach, due to the action of a surface-exposed urease that transiently buffers the acidic environment by catalyzing the hydrolysis of the urea into carbon dioxide and ammonia. These two products serve as substrates for other microbes and can change the gastric microbiota. It is now well established that gastric cancer is associated with bacterial dysbiosis within the stomach and that chronic infection with H. pylori is a major risk factor for the development of gastric cancer. Although H. pylori infects >50% of the world's population and only ∼1% of the infected individuals develops gastric cancer, it is estimated that 75% of all gastric cancer cases are caused by infection with H. pylori, which is a class I carcinogen according to the World Health Organization. Colonization of the human stomach by H. pylori induces a complex inflammatory and immunological response, with production of TNFα and IL-1β, which is also a powerful inhibitor of gastric acid secretion [H. pylori-dependent gastric cancer have been elucidated in detail [H. pylori localizes in close proximity to the apical cell-cell junctions and drives EMT through disruption of tight and adherens junctions [H. pylori cytotoxin-associated gene (cag) pathogenicity island encodes a type IV secretion system (T4SS) that translocates the effector protein CagA into the host cells after bacterial attachment to host apical or basolateral integrins [β-catenin is increased within gastric cancer precursor lesions such as gastric adenomas [β-catenin with gastric cancer plasticity. Activation of β-catenin by CagA occurs through multiple mechanisms, including destabilization of E-cadherin/β-catenin complexes and activation of the Wnt signaling pathway [β with activation of β-catenin and Snail [H. pylori's vacuolating cytotoxin A (VacA) and outer inflammatory protein A (OipA) can activate the epidermal growth factor receptor (EGFR) and the downstream PI3K-Akt signaling pathway, which results in GSK3β degradation and β-catenin stabilization [cagA-positive H. pylori strain induces morphological modifications characterized by a loss of polygonal shape, cell cluster disruption, elongated shape, stimulation of mesenchymal  and stem cell (CD44) markers, and inhibition of epithelial markers (cytokeratin 7 and osteopontin) [low cells, H. pylori-infected CD44high cells show mesenchymal phenotype and stem cell properties in vitro, high sphere-forming potential, enhanced migration and invasion capacities, and high tumorigenic capacity in xenografted mice [H. pylori infection promotes gastric tumorigenesis [Before the discovery of ecretion . This drecretion , 75. Then detail –82. In tunctions –86. The ntegrins . Here, Cntegrins . Once trntegrins . This phntegrins . The phontegrins . Nuclearadenomas , suggest pathway , phospho pathway , and degnd Snail . H. pylolization , 94. Theopontin) . Compareted mice . The abiigenesis .Bacteroides fragilis, barely detectable in the normal intestinal microbiota may exhibit pro-oncogenic activity in CRC [Il10−/−) mouse model [Il10−/− mice mono-associated with the mouse adherent-invasive commensal E. coli NC101 promoted invasive carcinoma in 80% of the hosts, while the human commensal Enterococcus faecalis OG1RF, which also caused aggressive colitis in Il10−/− mice, was unable to induce malignant progression [pks) genotoxic island, which encodes a multienzymatic machinery that synthesizes colibactin, a peptide-polyketide hybrid [Various models of bacteria-induced carcinogenesis have been postulated, suggesting how intestinal microbe-microbe and microbe-host interactions can contribute to development and progression of CRC . A receny in CRC , 97. It se model . Adminisgression . The mole hybrid , 100. Coe hybrid .B. fragilis in inducing tumorigenesis in human and animal models of CRC. A recent study in germ-free ApcMin/+ mice colonized with B. fragilis showed that the B. fragilis toxin (BFT) induces the activation of a procarcinogenic, inflammatory cascade in intestinal epithelial cells, which requires the combined activation of the IL-17R and Stat3 signaling pathways [β-catenin/TCF signaling pathway, and enhanced cell proliferation [Citrobacter rodentium infection [β-catenin, Notch, and TGFβ pathways, together with epigenetic changes such as histone modification and chromatin remodeling. This triggers trans-differentiation of crypt cells into fibroblast-like mesenchymal cells [The impact of the microbiota on the innate and adaptive immune system is well established , 102. Inpathways . IL-17R pathways , which lpathways . BFT wasferation , 106. Chnfection . A key mal cells .Lactobacilli and Bifidobacteria, can be outcompeted by opportunistic/passenger bacteria better adapted to the tumor microenvironment of human CRC [F. nucleatum. Its mechanistic contribution to CRC has been extensively documented, at variance with other human conditions such as pericarditis, brain abscesses, osteomyelitis, and other cancer types , where F. nucleatum appears to play a secondary role [F. nucleatum is the most abundant bacterial species in the oral cavity, but it is a weak colonizer of the gut. The abundance of F. nucleatum in CRC tissues has been demonstrated using orthogonal approaches, such as 16S ribosomal RNA (rRNA) gene sequencing and fluorescent in situ hybridization [B. fragilis and E. coli, that produce toxins able to change the immune response or to induce DNA damage, F. nucleatum is not known to produce toxins. Its major pro-oncogenic activities are due to the virulence factor Fusobacterium adhesin A (FadA), which is expressed on the bacterial surface, and whose corresponding gene fadA is strongly upregulated in colon tissue samples from patients with adenomas and adenocarcinomas as compared with healthy subjects [β-catenin from the plasma membrane and activation of Wnt signaling then occur, leading to enhanced cancer cell EMT and plasticity [F. nucleatum can influence cancer progression through the creation of a proinflammatory niche. ApcMin/+ mice fed F. nucleatum developed more colorectal and small-intestinal tumors than their sham-fed counterparts [F. nucleatum induces intratumoral myeloid cells, including macrophages, dendritic cells, and myeloid-derived suppressor cells [F. nucleatum also activates the NF-κB pathway and induces expression of the genes encoding several pro-inflammatory cytokines, such as TNFα, IL-6, IL-8, and IL-1β [F. nucleatum are significantly higher in tumor tissues than in adjacent normal tissues, and correlate with tumor invasion as well as with lymph node and distant metastasis [F. nucleatum in promoting CRC progression, possibly through the formation of an oncogenic biofilm [F. nucleatum induces loss of miR-4802 and miR-18a, with consequent increased expression of the autophagy signaling elements ULK1 and ATG7 [F. nucleatum levels in CRC tissues and the expression levels of Nanog was observed, supporting a role of F. nucleatum in modulation of cancer plasticity and EMT [Nonpathogenic intestinal commensals, such as uman CRC , 97. Theary role . F. nucldization . At varisubjects . FadA bisubjects . Releaseasticity . F. nuclterparts . It was or cells . F. nuclnd IL-1β . Analysitastasis . This su biofilm . Recent and ATG7 . This reand ATG7 . A posit and EMT .Atopobium vaginae and Porphyromonas sp. in the gynecologic tract, in combination with a high vaginal pH (>4.5), are associated with the presence of endometrial cancer [Lactobacillus spp. causes increased production of several inflammatory cytokines, such as IL-36γ, MIP-1β, RANTES, IP-10, IL-2, IL-4, Flt-3L, and sCD40L [The female reproductive tract  harbors complex and diverse bacterial communities that are associated with different physiological and pathological conditions , 115. Mel cancer . Thus, vd sCD40L . Alteratd sCD40L . A corred sCD40L –123. In d sCD40L , 123.Proteobacteria and Firmicutes may strongly reduce microbial diversity and lead to ovarian tumorigenesis by suppressing host inflammatory immune responses in the tumor microenvironment [Ovarian cancer accounts for about 3% of cancers among women and is the most lethal gynecological malignancy. Evidence from two different studies indicated that the ovarian cancer microbiota is distinctly different from that of the normal ovarian tissue. Persistent infection of ovarian tissues with ironment , 125.Cutibacterium and Staphylococcus are prevalent colonizers of sebaceous areas, while Corynebacterium, Staphylococcus, and beta-Proteobacteria are found in moist areas. Skin cancers are generally classified into two main groups, melanoma and non-melanoma skin cancers (NMSC), having different etiology and clinical behavior. NMSC arises from epidermal keratinocytes and can be divided into basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Melanoma develops from epidermal melanocytes. Overall, NMSC shows higher incidence than melanoma, but a better response to treatment and better long-term prognosis. While the role of viruses and environmental carcinogens (such as UV) in skin cancer progression has been deeply elucidated, the contribution of the bacterial microbiota remains controversial [Firmicutes and Actinobacteria [S. aureus have been implicated in the pathogenesis of atopic dermatitis [Lactobacillus paracasei have been found to be beneficial for the skin due to their immunomodulatory effects [S. epidermidis was found to protect against UVB-induced skin papillomas in preclinical models [S. epidermidis produces 6-N-hydroxyaminopurine (6-HAP), which showed a bactericidal activity against pathogens such as the group A Streptococcus (GAS) by inhibiting DNA polymerase [The skin is a complex ecosystem inhabited by bacteria, archaea, fungi, and viruses. The microbiota is fundamental to skin physiology and immunity, and interactions between skin microbes and the host are quite complex and context-dependent, ranging from mutualistic to pathogenic relationships . Types aoversial , though oversial . Chronicoversial . Injuredbacteria . Certainrmatitis , which wrmatitis . Interesrmatitis . Also, g effects , 134. Chl models . S. epidlymerase . In cultlymerase .Chlamydia trachomatis infection [The acquisition of motile/mesenchymal properties by epithelial cells is the phenotypic effect of a profound molecular and cellular reprogramming –139. Thenfection .P.  gingivalis leads to overexpression of EMT factors such as Zeb1 [F. nucleatum, which promotes CRC by modulating E-cadherin/β-catenin signaling through the adhesin FadA [H. pylori protein CagA can subvert multiple signaling cascades into host epithelial cells [A growing body of evidence suggests that the cancer microbiota can promote tumorigenesis and plasticity through metabolic reprogramming , 44, 141 as Zeb1 . Bacterisin FadA   and inflammatory cytokines  that can induce EMT in cancer cells. Also, myeloid cells have been shown to induce EMT-like properties in cancer cells via TGFβ, EGF, and HGF [As described in the previous sections, the role of the microbiota in cancer progression is largely connected to the modulation of host inflammatory responses in many body sites. In the lung parenchyma, the microbiota promotes production of IL-1 T cells . Gram-nend TNF-α –61, 74. nd Stat3 . This stnd Stat3 , which rnd Stat3 . Moreovend IL-1β , and infand IL-4 . Activatand IL-4 . TAMs pr and HGF . Chronic and HGF . It has  and HGF , thus in and HGF . The mol and HGF .Clostridium difficile infection [Dysbiotic states where the amount of pathogenic inhabitants overrides or even replaces the non-pathogenic commensals can have detrimental effects on the physiological processes and lead to various types of diseases, including cancer . Two comnfection  or lymphnfection . This ap


Propionibacterium acnes (P. acnes) is the main anaerobic bacteria that cause acne. It is known to proliferate within sebum-blocked skin hair follicles. P. acnes activates monocytic cell immune responses to induce the expression of proinflammatory cytokines. Although the anti-inflammatory function of the Laurus nobilis (L. nobilis) extract (LNE) on several immunological disorders have been reported, the effect of LNE in P. acnes-mediated skin inflammation has not yet been explored. In the present study, we examined the ability of the LNE to modulate the P. acnes-induced inflammatory signaling pathway, and evaluated its mechanism. LNE significantly suppressed the expression of P. acnes-mediated proinflammatory cytokines, such as IL-1β, IL-6, and NLRP3. We also found that LNE inhibited the inflammatory transcription factor NF-κB in response to P. acnes. In addition, eucalyptol, which is the main constituent of LNE, consistently inhibited P. acnes-induced inflammatory signaling pathways. Moreover, LNE significantly ameliorated P. acnes-induced inflammation in a mouse model of acne. We suggest for the first time that LNE hold therapeutic value for the improvement of P. acnes-induced skin inflammation.Acne is an inflammatory skin disorder in puberty with symptoms including papules, folliculitis, and nodules.  Propionibacterium acnes, a commensal Gram-positive anaerobia, is thought to be the primary bacteria in acne development [P. acnes overgrows in hair follicles and acts as an immunostimulator to secrete various proinflammatory cytokines that are crucial for the development of cutaneous inflammation [P. acnes has been proposed to activate monocytes to produce cytokines such as IL-1β, IL-8, and TNF-α, which cause granulomatous responses in inflammatory skin disorder [P. acnes is recognized by the toll-like receptors TLR2 and TLR4 and triggers the expression of inflammatory mediators through activating MAP kinases (MAPKs) and nuclear factor-κB (NF-κB) [Acne is a common skin inflammatory disorder in sebaceous glands that causes inflammation in the face, neck, back, chest, and shoulders. Acne occurs mainly in adolescence due to hormone imbalance, bacterial infections, food, and psychological stress . Propionelopment . P. acneammation . The pepdisorder ,5. P. ac (NF-κB) ,7.P. acnes-induced inflammation [Antibiotics, including tetracycline, erythromycin, and clindamycin, are commonly used for the treatment of ammation . Benzoylammation ,10,11. HLaurus nobilis Linn. has been cultivated in the Southern Mediterranean sea region and in Europe, as a vegetable and a traditional medicine [L. nobilis has been reported to have antioxidant, antimicrobial, analgesic, anticonvulsant, and antifungal actions [medicine . L. nobi actions ,14,15. T actions ,17,18.L. nobilis extract (LNE) on diverse inflammatory responses have been reported, the role of LNE in P. acnes-mediated skin inflammation has not yet been reported. Therefore, we examined the potential effects of LNE in response to P. acnes, using in vitro and in vivo mouse experimental models. We found that LNE and its major constituent eucalyptol effectively exert anti-inflammatory functions by suppressing P. acnes-induced inflammatory signaling pathways.Although the inhibitory effect of P. acnes-induced inflammatory signaling pathways, we first determined the noncytotoxic concentration of LNE to BMMs. For this experiment, BMMs were incubated with increasing concentrations of LNE for 12 h, and cell viability was assessed using Trypan blue dye exclusion  transcription factor, which is known to be critical for the expression of various inflammatory mediators. We first monitored IκB phosphorylation, which unmasks NF-κB. LNE effectively suppressed IκB phosphorylation in BMMs, following mulation A,B. To ferformed C. ConsisP. acnes-primed BMMs were treated with the well-known NLRP3 inflammasome activator ATP, which opens the P2X7 cation channel, in the presence or absence of LNE. As shown in +/H+ exchanger on partitioning into intracellular organelles and plasma membranes (P. acnes-primed NLRP3 inflammasome activation.Nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome regulates inflammation by triggering the maturation and secretion of IL-1β . To inveembranes B. CollecP. acnes-mediated inflammation. BMMs were treated with P. acnes in the presence or absence of LNE, and the activation of MAPKs was monitored using phospho-specific antibodies , including surface receptors such as toll-like receptors (TLRs) and cytosolic pattern recognition receptors such as Nod-like receptors (NLRs). Several studies have shown that and TLR4 . Activatand TLR4 . TLR2 haand TLR4 . Jugeau and TLR4 . NLRs arand TLR4 . The NLRand TLR4 . Mutatedand TLR4 . In addiP. acnes-induced NF-κB activation. However, MAPK activation was not altered by LNE. We, therefore, suggest that the anti-inflammatory effect of LNE occurs by inhibiting the NF-κB signaling cascade in BMMs.Most TLRs lead to the activation of common downstream innate signaling cascades, such as MAPKs  and NF-κB transcription . MAPKs aL. nobilis leaf has been reported in previous studies [L. nobilis [P. acnes-induced skin inflammatory signaling in BMMs.The chemical composition of  studies ,31. Acco studies . In a st nobilis . Many re nobilis ,33,34. E nobilis . Eucalyp nobilis . In addiP. acnes-induced inflammation, we used a well-known mouse model of acne, in which the pathological pattern is similar to that of human acne lesions [P. acnes-induced inflammation, such as the granulomatous response, ear thickening, and corresponding inflammatory cytokine expression. We monitored the most common inflammation markers expressed in macrophages because tissue macrophages surrounding the pilosebaceous unit in acne lesions expressed high levels of TLR2 and correlated with the degree of inflammatory nature of the clinical lesions [P. acnes-mediated skin inflammation, using a mouse model. Based on our results, further studies could possibly check its application in clinical usages by comparing it with well-known antibiotics and standard materials.To determine the physiological effect of LNE in  lesions . We obse lesions . Our stuP. acnes-induced skin inflammation. LNE significantly reduced the expression of P. acnes-induced proinflammatory cytokine expression by suppressing NF-κB signaling pathways  and anti-phospho-IκB antibodies were purchased from Cell Signaling Technology . The anti-GAPDH antibody was purchased from Santa Cruz Biotechnology . Nigericin was purchased from Tocris . Eucalyptol, ATP, and other chemicals were purchased from Sigma-Aldrich .L. nobilis were purchased from the Hub Village  and extracted with 70% ethanol for 3 h at room temperature. The filtered extract was then evaporated using a rotary evaporator  to remove the solvent. The lyophilized sample was stored at –20 °C and was used after dissolving in dimethyl sulfoxide (DMSO) before each experiment. The presence of eucalyptol in the extract was identified by gas chromatography–mass spectrum (GC–MS) chromatogram .LNE was prepared as described previously . BrieflyP. acnes (KCTC3314) was obtained from the Korean Culture Center of Microorganisms  and cultured in Reinforced Clostridial Medium  under anaerobic conditions, using a Gas-Pak at 37 °C for 72 h. P. acnes were collected by centrifugation at 4500 rpm for 20 min at 4 °C, and the pellets were washed with PBS .P. acnes.Bone marrow-derived macrophages (BMMs) were prepared as described previously . BrieflyThe cells lysates were separated on SDS-PAGE, and then transferred to nitrocellulose membrane. The membranes were blocked with 5% non-fat dried milk, and incubated with the indicated primary antibodies for 12 h. The bands were visualized with enhanced chemiluminescence system, and then quantified using ImageJ software 1.52a .NF-κB-Luc reporter and control Renilla-Luc reporter were transfected into HEK293 cells using a Lipofectamine . Luciferase activity was measured using a Dual-Luciferase Assays System .BMMs were plated in 12-well plates. The cells were treated with LNE for the indicated concentrations and then collected by centrifugation. The collected cells was mixed with a Trypan blue solution  and counted using a hemocytometer.The cells were incubated with MTT  at a final concentration of 1 mg/mL for 1 h at 37 °C and then lyzed in the solubilizing solution , overnight. Absorbance was measured at 570 nm and the viability of the cells was calculated as the percent relative to the control cells.The culture supernatants were collected and the concentrations of IL-1β were measured in accordance with the manufacturer’s instruction .TM . All samples were run in triplicates and changes in target mRNA expression were normalized to β-actin.Real-time PCR was performed as described previously . cDNA waP. acnes (1 × 108 CFU per 20 μL in PBS) intradermally into the ears with or without LNE (100 μg/mL). The mice were sacrificed at 24 h after the injection, and ear tissues were then collected for further analysis.ICR mice were purchased from Doo Yeol Biotech , and the mice were bred at the Animal Center of the Kangwon National University in a controlled environment. All procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee . Eight-week-old mice (five per group) were injected with live Isolated ear tissues were fixed in 4% formalin and embedded in paraffin. The sections (2–3 μm) were then stained with hematoxylin and eosin (H&E). Pathological changes were monitored by light microscopy  and photographed.t-test. Analysis of variance (ANOVA) followed by Bonferroni post hoc tests was performed for the multiple groups. A p < 0.05 was considered significant.The statistical data were analyzed by SPSS . The values are presented as the means ± standard deviations (SD) of three independent experiments. Comparisons between two groups were analyzed using Student’s


Plasmodium knowlesi is now the main cause of human malaria within Malaysian Borneo. While data is increasingly available on symptomatic cases, little is known about community-level patterns of exposure and infection. Understanding the true burden of disease and associated risk factors within endemic communities is critical for informing evidence-based control measures.Primarily impacting poor, rural populations, the zoonotic malaria P. knowlesi transmission is reported: Limbuak, Pulau Banggi and Matunggung, Kudat, Sabah, Malaysia and Bacungan, Palawan, the Philippines. Infection prevalence was low with parasites detected by PCR in only 0.2% (4/2503) of the population. P. knowlesi PkSERA3 ag1 antibody responses were detected in 7.1% (95% CI: 6.2–8.2%) of the population, compared with 16.1% (14.6–17.7%) and 12.6% (11.2–14.1%) for P. falciparum and P. vivax. Sero-prevalence was low in individuals <10 years old for P. falciparum and P. vivax consistent with decreased transmission of non-zoonotic malaria species. Results indicated marked heterogeneity in transmission intensity between sites and P. knowlesi exposure was associated with agricultural work  and higher levels of forest cover  and clearing  around houses. Spatial patterns of P. knowlesi exposure differed from exposure to non-zoonotic malaria and P. knowlesi exposed individuals were younger on average than individuals exposed to non-zoonotic malaria.We conducted comprehensive surveys in three areas where P. knowlesi and associated risk factors within endemic communities. Results indicate community–level patterns of infection and exposure differ markedly from demographics of reported cases, with higher levels of exposure among women and children. Further work is needed to understand these variations in risk across a wider population and spatial scale.This is the first study to describe serological exposure to  Plasmodium knowlesi is a species of malaria parasite found in wild macaque populations which is now the main cause of human malaria in Malaysian Borneo. Spread from macaques to people through infected mosquitoes, human P. knowlesi malaria cases have primarily been reported in adult men working in forests or plantations. However, little data is available on the extent of asymptomatic infections or people exposed to P. knowlesi not reporting to clinics. We conducted comprehensive surveys of three case study communities in Malaysian Borneo and Palawan, the Philippines with varying numbers of P. knowlesi cases reported. In addition to testing for infection, we measured species-specific antibody responses to P. knowlesi and other malaria species to identify exposed individuals. Few asymptomatic infections were detected and varying levels of P. knowlesi exposure was detected between sites. P. knowlesi exposure was identified in both men and women and associated with farm work and forest and clearing around the house. Spatial patterns and risk factors for P. knowlesi differed from other malaria species, highlighting the need for knowlesi specific disease control measures. Results suggest more people are exposed to P. knowlesi than are identified at clinics and exposure to P. knowlesi may occur in different demographic groups and geographic areas than previously reported. Plasmodium knowlesi in 2004 and advent of routine diagnosis of malaria cases by molecular methods, increasing numbers of human P. knowlesi cases have been reported in Southeast Asia and P. knowlesi is now the most common cause of human malaria in Malaysian Borneo [P. falciparum and vivax, the emergence of P. knowlesi presents a challenge to malaria elimination programmes. Despite increasing amounts of data available for symptomatic malaria cases presenting at hospital facilities, little is known about patterns of P. knowlesi exposure and infection at a community level [After the initial recognition of a large number of human cases of the zoonotic malaria n Borneo –3. AlthoP. knowlesi requires a detailed understanding of environmental and social risk factors. Carried by long and pig-tailed macaques (Macaca fasicularus and M. nemestrina), environmental changes affecting contact between people, mosquito vectors and simian hosts are believed to contribute to this apparent emergence of P. knowlesi in people [Anopheles balabacensis, the main knowlesi vector, has been associated with forest environments but is also found in peridomestic and agricultural areas [P. knowlesi infections, including in women and children, demographic groups comprising a minority of cases reported to facilities [Effectively targeting resources to identify and control n people , 6. Anopal areas , 8. Assoal areas . Additiocilities –14.P. falciparum and P. vivax e.g. [P.knowlesi have only recently been described[Patterns of community-level exposure can be assessed by the prevalence of specific antibodies against malaria parasites; these antibodies reflect exposure to previous infection and can be used to characterise the level of transmission and identify areas or groups with higher transmission . These svax e.g. , 20, 21bdescribed.P. knowlesi and other malaria species in three case study communities where P. knowlesi transmission has been reported; a largely deforested and highly fragmented site at Matunggong, Kudat, an area with large patches of secondary forest bordering large scale clearing for an oil palm plantation in Limbuak, Pulau Banggi in Sabah, Malaysia and an area with intact secondary forest and some remaining primary forest in Bacungan, Palawan, The Philippines . P. knowlesi is the main cause of reported human malaria in both the Matunggong and Limbuak sites while only few sporadic P. knowlesi cases have been reported from Bacungan [P. knowlesi in 2014 was 12 for Matunggong, 2 for Limbuak and 0 for Bacungan.This study aimed to characterise these community level patterns of serological exposure to and prevalence of asymptomatic parasitemia of lippines . These aBacungan –25. BaseThis study was approved by the Medical Research Sub-Committee of the Malaysian Ministry of Health (NMRR-14-713-21117), the Institutional Review Board of the Research Institute for Tropical Medicine, Philippines and the Research Ethics Committee of the London School of Hygiene and Tropical Medicine (8340). Written informed consent was obtained from all participants or parents or guardians and assent obtained from children under 18 in this study and all methods were performed in accordance with relevant guidelines and regulations.P. knowlesi cases and all households within these communities were enumerated and geo-located. All individuals were asked to participate in the study and consenting individuals were interviewed on demographic characteristics, movement patterns, malaria prevention methods and land use practices. Individuals were excluded if they were less than 3 months old, had not primarily resided in the area for the past month or could not be reached after three attempts to contact them, including during evenings and weekends. Finger-prick blood samples were collected to test for malaria infection and exposure; these included blood smears to detect malaria parasites by microscopy and approximately 200μl whole-blood specimens collected in a tube containing EDTA  and three 20μl spots stored on filter paper . Filter paper was dried and stored with desiccant at 4°C.This study involved comprehensive sampling of all individuals residing within the study areas. Study sites were selected based on the locations of previously reported clinical Plasmodium small subunit ribosomal RNA (ssRNA) was used to identify malaria infected individuals, as described by [Plasmodium genus were then screened using species specific primers targeting the ssRNA region; for P. knowlesi these included PkF1140 (5’-GATTCATCTATTAAAAATTTGCTTC-3’) and PkR1150 (5' GAGTTCTAATCTCCGGAGAGAAAAGA 3') for 35 cycles at 50°C, 72°C and 94°C. All products were visualised on a 2% agarose gel. PCR for malaria infection was performed at laboratories at the Universiti Sabah Malaysia in Malaysia and Research Institute for Tropical Medicine in the Philippines, with PCR validation of a subset of samples at the London School of Hygiene and Tropical Medicine in the UK.All blood smears were examined by trained malaria microscopists. DNA was extracted from filter paper or 10 μl blood pellets using the Chelex-100 boiling method and a nested polymerase chain reaction (PCR) method targeting the ribed by . This asP. vivax , P. falciparum  and PfMSP-119 (PMID: 8078519) and P.knowlesi SERA3 antigen 2[Pk serine repeat antigen (SERA) 3 antigen 2  is a novel recombinant protein, N-terminally located between positions 826–998 aa, inclusive. SERA3 (1079 aa) belongs to a multigene family whose members encode a papain-like cysteine protease domain (ref: PMID: 21423628). In P falciparum, the N-terminal domain of SERA 5 is showing promise as a potential vaccine candidate . The recombinant protein was expressed in Escherichia coli and affinity purified by a GST tag. Knowlesi -exposed hospital clinical case control samples showed antigen specific reactivity to the SERA3 antigen 2 recombinant when compared to responses from European malaria naïve and Ethiopian vivax-exposed serum samples  Eluates were tested in duplicate at a final concentration of 1:1000 for all antigens except 1:2000 for PfAMA-1. In addition, blank wells and a dilution series of the appropriate positive plasma pool were added per plate. Positive controls based on a hyper-immune endemic adult Tanzanian pool (PMID: 15792998), a lyophilised anti-malaria patient sample  and pooled Pk-exposed hospital serum samples were used to assay for P. falciparum, P. vivax and P. knowlesi antigens, respectively. Polyclonal rabbit anti-human IgG-HRP  was used at 1/15,000 dilution and plates were developed using TMB . Optical density (OD) values were measured at 450 nm with a microplate reader. Values in excess of 1.5 CV between duplicates were considered fails and re-ran. OD values were corrected by subtracting the background of the blank well per plate. For P. falciparum and P. vivax OD readings, values were normalised between plates using a standardised control. Normalisation was not done for P. knowlesi results due to the lack of standard control. All serological analysis was performed at the Universiti Malaysia Sabah and the London School of Hygiene and Tropical Medicine.Enzyme-linked immunosorbent assays (ELISA) were performed as previously described . BrieflyAll households and roads within these areas were geo-located using a hand-held GPS . Land cover maps were derived from LANDSAT 8 30m resolution satellite images  and supehttp://www.R-project.org).These classified land cover maps were used to calculate distance from the household to the forest edge. The proportions of different land types surrounding all households were evaluated for 100m, 500m and 1000m buffer radii. Additionally, the level of forest fragmentation was assessed within 500m and 1000m of each household; this was represented as the ratio of forest perimeter to forest area as described by . All geoP. falciparum and P. vivax as described by [P.knowlesi antigen a more parsimonious cutoff value was defined as the mean OD plus 5 standard deviations due to a lack of prior data. Because the assays were run in different laboratories, cut off values were defined separately for each antigen, malaria species and location . For P. falciparum and P. vivax, individuals were considered positive if they were positive for either MSP-1 and/or AMA-1. Reversible catalytic models were fit to age sero-prevalence data using maximum likelihood methods; these models were then used to generate age sero-prevalence curves and estimate the seroconversion rate (SCR) [Questionnaire data was collected electronically using Pendragon Forms VI  and analysed using R statistical software. To define sero-positive individuals, mixture models were fit for normalised optical densities (ODs), with the distribution of ODs modelled as two Gaussian distributions. Cut off values to define sero-prevalence for each antigen were defined as the mean OD of the sero-negative population plus 3 standard deviations for ribed by . For thete (SCR) . Evidencte (SCR) . Models P. knowlesi sero-positivity were evaluated using multivariate logistic regression, with household included as a random effect to account for correlation between individuals from the same household. An additional model was developed to compare individuals sero-positive for P. knowlesi with those sero-positive for non-zoonotic malaria species. Explanatory variables included age, gender, site, individual and household level farming activities, residence in the area, elevation and distance to forest. Additionally, the proportions and configuration of different land types were extracted for each household at 100m, 500m and 1000m radii and categorised as greater or less than 30% coverage within a specific radius in the final model. Univariate analysis was conducted for all explanatory variables and variables with p < 0.2 were included in multivariate analyses. For highly correlated variables (such as land cover proportions at different radii), single variables were selected based on marginal increases in Akaike Information Criterion (AIC). The final adjusted models were developed by retaining all variables significant at a 0.05 level and variables were added in a forward stepwise fashion to check for interactions. Potential residual spatial autocorrelation of exposure to P. knowlesi was assessed separately for all sites using Moran’s I.Risk factors associated with P. knowlesi and nonzoonotic malaria species was explored through correlograms, plots of spatial autocorrelation with lag distances. First, ODs were log-transformed and adjusted for age by linear regression as described by [P.knowlesi and each other antigen were plotted. Correlation ranges were determined by significance values (p < 0.05) of individual bins of lag distances of 500m. Pairwise correlation between antibody responses was determined using a simple Mantel test to test the significance of associations [Correlation between spatial patterns of exposure to ribed by . For eacciations , 36.P. knowlesi cases were reported from the Bacungan study site while one P. knowlesi case was reported in the Limbuak site and two cases were reported in Matunggong site. The median age of participants was 24 years (age range 3 months– 99 years) and similar proportions of men and women were sampled in all study sites. While only 22% (538/2503) of individuals reported their primary occupation as farming or plantation work, the majority of individuals  reported their household engaged in some agricultural activities  and Matunggong (n = 1162) sites in Sabah and in September 2014 in Bacungan, Palawan (n = 546). During this time, no clinical tivities . The protivities . The MatP. knowlesi mono-infections by PCR. All PCR infections were re-confirmed at the laboratory in the U.K. Both of these individuals were male plantation workers (ages 21 and 25) residing in the same household. An additional two individuals in Matunggong were microscopy negative but identified as P. knowlesi infected by molecular methods; these included a three year old girl and 33 year old woman residing in different villages within the study site. Only one out of these four infected individuals identified self-reported history of fever. None of the survey participants in either the Limbuak or Bacungan sites were positive by microscopy or PCR and no infections with any other malaria species were identified in any participants.Two microscopy positive individuals were identified from the Matunggong, Kudat site; these were both subsequently identified as P. knowlesi  followed by 6.8% (79/1162) in Matunggong Kudat. Bacungan, Palawan had the lowest sero-prevalence . Similar reactivity to P. knowlesi was observed in men : med: 0.035, IQR: 0.006–0.094) and women  and gender was not significantly associated with P. knowlesi sero-positivity .Overall, 7.1% (178/2503) of the population surveyed was seropositive to knowlesi . ExposurP. falciparum and P. vivax were higher in all sites, with 16.1% (364/2266) of individuals sero-positive to one or both P. falciparum antigens and 12.6% (270/2141) positive for one or more P. vivax antigens. Sero-prevalence to P. falciparum was 16.9% (196/1162) in Matunggong, 13.5% (107/795) in Limbuak and 10.4% (61/587) in Bacungan. In contrast, reactivity to P. vivax was highest in Limbuak  with sero-prevalences of 6.9% (80/1162) and 9.7% (57/587) in Matunggong and Bacungan respectively. Due to insufficient samples and non-systematic errors in labelling, results for all antigens were not available for all individuals. Out of individuals with complete test results for all antigens, 25.7% (499/1941) of participants were sero-positive to at least one species of malaria and 7.9% (54/1941) were sero-positive for two or more malaria species. Of individuals exposed to P. knowlesi, 29.7% (53/ 178) were also positive for P. falciparum or P. vivax antigens. There was no evidence of correlation between P. knowlesi and other antigens tested  had antibodies to P. falciparum and 2.9% (23/792) to P. vivax. Changes in age sero-prevalence were more pronounced for P. falciparum and P. vivax, with 32.9% (78/237) and 28.1% (64/228) reactivity to P. falciparum and P. vivax in individuals over the age of 60 years. In contrast, antibodies for P. knowlesi were detected in 9.4% (25/265) of individuals over 60 years old and the highest sero-prevalence was detected in adults from 45–60 years old . As reactivity to P. knowlesi was low and not evenly distributed through the population, seroconversion rates (SCR) for P. knowlesi could not be calculated. Historical changes in falciparum transmission intensity were apparent in all sites and SCR models fitted with two forces of infection suggest substantial reductions in P. falciparum transmission occurred 18–30 years ago (p < 0.05)  . Strong P. knowlesi sero-positivity . There was no significant spatial correlation detected between age-adjusted antibody responses for P. knowlesi and other malaria species for either Matunggong or Limbuak . Comparisons between P. knowlesi and other malaria species could not be evaluated for Bacungan due to the low prevalence of P. knowlesi sero-positivity.Based on Moran’s I, there was no evidence of residual spatial autocorrelation for P. knowlesi through antigen specific antibody responses and associated risk factors and is one of few studies to assess P. knowlesi carriage prevalence at a community level. Results indicate spatial and temporal patterns of P. knowlesi transmission differ markedly from other non-zoonotic malaria species within the region. Although P. knowlesi sero-positivity was associated with some landscape attributes within these communities, extensive cross sectional surveys are needed to identify ecological risk factors across a broader geographic scale.This is the first study to describe exposure to P. knowlesi transmission intensity between sites. Although formal comparisons between P. knowlesi infection and exposure could not be undertaken due to the low prevalence of parasite carriage, these geographical differences in transmission mirror hospital-based reporting rates in the study sites at Kudat, Pulau Banggi and Palawan [P. knowlesi and P. vivax indicate the possibility of cross reactivity between antigens, relatively low numbers of individuals were identified as sero-positive for both knowlesi and vivax  and individuals could have been plausibly exposed to both species due to the co-endemicity of these species within this region. Additional work has been done to characterise response to P. knowlesi in vivax-exposed individuals and validate these antigens for population-based studies[Sero-prevalence data indicate distinct heterogeneities in  Palawan –25. Thesd studies.P. falciparum and P. vivax cases within the past 20 years following the scale up of malaria control and elimination programmes [P. falciparum[P. knowlesi exposure was identified in children under 5 in all sites, suggesting recent or on-going transmission albeit at a low level. Further work is needed to refine P. knowlesi serological analysis to allow for antigenic variation, identify further antigenic targets and assess the differential responsiveness of individuals and longevity of antibody responses [Changes in seroconversion rates can also reflect temporal changes in malaria transmission. In Sabah, state-wide malaria notification records describe dramatic decreases in clinical alciparum. These cesponses .P. knowlesi, levels of exposure between different demographic groups varied markedly from clinical case reports. While clinical P. knowlesi has been commonly reported in adult men, men and women had similar antibody reactivity to P. knowlesi antigens in all sites [P. knowlesi infection, more men could develop clinical infections due to higher exposure or number of bites; however, this requires further research to assess [P. knowlesi affects groups which may be underrepresented by current passive surveillance systems.Despite these similarities between existing case data and community-level exposure to ll sites , 39. Witll sites . Asymptoll sites , 12. As o assess . Larger P. knowlesi exposure was also associated with landscape factors. Both the proportion of forest cover and cleared areas around the household were positively associated with knowlesi sero-positivity, potentially reflecting the higher vectorial capacity and sporozoite rates reported in secondary forest within these study sites [P. knowlesi and forest activities, data on movement into different environments was not available for all survey participants [dy sites . Althougicipants , 41. Insicipants , 42, 43.P. knowlesi were more likely to reside in areas with higher proportions of forest cover; this may reflect differences in disease dynamics between species or temporal changes in transmission. Because of the longevity of antibody responses and the rapid rates of land use change within these areas, seroreactivity to non-zoonotic species is probably more likely to be associated with past rather than current environmental factors. The main mosquito vector species of P. knowlesi, Anopheles balabacensis, was historically incriminated as the main vector of other human malaria species within these same areas [These spatial patterns differed markedly from exposure to other non-zoonotic malaria species. Individuals with antibodies to me areas , 45. Whime areas , 42, 46.me areas .P. knowlesi risks across wider populations. As this study surveyed three relatively homogenous populations, there was minimal variation in environment, ethnicity, socioeconomic status and access to healthcare within each site. Identifying environmental and population-level risk factors will require randomised sampling across a wider ecological gradient; community level data on presence and absence of exposure and infection are required to understand spatial heterogeneity of disease transmission and develop and refine predictions of disease risk [The main limitations of this study are the non-randomised population sampling approach and limited geographical scale. While this study describes fine scale patterns of malaria exposure and infection within these three case study communities, these results cannot be generalised to extrapolate ase risk . AdditioP. knowlesi infection and exposure within these communities and illustrates how serologic markers can be used to describe differences in transmission intensity between malaria species in low transmission settings. Results from these surveys indicate patterns of P. knowlesi exposure and infection within the community may be substantially different from cases detected by passive surveillance systems. Cross sectional surveys across a broader geographical scale are needed to describe spatial variation in transmission intensity and identify associated environmental and population-based risk factors. Integration of serology into these surveys would provide vital information on rare infections for control programmes [Despite these limitations, this study describes ogrammes .S1 Checklist(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigP. falciparum AMA-1 antibody density; b. P. falciparum MSP-1 antibody density.a. (TIF)Click here for additional data file.S4 FigP. vivax AMA-1 antibody density; b. P. vivax MSP-1 antibody density.a. (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Background: The mechanisms underlying clustering of longevity in families are unclear. We have previously shown a low cancer incidence in offspring of long-lived siblings, i.e. cancer robustness. Here we test whether such offspring are also more resilient in terms of survival after cancer diagnosis. Methods: Identification of offspring from long-lived families was undertaken in three nationwide, consecutive Danish studies . Cancer cases were identified through linkage with the Danish Cancer Registry. Each offspring cancer case was matched with two control cancer cases from the 5% random sample of the Danish population. Matching criteria were birth year, sex, year of diagnosis and cancer site. The main outcome was overall survival. Factors studied were sociodemographic, health-related and cancer-related. Survival analyses were performed using stratified Cox proportional hazards models based on the matching data. Results: Among the 5,377 offspring of the 634 families, 465 offspring of long-lived siblings with first primary cancer were included, along with 930 controls. Offspring of long-lived siblings had a significantly better survival than controls (HR=0.64 95%CI=[0.52-0.78]). The association attenuated only slightly after adjustment of marital status, education, Charlson Comorbidity Index, and number of prescribed drugs (HR=0.66 95%CI=[0.54-0.81]). Conclusion: Our results suggest that in addition to being more robust to cancer risk, offspring of long-lived siblings are also more resilient to cancer after its diagnosis and show better overall survival compared to individuals with cancer from general Danish population. Funding: The LLFS study is funded by the US National Institute on Aging / National Institutes of Health.