Plasmodium falciparum malaria. We found that the high prevalence of severe and profound thrombocytopenia in patients with severe non-P. falciparum malaria at 47% and 20%, respectively. Moreover, deaths were seen in patients with severe and profound thrombocytopenia at 11%. Therefore, the present systematic review provided the new insight into the clinical relevance of severe and profound thrombocytopenia in diagnosing and managing non-P. falciparum malaria. Severe thrombocytopenia should serve as a warning sign of increased chances of complications. Meanwhile, profound thrombocytopenia should serve as a marker of mortality among patients with non-P. falciparum malaria.Alteration of platelet count is frequently seen in patients with malaria. However, the platelet biology under severe pathological conditions like severe malaria remains unclear. The present systematic review collated literatures and synthesized the evidence regarding the risks of severe and profound thrombocytopenia in patients with severe non-Plasmodium falciparum malaria. Our overall aim was to identify potential indicators of severe non-P. falciparum malaria and the Plasmodium species that cause severe outcomes. This systematic review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) under registration ID CRD42020196541. Studies were identified from previous systematic reviews (n = 5) and the MEDLINE, Scopus, and Web of Science databases from 9 June 2019 to 9 June 2020. Studies were included if they reported the outcome of severe non-Plasmodium species infection, as defined by the World Health Organization (WHO) criteria, in patients with known platelet counts and/or severe and profound thrombocytopenia. The risk of bias was assessed using the Newcastle–Ottawa Scale (NOS). Data were pooled, and pooled prevalence (PP) and pooled odds ratios (ORs) were calculated using random effects models. Of the 118 studies identified from previous meta-nalyses, 21 met the inclusion criteria. Of the 4807 studies identified from the databases, three met the inclusion criteria. Nine studies identified from reference lists and other sources also met the inclusion criteria. The results of 33 studies reporting the outcomes of patients with severe P. vivax and P. knowlesi infection were pooled for meta-analysis. The PP of severe thrombocytopenia (reported in 21 studies) was estimated at 47% : 33–61%, I2: 96.5%), while that of profound thrombocytopenia (reported in 13 studies) was estimated at 20% . The pooled weighted mean difference (WMD) in platelet counts between severe uncomplicated Plasmodium infections (reported in 11 studies) was estimated at −28.51% , while the pooled WMD in platelet counts between severe non-Plasmodium and severe P. falciparum infections (reported in eight studies) was estimated at −3.83% . The pooled OR for severe/profound thrombocytopenia comparing severe to uncomplicated Plasmodium infection was 2.92 . The PP of death from severe and profound thrombocytopenia was estimated at 11% (95% CI: 0–22%). These results suggest that individuals with severe non-P. falciparum infection (particularly P. vivax and P. knowlesi) who exhibit severe or profound thrombocytopenia should be regarded as high risk, and should be treated for severe malaria according to current WHO guidelines. In addition, severe or profound thrombocytopenia coupled with other clinical and microscopic parameters can significantly improve malaria diagnosis, enhance the timely treatment of malaria infections, and reduce the morbidity and mortality of severe non-P. falciparum malaria. The understanding of platelet biology under physiological and pathological conditions like malaria infection is critical importance in the context of the disease outcome or model systems used. The importance of severe thrombocytopenia  and profound thrombocytopenia  in malaria patients remains unclear. This study aimed to synthesize evidence regarding the risks of severe and profound thrombocytopenia in patients with severe non- Plasmodium falciparum and Plasmodium vivax infections, thrombocytopenia is associated with increased mortality [P. falciparum malaria are increased platelet activation and consumption by factors such as the cytoadherence of infected erythrocytes and subsequent endothelial damage [P. falciparum and P. vivax malaria and is associated with bleeding [An understanding of platelet biology under physiological and pathological conditions like malaria infection is of critical importance in the context of the disease outcome or model systems used. Platelets are produced from megakaryocytes and play a crucial role in hemostasis and thrombus formation . Thromboortality ,6. The pl damage , platelel damage , bone mal damage . Severe l damage . Severe bleeding  and dissbleeding .Plasmodium parasitemia and one or more of the following manifestations: impaired consciousness, prostration, acidosis, hypoglycemia, severe malarial anemia, renal impairment, jaundice, pulmonary edema, significant bleeding, shock, or hyperparasitemia [P. falciparum malaria, although it is not included in the current World Health Organization (WHO) criteria for severe P. falciparum malaria [P. falciparum malaria and profound thrombocytopenia were five times more likely to die than patients with higher platelet counts [P. falciparum infection, information on the prevalence of severe or profound thrombocytopenia in patients with non-P. falciparum malaria  is limited. The most recent systematic review on severe thrombocytopenia in P. vivax malaria demonstrated that severe thrombocytopenia was equally common in P. vivax and P. falciparum malaria [P. vivax infection. To clarify this issue, we conducted a meta-analysis of studies reporting severe or profound thrombocytopenia in patients with severe P. vivax, P. malariae, P. ovale, and P. knowlesi malaria. Our goal was to assess whether severe or profound thrombocytopenia could inform clinical decision-making, and whether the degree of thrombocytopenia had predictive utility independent of current clinical and laboratory prognostic indices.Severe malaria is defined as the presence of asitemia . Severe  malaria . A previt counts . Althoug malaria . HoweverThis systematic review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) under registration ID CRD42020196541.Plasmodium falciparum including P. vivax, P. malariae, P. ovale, and P. knowlesi infections; (3) severe thrombocytopenia  or profound thrombocytopenia ; (4) severe and uncomplicated malaria with known platelet counts; or (5) severe P. falciparum and non-P. falciparum infections with known platelet counts. The following types of studies were excluded: (1) studies examining severe non-Plasmodium infection co-morbidity with other diseases, (2) studies conducted among pregnant patients, and (3) studies in which platelet counts could not be extracted. The systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (see PRISMA Checklist S1).Original studies were included if they were observational, prospective, or retrospective in design, and reported outcomes of patients with: (1) severe malaria as defined by the WHO ; (2) non-n = 5) reporting outcomes of severe P. vivax, P. malariae, P. ovale, and P. knowlesi infections. The second source of studies was a systematic search of relevant databases  between 1 June 2020 and 9 June 2020 using MeSH headings (Severe OR complicated OR complication) AND  AND (thrombocytopenia OR “low platelet”), with no restrictions on language. The third source of studies was the reference lists of the included studies as well as other sources . Two authors (AM and MK) independently screened the titles and abstracts. Any disagreements in full-text article selection were resolved by consensus. Potentially relevant full-text articles were obtained, read, and extracted by two independent authors concerning the inclusion criteria. Data were extracted into a standardized form using an Excel spreadsheet. For all studies, data on authors, year of publication, study location, year of publication, and study design were extracted. In addition, data on mean/median age of participants, male percentage, mean/median platelet count, parasitemia level, number of patients with severe and profound thrombocytopenia, Plasmodium species, number of patients with severe malaria, number of patients with severe or profound thrombocytopenia, and number of deaths were extracted. Data extraction was undertaken by one author and checked by all authors.Potentially relevant studies were identified from three sources. The first source of studies was previous systematic reviews  and not the comparator groups. Therefore, only three domains were used to assess the included studies: case definition, representativeness of cases, and ascertainment of the outcome. The risk of bias was reported as low (1 star), moderate (2 stars), or high (3 stars) based on the total of the three scores retrieved from each domain. Any discrepancies in ratings were addressed in the discussion.The risk of bias was independently assessed by two authors (A.M. and M.K.). As the included studies were observational retrospective, prospective, and case-control studies, the Newcastle–Ottawa Scale (NOS) tool was used . The NOSP. falciparum infection, estimated using the random-effects DerSimonian and Laird models [P. falciparum infection compared with individuals with uncomplicated non-P. falciparum infection, estimated using fixed- or random-effects models depending on the level of heterogeneity; (3) pooled ORs in individuals with severe non-P. falciparum infection compared with individuals with severe P. falciparum infection, estimated using fixed- or random-effects models depending on the level of heterogeneity. The heterogeneity and level of heterogeneity of studies were assessed using Cochran’s Q and I2 (inconsistency) statistics, respectively. Publication bias was evaluated using visual inspection of funnel plots and Egger’s tests. If any small-study effects were observed, a further contour-enhanced funnel plot was generated to assess if asymmetry resulted from publication bias or other factors. The meta-analysis was carried out using Stata software. To explore whether a single study affected the heterogeneity or the conclusions of the meta-analysis, sensitivity analyses omitting each study were performed in turn. Subgroup analyses were conducted to evaluate potential sources of heterogeneity.The following three outcomes were analyzed by meta-analysis: (1) PP and 95% confidence interval (CI) of severe and profound thrombocytopenia in individuals with severe non-d models ; (2) pooP. falciparum malaria were retrieved from previous systematic reviews and meta-analyses [Twenty-one studies reporting the outcomes of severe non-analyses ,20,21,22analyses ,52,53,54P. falciparum malaria. The included studies were prospective or retrospective observational studies, case-control studies, and cohort studies. All studies excluded individuals with P. falciparum single or mixed infections. Most of the included studies reported the outcomes of severe non-P. falciparum malaria in adults, while Gupta et al., 2016 [P. falciparum malaria in children younger than 7 years. Most of the included studies reported outcomes of predominantly male individuals who developed severe non-P. falciparum malaria, while Sharma et al., 2012 and Singh et al., 2011 reported a higher proportion of women who developed severe non-P. falciparum malaria. All non-P. falciparum malaria cases reported in the included studies involved P. vivax and P. knowlesi malaria. Of the included studies, 30 [P. vivax single infections, while four [P. knowlesi infections. Barber et al., 2013 [P. vivax and severe P. knowlesi infections. Other characteristics of the included studies are shown in Thirty-three studies from Asia  from 18 studies [P. vivax malaria was 42% . The prevalence reported in individual studies varied highly (range: 3–86%). Data on severe thrombocytopenia in patients with severe P. knowlesi malaria were available from three studies [P. knowlesi malaria (77 cases) was 80% . The prevalence reported in the three individual studies of severe P. knowlesi malaria was consistent, ranging from 71% to 84%. Overall, the PP of severe thrombocytopenia (440 cases) in patients with severe P. vivax and severe P. knowlesi malaria (885 cases) was 47%  (Data on severe thrombocytopenia 349 cases) in patients with severe 9 cases i studies ,50,51,52 studies ,53,54. T: 96.5%) .P. vivax malaria (954 cases) from 12 studies [P. vivax malaria was 21% . The prevalence reported in individual studies varied highly (range: 2–67%). Data on profound thrombocytopenia (13 cases) in patients with severe P. knowlesi malaria (60 cases) were available from two studies [P. knowlesi malaria (77 cases) was 17% . The prevalence reported in individual studies varied highly (range: 6–26%). Overall, the PP of profound thrombocytopenia (202 cases) in patients with severe P. vivax and severe P. knowlesi malaria (1014 cases) was 20%  (Data on profound thrombocytopenia (189 cases) in patients with severe  studies ,45,48,49 studies ,53. The : 85.2%) .P. vivax malaria (557 cases) and uncomplicated P. vivax malaria (2451 cases) were available from eight studies [P. vivax malaria were significantly lower than those of patients with uncomplicated P. vivax malaria . Data on mean platelet counts in patients with severe P. knowlesi malaria (124 cases) and uncomplicated P. knowlesi malaria (318 cases) were available from four studies [P. knowlesi malaria were significantly lower than those of patients with uncomplicated P. knowlesi malaria . Overall, the pooled WMD showed the platelet counts of patients with severe non-P. falciparum malaria (681 cases) were significantly lower than those of patients with uncomplicated non-P. falciparum malaria (2769 cases)   .P. vivax malaria (252 cases) and severe P. falciparum malaria (576 cases) were available from seven studies [P. vivax malaria and severe P. falciparum malaria were comparable . Data on mean platelet counts in patients with severe P. knowlesi malaria (85 cases) and severe P. falciparum malaria (26 cases) were available from two studies [P. knowlesi and severe P. falciparum malaria were comparable . Overall, the pooled WMD showed that the platelet counts of patients with severe non-P. falciparum (337 cases) and severe P. falciparum malaria (610 cases) were comparable   .P. falciparum malaria was performed using age, male ratio, and parasite counts as co-variates. The results of the univariate meta-regression analysis showed that the difference in platelet counts between the two groups was not confounded by age, male ratio, or parasite counts  and uncomplicated P. vivax malaria (1647 cases) were available from five studies [P. vivax malaria had significantly higher odds of severe or profound thrombocytopenia than those with uncomplicated P. vivax malaria . Data on severe thrombocytopenia in patients with severe P. knowlesi malaria (55 cases) and uncomplicated P. knowlesi malaria (185 cases) were available from two studies [P. knowlesi malaria had significantly higher odds of severe or profound thrombocytopenia than those with uncomplicated P. knowlesi malaria . Overall, the odds of severe or profound thrombocytopenia were significantly higher in patients with severe non-P. falciparum malaria compared with those with uncomplicated non-P. falciparum malaria   .P. vivax malaria (381 cases) and severe P. falciparum malaria (396 cases) were available from seven studies [P. vivax and severe P. falciparum malaria were comparable . Data on severe thrombocytopenia in patients with severe P. knowlesi malaria (32 cases) and severe P. falciparum malaria (10 cases) were available from one study [P. knowlesi and severe P. falciparum malaria . Overall, no significant difference was observed in the odds of severe or profound thrombocytopenia between patients with severe non-P. falciparum and severe P. falciparum malaria   .P. vivax malaria were available for extraction. Deaths resulting from severe P. vivax malaria in patients with severe or profound thrombocytopenia were investigated. Mortality related to severe thrombocytopenia in patients with severe P. vivax malaria  was 24% (95% CI: 0–45%) [Data on deaths from severe  studies ,39,50) w: 0–45%) .p = 0.041, t-statistic: 2020, coefficient: 4.48, standard error: 2.04). A contour-enhanced funnel plot was generated and showed that the asymmetry of the funnel plot resulted from publication bias in most studies  and severe P. knowlesi malaria (80%). In addition to severe thrombocytopenia, we found that the PP of profound thrombocytopenia was low in patients with severe P. vivax malaria (21%) and severe P. knowlesi malaria (17%). The PP of severe and profound thrombocytopenia in patients with severe P. vivax malaria could not be precisely estimated because of the high heterogeneity among the included studies (I2 > 86%). In addition, the PP of severe thrombocytopenia in patients with severe P. knowlesi malaria could be reliably estimated because of the homogeneity of the included studies (I2: 0%). Nevertheless, we caution that the homogeneity among studies reporting the outcomes of patients with severe thrombocytopenia in P. knowlesi malaria could result from the relatively small number of included studies. Severe thrombocytopenia is not currently considered an indicator of severe malaria according to the WHO criteria [P. falciparum malaria, it may be worth investigating individuals with severe malaria and severe or profound thrombocytopenia. Clinicians managing patients with malaria should be aware of the risks, as these individuals are more likely to die than patients with higher platelet counts [Thrombocytopenia frequently occurs in individuals with  malaria ,56. The of cases . In addi malaria . Moreoveytopenia ,58,59. Tcriteria . Interest counts . Furthert counts .P. falciparum, P. vivax, and P. knowlesi infection [Plasmodium species [Plasmodium infections, significant bleeding is often unrelated to the level of thrombocytopenia [P. vivax and severe P. knowlesi malaria were lower than those of patients with uncomplicated P. vivax and P. knowlesi malaria. Although high heterogeneity was present in the pooled analysis, these results demonstrated the significant diagnostic value of lower platelet counts in patients with severe non-P. falciparum malaria compared with patients with uncomplicated malaria. In the two-by-two meta-analysis of proportion, individuals with severe P. vivax malaria had more than three times the odds of developing severe or profound thrombocytopenia than those with uncomplicated P. vivax malaria. Lower odds of severe or profound thrombocytopenia were observed in patients with severe P. knowlesi malaria. The pooled ORs from the meta-analysis were reliable because of the low heterogeneity among the included studies. From the results of meta-analysis, most of the included studies showed lower mean platelet counts in patients with severe P. vivax malaria than in those with uncomplicated malaria [P. vivax malaria. Arévalo-Herrera et al., 2015 reported a wider range of platelet counts in patients with severe P. vivax malaria  than in those with uncomplicated P. vivax malaria, but no difference in the risk of spontaneous bleeding associated with thrombocytopenia [P. knowlesi malaria were similar to those in patients with severe P. vivax malaria: all included studies reported a significantly lower mean platelet count in these patients than in those with uncomplicated P. knowlesi malaria. Thrombocytopenia in patients with P. knowlesi infections is common, and platelet counts were lowest in cases of severe disease [P. knowlesi malaria exhibited higher mean platelet counts than those with severe P. vivax malaria.Thrombocytopenia is a common feature of malaria resulting from nfection ,30,55. T species . Althougytopenia . Therefoytopenia  and not ytopenia . The potytopenia . The pre malaria ,38,43,45 malaria  demonstrytopenia . Trends  disease . HoweverP. vivax/P. knowlesi malaria and severe P. falciparum malaria. The two-by-two meta-analysis of proportion confirmed that neither severe nor profound thrombocytopenia was significantly different in patients with severe P. vivax/P. knowlesi malaria and severe P. falciparum malaria. The meta-analysis results were reliable because of the low heterogeneity in the pooled analysis of proportion. Our results demonstrated that severe and profound thrombocytopenia might affect the risk of death in individuals with severe P. vivax malaria. The data reported by four included studies [P. vivax malaria exhibited severe and profound thrombocytopenia. Infection by other Plasmodium species, including P. malariae and P. ovale malaria, resulted in severe thrombocytopenia less frequently than P. vivax malaria [P. falciparum malaria [P. ovale wallikeri infection compared with those with P. ovale curtisi infection [P. falciparum infection, patients with profound thrombocytopenia were five times more likely to die than patients with higher platelet counts, suggesting that thrombocytopenia might be a marker of disease severity in patients with falciparum malaria [P. falciparum and P. vivax suggested that severe thrombocytopenia had a sensitivity and specificity of 65.6% and 70.6%, respectively, to discriminate against severe malaria. Therefore, it is valuable to further investigate the sensitivity and specificity of thrombocytopenia using large longitudinal studies to conclude the performance of using platelet as an indicator for severe non-P. falciparum malaria. In light of our analyses for non-P. falciparum malaria, the mortality rate in patients with profound thrombocytopenia was higher than those with severe thrombocytopenia, suggesting the possibility of using thrombocytopenia to identify the risk of death in patients with severe non-P. falciparum malaria.We found no significant differences in the mean platelet counts of patients with severe  studies ,39,49,50 malaria ,65 and Pnfection . In P. f malaria ,57. The P. falciparum malaria deficient in a disintegrin and metalloprotease with thrombospondin type I repeats, member 13 (ADAMTS13), had higher frequencies of severe thrombocytopenia and other complications [P. falciparum molecules and release prepackaged inflammatory mediators, leading to decreased signaling and inflammatory responses [The pathogenic mechanisms underlying thrombocytopenia and the mechanisms through which platelet numbers affect severe malaria and death remain unclear. However, previous studies have shown that endothelial activation  results ications . The higesponses ,71. Anotesponses .In addition to identifying new candidate makers for severe malaria, new diagnostic tools for detecting malaria parasites are challenging. Recently, magnetic resonance relaxometry (MRR) ,74,75, oP. falciparum malaria, particularly of P. knowlesi are fragmented, and is a research gap for malaria studies. The present study, P. vivax studies and two P. knowlesi studies. P. vivax studies and one P. knowlesi study, while P. vivax studies. It would be interesting to determine whether such comparability in the severity or profoundness of thrombocytopenia and related outcomes was due to the limitations in the available studies or unique biological traits that exist within non-P. falciparum species that induce host responses. The detection of human cases of P. knowlesi demonstrated an ongoing increase despite effective control programs to disrupt the transmission of human-only Plasmodium species [P. knowlesi malaria can be found in the changing patterns in land use where spillover opportunities for human infection due to increased contact with natural reservoirs and infected vectors have been aggravated. In addition, the range and distribution of primary hosts and vectors including their bionomics are still lacking in data [P. falciparum malaria and host immunological responses to the same.Biological and observational studies of non- species . Some no in data . These aP. vivax and severe P. knowlesi malaria. Therefore, the prevalence of severe or profound thrombocytopenia was not assessed among patients with severe P. malariae, P. ovale, or mixed malaria. Second, the prevalence of severe or profound thrombocytopenia-related death could not be precisely estimated because of the limited number of included studies. Despite these limitations, the presence of severe or profound thrombocytopenia in malaria patients may support the diagnosis of malaria, particularly in patients with severe non-P. falciparum malaria, because low levels of parasitemia are frequently encountered in routine diagnosis. In addition, severe or profound thrombocytopenia could be used as an indicator of increased risk of poor outcome and mortality if there are delays in diagnosis. Our results suggest that severe and/or profound thrombocytopenia should be included in the WHO criteria for severe non-P. falciparum malaria because a high prevalence of severe thrombocytopenia was observed in these patients (47% for severe thrombocytopenia and 20% for profound thrombocytopenia).Our study had several limitations. First, the data on platelet counts and the frequency of severe or profound thrombocytopenia could only be extracted from studies of patients with severe P. falciparum malaria. Severe thrombocytopenia should serve as a warning sign of increased chances of complications. Meanwhile, profound thrombocytopenia should serve as a marker of mortality among patients with non-P. falciparum malaria.The present systematic review provided the new insight into the clinical relevance of severe and profound thrombocytopenia in diagnosing and managing non-P. falciparum malaria and thrombocytopenia. Our results suggest that individuals with severe non-P. falciparum malaria, particularly P. vivax and P. knowlesi malaria, with severe or profound thrombocytopenia should be regarded as high-risk patients and should subsequently be treated for severe malaria according to the current WHO guidelines. In addition, severe or profound thrombocytopenia coupled with other clinical and microscopy parameters, may significantly improve malaria diagnosis, provide timely treatment for malaria infections, and reduce the morbidity and mortality of severe non-P. falciparum malaria. Observational studies and research on the parasite, vector, and host biology and outcomes for non-P. falciparum malaria is still fragmented. Moreover, the changing patterns of land use that increase the exposure of natural reservoirs and infected vectors, range distribution of hosts, and vectors, including the biomics of non-P. falciparum malaria, require further and more in-depth exploration.A meta-analysis provides enhanced statistical power, thereby providing more robust and reliable knowledge of the effects of severe non-


Plasmodium growth in vitro and in mammalian hosts. The parasite alters metal homeostasis in red blood cells to its favor, for example metabolizing hemoglobin to hemozoin. Metal interactions with the mosquito have not, however, been studied. Here, we describe the metallomes of Anopheles albimanus and Aedes aegypti throughout their life cycle and following a blood meal. Consistent with previous reports, we found evidence of maternal iron deposition in embryos of Ae. aegypti, but less so in An. albimanus. Sodium, potassium, iron, and copper are present at higher concentrations during larval developmental stages. Two An. albimanus phenotypes that differ in their susceptibility to Plasmodium berghei infection were studied. The susceptible white stripe (ws) phenotype was named after a dorsal white stripe apparent during larval stages 3, 4, and pupae. During larval stage 3, ws larvae accumulate more iron and copper than the resistant brown stripe (bs) phenotype counterparts. A similar increase in copper and iron accumulation was also observed in the susceptible ws, but not in the resistant bs phenotype following P. berghei infection. Feeding ws mosquitoes with extracellular iron and copper chelators before and after receiving Plasmodium-infected blood protected from infection and simultaneously affected follicular development in the case of iron chelation. Unexpectedly, the application of the iron chelator to the bs strain reverted resistance to infection. Besides a drop in iron, iron-chelated bs mosquitoes experienced a concomitant loss of copper. Thus, the effect of metal chelation on P. berghei infectivity was strain-specific.Iron and copper chelation restricts  Plasmodium have not been performed. Here, we address such micronutrient relationships in three steps. First, we characterized how the metallome fluctuates during development in two species of mosquito. Second, we asked whether susceptibility to Plasmodium infection correlated with a differential response in mosquito metal homeostasis. Third, we tested the effects of iron and copper chelation treatment of adult mosquitoes concerning propensity of infection and mosquito reproduction. Metal ions offer a promising target in the ongoing efforts to control the mosquito-borne disease.To establish a life cycle between insect and mammalian hosts, the malaria parasite has evolved mechanisms to manage metal ions from the distinct microenvironments it encounters. Previous work has addressed how interference using metal chelation affects parasite development in human, primate, and rodent hosts. Similar studies in mosquito species that harbor  Wolbachia endosymbiont [Drosophila melanogaster [Aedes aegypti. Αn iron regulatory protein has also been characterized in Culex pipiens [Anopheles albimanus [Ae. aegypti, where instead homologues of the Zip/ZnT families of transporters may have taken over cellular iron import [Anopheles gambiae [Ae. aegypti [The role of mosquitoes as vectors of different diseases has long been recognized . Many stsymbiont , and prosymbiont . A thorosymbiont ,8. One snogaster –13–is menogaster ), includnogaster , the ironogaster , and ironogaster  in Aedes pipiens . A divallbimanus , but appn import ,21. Heme gambiae  and Ae.  aegypti .Ae. aegypti, the fate of blood meal iron was traced showing iron accumulation in female ovaries [Wolbachia interferes with insect iron metabolism [Plasmodium gametogenesis in the intestinal lumen of Anopheles stephensi mosquitoes [D. melanogaster see [Culex quinquefasciatus [An. gambiae [Ae. aegypti [Ae. aegypti and Aedes albopictus mosquitoes [Ae. albopictus, An. stephensi, and C. pipiens, respectively–resulted in developmental delays [Ae. aegypti and An. albimanus throughout the different stages of their life cycle.In  ovaries ,25. It itabolism ,27. The tabolism . It is asquitoes . Amongstsquitoes , recent squitoes  and unrasquitoes ,33 in irster see –36). Beyasciatus , An. gam gambiae  and Ae.  aegypti , whereassquitoes –44. Lowel delays . ConsideAe. aegypti strains, one originally collected in the Carribean in the 1930s known as the Rockefeller (Rock) strain [in vitro arboviral infection [An. albimanus phenotypes, both isolated through inbreeding from a parental strain collected on human bait in the coastal plains of Southern México [P. vivax [P. berghei [white stripe (ws) due to the stripe’s presence, whereas the refractory (resistant) phenotype has been called brown stripe (bs). Field studies suggest that An. albimanus is only second to Anopheles pseudopunctipennis in Anopheles species population density in México, the two species together accounting for 92% of all individuals sampled [We opted to work with two laboratory-maintained ) strain ,47 and a) strain . The Roc) strain . Recentl) strain . Differe) strain  and follnfection , consistnfection ,53 playinfection ,54,55. Wn México . The twon México  and thoun México . The pheP. vivax  and P. b berghei . We refe sampled .in vitro [Plasmodium may itself cause iron deficiency to the human host [et al. studied P. falciparum in human erythrocytes, assessing copper’s role during the trophozoite stage of the parasite [et al. demonstrated that neocuprine blocked sexual differentiation of P. berghei and, hence, transmission to the mosquito vector [P. berghei mutant for a copper P-type ATPase transporter offered additional evidence in support of the conclusion that parasite copper homeostasis is critical to the parasite in the mosquito vector and to a smaller extent in a rodent host [Plasmodium protists require iron and copper to grow and differentiate; however, metal interactions have not been studied in the mosquito host. Here, we have assessed how the An. albimanus metallome is affected by P. berghei, using the susceptible ws and refractory bs phenotypes of the An. albimanus Tapachula strain [P. berghei for intestinal colonization.In malaria, the role for iron has long been studied and discussed, although the emphasis has been placed on the parasite and its interaction with the human host . Thein vitro ,64, in rin vitro ,66, and in vitro  hosts. Tin vitro ,62,68,69man host . On the parasite . These ao vector . Study oent host . Thus, aa strain , and appThe mosquito life cycle is divided into different stages of development . Adult fAn. albimanus mosquitoes used in this study originate from the Tapachula strain, which shows phenotypic variability to dorsal cuticle pigmentation  in the fe cycle . The datfe cycle .Ae. aegypti, the average increase  was five-fold for sodium; three-fold for copper, and two-fold for iron and potassium; in this species, calcium also showed a two-fold increase. For An. albimanus, the average increase was four-fold for sodium and 1.5-fold for iron, potassium, and copper. Little difference was observed between larvae and adult mosquitoes, adapted to different environments and physiologies, for the rest of the elements  feeding and same age mosquitoes for the sugar-fed group , magnesium (-19%), and manganese (-25%), and to an increase of potassium (+36%). Phosphorus was the only measured element that showed no change upon blood feeding  or 200 μM bathocuproine sulfate (BCS) from the emergence of the mosquitoes until the day of oocyst counting (same timeline as above) to chelate iron and copper, respectively. Mosquitoes were fed on P. berghei infected blood on day five of adulthood as in the previous experiments. These metal chelators have been used before in D. melanogaster at dietary concentrations between 100 and 300 μM, acting with proven metal specificities [Anopheles species [P. berghei infection affected follicular development in 51% (n = 47) of the susceptible ws female mosquitoes and 22% (n = 41) of the resistant bs counterparts  and 54% , respectively. This result is consistent with the previously shown requirement of iron for oogenesis in Ae. aegypti [p = 0.287 for ws and p = 0.076 for bs comparisons, respectively).We decided to explore the role of iron and copper during ficities ,75. As p species –78, P. bterparts . Applica aegypti ,25. EachAn. gambiae [C. pipiens [An. albimanus [P. berghei oocysts colonizing the An. albimanus midgut and the inhibition of FD. Therefore, we compared infection intensity considering the presence or absence of FD in each mosquito group (P. berghei infection in the otherwise susceptible ws phenotype (p = 0.0004) and five with the copper chelator (p = 0.01). The bs phenotype under control conditions showed a median infection of three and no statistically significant difference to the BCS-treated bs mosquitoes (median of five). Surprisingly, BPS-treated bs mosquitoes showed a median value of six (p = 0.03), reversing the normally resistant bs phenotype into a susceptible condition to infection . BCS had a clear and specific effect lowering copper accumulation by 33% in the bs phenotype. Although iron was not statistically significant different in bs mosquitoes fed with BPS, this treatment unexpectedly decreased copper by 43%  or inherent biological variance between generations [Plasmodium infection in Anopheles are called for. Further work with different mosquito species would also contribute to generalizations or identifying species-specific patterns in mosquito metallomics. Overall, the data reported here suggest similar metallome patterns between An. albimanus and Ae. aegypti with only few exceptions that were already described in detail above.Previous studies of how insect metallomes vary with respect to their evolutionary histories and specific ecology suggested that transition metal ions accumulate with the enzyme systems that require them as cofactors; variation amongst species was mostly attributable to changes in storage mechanisms or differing diets ,80. HemaO strain ; in conterations ,48. The erations –51, simiP. berghei infection between ws and bs phenotypes are unknown [An. gambie to P. bergei transmission identified the complement-like protein TEP1 interacting with the leucine-rich protein complex LRIM1 and APL1C for the parasite’s killing, followed by parasite elimination at a later step with melanization reactions under the control of NFkB signaling [bs strain, whereas melanization genes, such as PPO1, showed reduced expression [ws and bs phenotypes (discussed in the following paragraph). Of interest in respect to melanization is a recent discovery of a conserved transporter gene, which turns pupae of Tephritidae flies white when mutated [The genetic and physiological reasons that underlie the different susceptibility to  unknown –59. Prevignaling –86. Intepression , as obse mutated .An. albimanus. First, there is a moderate (10–30%) increase in zinc concentration in ws adult mosquitoes than similarly grown and aged bs counterparts  for 2–12 months kitten (Whiskas) [ws and bs phenotypes have been reared independently for more than sixty generations resulting in stable colonies  0.05% and antibiotic-antimicotic 1X (GIBCO). The An. albimanus colonies and the Ae. aegypti Rockefeller strain were reared at the insectary of the National Institute of Public Health (INSP) in Cuernavaca, Morelos, México. The Ae. aegypti New Orleans strain was reared at the insectary of the Collaborative Unit for Entomological Bioassays (UCBE) of the Autonomous University of Yucatán (UADY) in Mérida, Yucatán, México. Larval stages were fed with a meat flour and yeast (8:2) solution. Adult mosquitoes were fed with 10% sugar solution using a felt strip device. The metal ion content of the two diets was determined (The Whiskas) . The ws colonies . Adult mtermined .Plasmodium berghei ANKA strain, which constitutively expresses the green fluorescent protein [5 parasites were propagated IP up to the eighth pass. For ookinete culture, the mice were treated with 200 μl of phenylhydrazine at 6 mg/ml three days before the inoculation of 5 x 107 parasites. Three days post-inoculation the infected blood was extracted via cardiac puncture of a CO2 euthanized mice, using a heparinized syringe (250 IU of heparin / ml of blood). Blood was diluted 1:5 in ookinete medium  and incubated for 18 h at 20°C to allow ookinete formation. Only mice with a parasitaemia between 15 to 25% and with more than 7 exflagellation centers in a 400X field were used. The exflagellation was evaluated prior to mouse sacrifice by diluting tail-blood in ookinete medium (1:4), incubated at 20°C for 15 minutes.The clone 2.34 of the  protein , was use protein . Briefly5 ookinetes per ml in all experiments. Ookinetes were centrifuged at 1500g and re-suspended using blood of an uninfected mouse. After feeding, mosquitoes were kept at 21°C to permit Plasmodium development. Seven days after feeding, mosquitoes were dissected in phosphate buffered saline; midguts were collocated on slides over RPMI medium drops to permit oocyst counting using an epi-fluorescence microscopy (Leica DM 1000). Additionally, during midgut dissections, we obtained ovaries to determine the presence or absence of follicular development, as was previously reported in An. albimanus during P. berghei infection [For infection, four days-old mosquitoes were fed with 8 x 10nfection .i.e. for five days prior to and seven days after the Plasmodium infected blood meal.Bathophenanthrolinedisulfonic acid disodium salt hydrate  and bathocuproinedisulfonic acid disodium salt  were used as extracellular iron and copper chelators, respectively ,74. ChelAnopheles and Aedes mosquitoes were collected at different larvae instars. Three days-old male and female mosquitoes feeding exclusively on 10% sugar solution were used for the adult stages. Each sample was collected and dried in a precipitate vase glass (5 ml) during 72 hours at 50°C in order to remove water. Dried samples were collected using a flat spatula, weighed and placed in Eppendorf tubes. For the Plasmodium infection assays, mosquitoes were collected at day seven post-treatment. Each sample consisted of 30 females dried in a glass Petri dish and treated as explained above.For mosquito development assays, samples from Inductively coupled plasma optic emission spectrometry (ICP-OES) was used for metal determinations in all samples. Between 10 and 20 mg of dry sample was digested with 1 mL of nitric acid at 200°C for 15 min in a MARS6 microwave digestion system . Samples were adjusted to 5mL volume with water. Total metal concentrations were measured against a calibration curve in the PerkinElmer Optima 8300 ICP-OES  instrument and presented as mg metal per g sample dry weight.versus instar/stage) was used for analysis of the metallomics results during development (ws or bs) and treatment  as variables for the analysis of the metallomes after blood feeding and infection (ws versus bs) and Tukey’s test was used to determine differences between treatments in the same phenotype . The chi-squared test was used to determine the probability of follicular development between groups treated with chelators and control groups . Following inbreeding for over sixty generations, the two phenotypes have been successfully separated (lower images).The parental strain contains both (TIF)Click here for additional data file.S2 Figws (grey circles) and bs (brown diamonds) mosquitoes were determined at seven days post-feeding with P. berghei-infected blood. Data were analyzed with the Mann-Whitney U test, statistical difference is indicated by asterisks, ***p = 0.0006. Medians with interquartile range are indicated in black. Infection distributions for each phenotype are described in the table.Oocyst number per midgut of (TIF)Click here for additional data file.S3 Figws (left) and bs (right) phenotypes of An. albimanus. After blood-feeding, ovarian development was observed in both phenotypes. Ovaries from blood-fed (BF) or sugar-fed (SF) mosquitoes are indicated with arrows. Scale bar = 500 μm.Dissected ovaries from (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableAn. albimanus larvae.Results are given in mg element per g of diet. The diet used for NO was also used for rearing of (DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Fusobacterium nucleatum in human colorectal specimens has been demonstrated by RNA-sequencing. F. nucleatum has been shown to express high levels of virulence factors such as FadA, Fap2 and MORN2 proteins. Our review of the published data suggest that F. nucleatum may be a prognostic biomarker of CRC risk, and hence raises the potential of antibiotic treatment of F. nucleatum for the prevention of CRC.Colorectal cancer (CRC) is the third most common cause of cancer globally and the fourth attributable cause of mortality and morbidity due to cancer. An emerging factor contributing to CRC is the gut microbiota and the cellular changes associated with it. Further insights on this may help in the prevention, diagnosis and new therapeutic approaches to colorectal cancer. In most cases of CRC, genetic factors appear to contribute less to its aetiology than environmental and epigenetic factors; therefore, it may be important to investigate these environmental factors, their effects, and the mechanisms that may contribute to this cancer. The gut microbiota has recently been highlighted as a potential risk factor that may affect the structural components of the tumor microenvironment, as well as free radical and enzymatic metabolites directly, or indirectly. Many studies have reported changes in the gut microbiota of patients with colorectal cancer. What is controversial is whether the cancer is the cause or consequence of the change in the microbiota. There is strong evidence supporting both possibilities. The presence of  Lactic acid bacteria and Lactobacilli, streptococci, for example bifidobacterium. The gut microflora changes with changes in dietary habits and the selection of adult dietary patterns  OR F. nucleatum[Title/Abstract])] AND [Microbio*[Title/Abstract] OR Microbial[Title/Abstract] OR Diet[Title/Abstract] OR Dysbios*[Title/Abstract] OR Dysbacterios*[Title/Abstract] OR MicroRNA[Title/Abstract] OR miRNAs[Title/Abstract] OR “Micro RNA” [Title/Abstract] OR miRNA[Title/Abstract]] OR [Marker*[Title/Abstract] AND (Tumor[Title/Abstract] OR Carcinogen*[Title/Abstract] OR Neoplasm*[Title/Abstract] OR Cancer[Title/Abstract])) OR (Biomarker*[Title/Abstract] AND (Tumor[Title/Abstract] OR Carcinogen*[Title/Abstract] OR Neoplasm*[Title/Abstract] OR Cancer[Title/Abstract])) OR immunomodulator*[Title/Abstract]].The protocol was performed in accordance with the preferred reporting items for Systematic reviews and Meta-Analyses (PRISMA) guidelines , outlineFusobacterium nucleatum with colorectal cancer in patients and the published studies in English language. The exclusion criteria were: 1—No access to full-text articles 2—Case reports, randomized clinical trials and review articles 3—Studies on teenagers and 4—Duplicate records were excluded. Figure Three hundred and ninety one unique records were checked by title and abstract to assess their eligibility for inclusion in the project after finding a total of 497 papers and deleting the duplicate records. The full texts of 202 publications were then checked and the related articles were chosen according to the study inclusion criteria Fig. . The incCancer usually arises due to the failure of mechanisms controlling cell growth and proliferation. This control system responds to growth inhibition, growth and death signals. Colorectal carcinogenesis involves a series of well-defined changes that begin with a benign mucosal lesion called a polyp and can progress to malignancy leading to cancerous changes such as hyperplasia, adenoma, carcinoma, and metastasis . The molEnterococcus faecalis, enterotoxin Bacteroides fragilis or FadA in F. nucleatum are capable of damaging DNA, they can induce proliferation of colon cells in studies on gut microbiota in cancer patients [F. nucleatum as a parameter in the development and diagnosis of colorectal cancer.Gut dysbiosis can promote CRC through various processes that include: the induction of a chronic inflammatory disease or immune response, biosynthesis of toxic metabolites and genotoxin and effect of host metabolism , 67. Altpatients . Gut bacpatients . Most capatients . Consequpatients . In the Fusobacterium species were found to be enriched in colorectal carcinomas [Fusobacteria were observed in colorectal tumors by FISH. According to the obtained results, there are some changes in the microbiota in CRC [F. nucleatum and some common bacteria were found in the primary tumors but also in distant metastases [F. nucleatum was reduced following treatment with antibiotics, consistent with the causal role played by bacteria in the development of tumors [Fusobacterium in human tumors by the RNA-seq method was similar to the one obtained from mice tumors using flow cytometry [Fusobacteria are not part of the natural bacteria of the large intestinal flora. Studies show that cancerous tissues contain significantly more Fusobacteria [Sequences of rcinomas . The resa in CRC . F. nucltastases . Prelimif tumors . Preclinf tumors . The freytometry . In mostbacteria . Previoubacteria .Firmicutes, Bacteriodetes, Proteobacteria and Actinobacteria [Bacteroides, Eubacterium, Bifidobacterium, Fusobacterium, Peptostreptococcus, Atopobium and optional anaerobes, including Lactobacilli, Enterococci and Enterobacteria. However, diet, age, gender, and ethnicity affect individual microbes, making its dynamic nature difficult investigate [Proteobacteria is increased, and Bacteroidetes decreased [Lactobacillus spp. [Lactobacillus acidophilus in different forms may increase the anticancer effects by different mechanisms such as downregulation of ErbB-2, activation of natural killer cells, dendritic cell maturation, and release of probiotic-derived ferrichrome (iron-scavenging peptide) [L. casei probiotic-derived ferrichrome has its anti-tumor effect through the pathway contributing to JNK-mediated apoptosis [F. nucleatum can cause cancer by activating cellular signals through various mechanisms. These mechanisms are important for causing cancer in terms of cell surface receptors and their effects on the immune system  , 79. Thepeptide) , 81. Micpeptide) , 83. On peptide) –87. Manypeptide) . The micpeptide) . Modificpeptide) , 91. Prepeptide) . L. casepoptosis . They arpoptosis , 93. Thepoptosis . Moreovepoptosis . The micpoptosis . Many gepoptosis . Most stNF-kB), a transcriptional regulator activating at least 125 genes, most of which are pro-inflammatory. Glucose intake also increases the two pro-inflammatory transcription factors; activated protein 1 (AP-1) and early growth response protein 1 (Egr-1) [Doll and Peto have previously argued that 30% of the risk of cancer might be attributed to diet. Since then, much available evidence has pointed out that several nutrients can change cancer growth and progression . Long-te (Egr-1) . AP-1as  (Egr-1) . Egr-1 i (Egr-1) , and mod (Egr-1) , 101. In (Egr-1) . Nutrien (Egr-1) . The evi (Egr-1) . Other s (Egr-1)  and play (Egr-1) . Althoug (Egr-1) . Statist (Egr-1) . These rbifidobacteria, lactobacillus, bacteroids are mainly found in the colorectal and are beneficial for human health. They control the population of pathogenic bacteria by producing short-chain fatty acids such as acetic, butyric and propionic acid. Prebiotics are also a substrate choice for the growth of beneficial bacteria like bifidobacteria. Prebiotic foods include sugars such as inulin and oligofructose (FOS). During breastfeeding, the major bacterium in neonatal feces is bifidobacteria, but during weaning, the level of bifidobacteria is decreased and other bacteria such as coliform, Clostridium and Streptococcus are increased [Fusobacterium, Alistipes, Porphyromonadaceae, Coriobacteridae, Staphylococca-ceae, Akkermansia, Lactobacillus, Faecalibacterium, Roseburia and Treponema are present in patients with CRC [TLR2 was identified in this process [TLRin the development of colorectal cancer.Most studies indicate that the composition of the intestinal microbiota is formed before the age of three and then has a constant composition throughout life , 107. Soncreased . As morencreased . The diswith CRC , 113–118with CRC . Using a process . These fFusobacterium is a genus of anaerobic, Gram-negative, non-spore forming bacteria, similar to Bacteroides. F. nucleatum and F. necrophorum are usually members of the Fusobacterium species. They usually reside in the oral cavity and sometimes cause periodontal and gum infections [Fusobacterium is considered a risk factor involved in CRC start and improvement. Immune modulation is considered to be the most important mechanism of Fusobacterium playing a role in CRC carcinogenesis  [TLR2/TLR4 [TLRs are highly important. Among them, TLR4 is considered a representative receptor for LPS. When TLR4 is activated by LPS, a series of intracellular events are triggered. This leads to nuclear translocation of NF-κB, thereby increasing the expression of IL-8 gene [F. nucleatum doesnot encode any known toxins, while only few common virulence factors are encoded by it. Adhesion protein FadA is a known virulence factor in F. nucleatum contributing to easier attachment and invasion of bacteria [FadA binds with an E-cadherin receptor and increases carcinogenesis. It causes activation of β-catenin and stimulates expression of transcriptional factors, Wnt genes, inflammatory genes, and related oncogenes  cells. F. nucleatum would also inhibit T and NK cell activities [PRRs). They include CLRs (C-type lectin receptors), LRR (nucleotide-binding domain leucine-rich repeat) containing (NOD-like) receptors (NLRs), RLRs (RIG-I-like receptors), ALRs (AIM2-like receptors) and cytoplasmic DNA sensors [PRRs in the whole range of inflammatory responses, including cancer development and inhibition [PRRs signaling impacts all stages of intestinal cancer, from the early stages of cancer to the metastatic stage and appearance of different cells in the tumor microenvironment, and from neoplastic cells to tumor and stromal cells [MDSCs), thereby inhibiting CD4+ T cell. Inhibition of immune cells would be desirable for tumor cells, since it will spread the tumor to other parts of the body [F. nucleatum levels in mucosal or fecal samples of CRC patients has shown that F. nucleatum levels in CRC tissue is associated with the tumor site of right-sided proximal colorectum and CpG island methylator phenotype (CIMP) status, microsatellite instability (MSI) and mutations in BRAF, KRAS, CHD7, CHD8 and TP53 genes [E. cadherin on epithelial cells activates B-catenin signaling, increases NF-κB, C-myc expression and proliferates tumor cells [F. nucleatum, due to activation of Toll-like receptors (TLR), cause more mRNA expression and release. F. nucleatum induces lymphocyte cell death and tumor progression by blocking G1 phase cell myeloid derivative suppressor cells (MDSCs) and TIGIT receptor inhibition [F. nucleatum also affects the IL-6-STAT3 axis signaling and induces tumorigenesis by directly interacting with epithelial cells through activating TLRs. The key molecules stimulating tumor growth and invasion induced by these bacteria include IL-6, cyclin D1, TNFα, MMP9 and heparanase [Tumorigenesis mechanism of ignaling . Recentland IL8) , 119. ThLR2/TLR4 , 119. InL-8 gene . Howeverbacteria , 125. Fa unknown . When Farium/WNT . FadA aninvasion . FadA ise models . Thus, Fironment . Among ter cells . Metagenbacteria , 129. Thtivities . The fol sensors . Recent hibition . PRRs sial cells , 130. Smthe body . Investi53 genes . Increasor cells . Cells ihibition , 132. F.paranase .F. nucleatum is likely to affect the proliferation of cancer cells in the colorectal. According to epidemiological associations, F. nucleatum can improve instability and mutation of genes [IL.10 receptor (antiIL10R) and CpG oligodeoxynucleotides with the increased Alistipes shahii. In this model, A. shahii caused an increase in the production of the tumor necrosis factor (TNF) by intrauterine myeloid cells, while TNF neutralization abolished the therapeutic effect [B. fragilis stimulate pro-inflammatory Th17 cells that accelerate carcinogenesis in mice prone to the tumor [F. nucleatum can correctly identify patients with CRC. Recent studies have shown that F. nucleatum DNA in the early stages of the disease has the diagnostic potential as a non-invasive primary biomarker for CRC from fecal samples [Fusobacterium is associated with the signature of human CRC gene expression. A correlation of immune cell marker genes, including tumor-associated macrophages (TAMs) , MDSCs (CD33 and IL6), dendritic cells (DCs)  and Fusobacterium has been found in humans [Fusobacteria, in particular Fusobacterium, is also prevalent in CRC tissues despite being predominant in the oral microbiome. Using tissues from CRC patients that were positive for the 16S ribosomal RNA gene Fuso sequence and Th1 and Th17 cell populations in CRC patients by flow cytometer, there was a positive relation with both IL-17+ and IFNγ+ cytokines. These findings suggest that immune responses in CRC patients (Th1 and Th17) correlate with the frequency of Fusobacterium, especially the nucleatum [Fusobacterium-related genes, including PTGS2 (COX-2), IL1b, IL6, IL8 and TNF, are expressed not only in colon cancer but also in cultures of human and mouse cell lines in vitro known as the central link between inflammation and cancer [Fusobacterium-dependent proinflammatory genes is higher in colorectal tumors than in small bowel tumors. This may be due to the anatomical location related to the fact that the listed genes are derived from human CRC [F. nucleatum cause resistance to chemotherapy [F. nucleatum binds with the host epithelial E-cadherin and stimulates colorectal carcinogenesis through Fusobacterial adhesion FadA [F. nucleatum targets micro-RNA and autophagy Signaling via upregulation of CARD3 expression causing resistance to chemotherapy [Fusobacterium with recurrent CRC has even been postulated as a way to predict disease outcomes or change chemotherapy regimens such as inclusion of capecitabine and oxaliplatin for patients with a high burden of F. nucleatum [Fusobacterium [Fusobacterium anaerobic bacteria. Fusobacteria are highly sensitive to metronidazole. Finally, oral administration of metronidazole to mice that were Fusobacterium- positive resulted in a significant decrease in tumor growth pathways. Treatment with metronidazole resulted in a significant reduction in the Fusobacterium burden [Bacteroides fragilis (ETBF) is a toxin-producing bacterium that can activate TH17-mediated colitis, with simultaneous colon-specific STAT3 activation and tumor stimulation in susceptible ApcMin  mice, which is reversed by IL-17 antibody blockade [Fusobacterial colonization of carcinomas in experimental mice models [Fusobacterium burden are likely to recur, implying that Fusobacterium-positive tumors may benefit from anti-Fusobacterial treatment [Cancer in its simplest form of uncontrolled cell growth in association with of genes . In the he tumor . Compare samples . Fusobacn humans . Some T ucleatum . Fusobacd cancer . In geneuman CRC . Mouse suman CRC . In patiotherapy . F. nuclion FadA . It has otherapy , 45. Theacterium . One conblockade . This ise models . A recenmiR-17-92a and miR-25-106b [miR-21, miR-224, miR-200c miR-96, miR-135, miR-31, and miR-155 are related to pathogenesis of CRC [miRNAs in F. nucleatum induced CRC, while in a Multi-Class-Dif analysis, there was a significant expression of 96 miRNAs in early and advanced stages of CRC with positive infection of F. nucleatum [miR-4474 and miR-4717 expressions were upregulated in CRC with positive infection of F. nucleatum [CREBBP (CREB-binding protein), STAT1, CAMK2B, PRKACB, JUN, TP53 and EWSR1, which were involved in cancer signaling pathways were dysregulated [MiR-4802 and miR-18a* are abnormally reduced in expression by F. nucleatum that has been also known to induce chemoresistance to oxaliplatin and 5-FU by reduction of apoptosis through the activation of autophagy and TLR4/MYD88 signaling [Enrichment of Fusobacterium species is observed in the microbiota in carcinomas near healthy colonic tissue. They are observed in stool samples obtained from CRC patients at a higher degree compared to healthy controls. In the early stages of tumorigenesis, F. nucleatum that is usually present in the oropharynx [MicroRNAs (MiRNAs) are non-coding molecules of RNA with approximately a length of 19–25 nucleotides. At the post-transcriptional level, they regulate target genes expression negatively. It has been shown that oncogenic miRNAs (clusters of -25-106b ) play an-25-106b . Moreoves of CRC , 137. Thignaling . Enrichmopharynx , is in n14 [E. coli strain as a probiotic-gut bacterium producing a network of nanofibers that were directly attached to the mucosa [F. nucleatum colonization is the outcome or a cause of carcinogenesis or inflammation in colorectal tissue. The results produced some intriguing results representing Fusobacterium sp. as a potential biomarker for colorectal carcinogenesis. Above all, these results provide a mechanistic insight indicative of the mediation of Fusobacterium sp. actions through FadA binding to host epithelial cells’ receptors in order to change the function of barrier, to increase inflammation through modulation of the microenvironment of tumor, and to activate pro-oncogenic signals for CRC promotion. These findings can affect the prevention, diagnosis and treatment of CRC. However, further studies are required for evaluation of the FadA diagnostic potentiality. More certain answers on the temporal order between F. nucleatum and CRC can be found in prospective studies. Even though F. nucleatum colonization may result from colorectal cancer, nevertheless it may play a key role in tumor malignancy increasing, metastasis promoting and antitumor immunity evasion. Some interesting questions have been raised on cancer causes based on the role of F. nucleatum in tumorigenesis. It is possible to reduce the development of cancer through manipulation of bacterial microbiota by fecal microbial implants, probiotics and using antibiotic treatments or vaccination. Although fecal F. nucleatum may be a considered as a proper measurable biological marker for detection of CRC; further research is required to make it clear how it changes in different stages of colon cancer. A combination of microbiome modulation and its products with simpler immunotherapy approaches directly targeting malignant cells could be used in the future for antineoplastic therapy. The importance of this method is in the new anticancer method or enhanced therapeutic drugs against cancer. This method will have positive clinical results for patients with cancer. Oncomicrobiotics  is a new supportive treatment for cancer improving immune responses by enhanced gut function. Therefore, much more research is needed to be conducted on cross talk between host–bacteria and their virulence proteins that play a role in colorectal carcinogenesis.The gut microbiota is the largest reservoir of human microbiota. They consist of species of microorganisms living in the gastrointestinal tract in coexistence with the host, reaching a population of tens of 1014 . They in14 –142. Alt14 . Microbi14 . They al14 . Other i14 –148. Rese mucosa . This ste mucosa . This mae mucosa . The cone mucosa . It is s


Nannochloropsis has broad applicability to produce biofuels, animal feed supplements and other value-added products including proteins, carotenoids and lipids. This study investigated a potential role of N. oceanica in the reversal of metabolic syndrome. Male Wistar rats (n = 48) were divided into four groups in a 16-week protocol. Two groups were fed either corn starch or high-carbohydrate, high-fat diets  for the full 16 weeks. The other two groups received C and H diets for eight weeks and then received 5% freeze-dried N. oceanica in these diets for the final eight weeks  of the protocol. The H diet was high in fructose and sucrose, together with increased saturated and trans fats. H rats developed obesity, hypertension, dyslipidaemia, fatty liver disease and left ventricular fibrosis. N. oceanica increased lean mass in CN and HN rats, possibly due to the increased protein intake, and decreased fat mass in HN rats. Intervention with N. oceanica did not change cardiovascular, liver and metabolic parameters or gut structure. The relative abundance of Oxyphotobacteria in the gut microbiota was increased. N. oceanica may be an effective functional food against metabolic syndrome as a sustainable protein source.The microalgal genus  Nannochloropsis can be defined as biorefineries to produce biofuels, animal feed supplements and pigments [Nannochloropsis species have a wide range of applications in biotechnology, using techniques to modify biomass composition [Nannochloropsis species contain 28.7–40.4% carbohydrates, 22.2–37.4% crude protein and 15.1–21.7% total lipids on dry weight basis [Nannochloropsis species contain polyunsaturated fatty acids (PUFA), mainly eicosapentaenoic acid (EPA), polyphenols, carotenoids and vitamins with toxicological tests on animals confirming the safety of this microalga for consumption in food [Nannochloropsis, including N. oceanica, N. gaditana and N. oculata. Baseline information on the biology of N. oceanica has been published for use in the risk analysis of genetically modified N. oceanica in Australia [N. oceanica strains isolated in Southeast Queensland, Australia, showed improved lipid characteristics [Microalgae are unicellular organisms that, in the presence of sunlight, convert carbon dioxide into biomass . The mempigments ,3. Nannoposition . Nannochht basis . Nannocheristics .Nannochloropsis has been proposed as an appropriate nutritional supplement to increase PUFA and EPA intake as its EPA content can be rapidly increased, for example by low-level UV-C radiation [Nannochloropsis species on metabolic syndrome. Species such as N. gaditana and N. oculata alleviated oxidative stress and inflammation in streptozotocin-induced diabetic rats [N. oculata showed antihypertensive effects [N. oceanica, a source of EPA, on the combination of pathological changes that define metabolic syndrome.Edible microalgae may have therapeutic potential such as the reduction of signs of metabolic syndrome including hypertension, obesity, fatty liver and systemic inflammation . Nannochadiation ,7. Its iadiation ,12. Few tic rats , and a p effects . Omega-3 effects . HoweverN. oceanica farmed locally in a covered pond under controlled conditions [N. oceanica could reverse the signs of diet-induced metabolic syndrome. We tested the microalga using a validated diet-induced model of metabolic syndrome in rats that closely mimics the symptoms of human metabolic syndrome [N. oceanica starting 8 weeks after initiation of the obesogenic diet. As the microalgal cells are difficult to lyse in vivo to release EPA, we used the whole biomass to test the effect of components other than EPA, which has already been tested in metabolic syndrome [This project has tested an Australian strain of nditions . The aimsyndrome . Cardiovsyndrome .N. oceanica supplementation for the last eight weeks of the protocol will reverse the changes induced by the high-carbohydrate, high-fat diet. The mechanisms of these hypothesised effects could include the provision of essential amino acids to provide an increased lean mass as well as the actions of carotenoids, chlorophyll a and the omega-3 fatty acid EPA to decrease infiltration of inflammatory cells into organs such as the heart and liver.We measured cardiovascular parameters including systolic blood pressure, left ventricular diastolic stiffness, cardiac inflammatory cells and collagen deposition in the heart; liver parameters including plasma liver enzyme activities, inflammatory cells and fat vacuoles in the liver; and metabolic parameters including body weight, total cholesterol and triglyceride concentrations and glucose and insulin tolerance tests. Further, as functional foods may reverse obesity-induced changes in the gut microbiota ,18, we cN. oceanica (strain CS-179) was cultivated and harvested by Teraform microalgae farm, Miles, QLD, Australia during June and July 2018. The harvested paste (40 L) was transported frozen to the University of Southern Queensland, Toowoomba, QLD, Australia in sealed plastic containers. The paste was kept frozen until it was freeze-dried . Batches of 5 L were freeze-dried for 36–48 h at 0.011 mbar and −60 °C. The powder was stored at 4°C in sealed plastic containers until feeding or analysis. The composition of N. oceanica was obtained as described previously [eviously .n = 48) were sourced from Animal Resource Centre, Murdoch, WA, Australia and housed at the University of Southern Queensland Animal Facility before being randomly divided into four groups (12 rats per group). Two groups received either corn starch (C) or high-carbohydrate, high-fat diets (H) for the full 16 weeks [N. oceanica for the final eight weeks. The composition of C and H diets is described in our previous study [w/v) along with H diet.Male Wistar rats , systolic blood pressure and abdominal circumference measurement . DXA meaFor oral glucose tolerance tests, all rats were food-deprived overnight (~12 h). H and HN rats were given normal tap water without fructose during the food deprivation period. After overnight food deprivation, basal blood glucose concentrations were measured in blood collected from the tail vein and analysed using glucometer . The rats were then gavaged with 2 g/kg aqueous glucose solution, and blood glucose measurements were performed at 30, 60, 90 and 120 min after glucose loading using tail vein prick method . For insFollowing euthanasia with intraperitoneal Lethabarb , heparin (~200 IU) was injected into right femoral vein before blood collection, centrifugation and plasma isolation. Following blood collection, hearts were removed to measure diastolic stillness using isolated Langendorff heart preparation . Organ w3-V4 regions of the 16S rRNA gene. Data were presented and analysed for statistical significance as detailed in previous studies [After euthanasia, two or three faecal pellets were collected from the colon of each rat and processed as described previously to obtain the gut microbiota composition ,21. Gut  studies ,21.N. oceanica was intact  compared to H and HN rats . An increase in the relative abundance of bacteria from the class Clostridia was observed in H and HN rats  compared to C and CN rats . An increase in the relative abundance of bacteria from the class Oxyphotobacteria was observed in CN and HN rats  compared to C and H rats  (p > 0.05) compared to CN rats (3.26%), while an increase was observed in H rats  compared to C rats (0.05%) (p > 0.05) compared to H rats (8.89%) and lower in CN rats  compared to HN rats (9.18%)  . A decre (0.05%) . Similar (9.18%) .N. oceanica on the ratio of Firmicutes and Bacteroidetes  for three weeks. N. oculata reduced serum concentrations of glucose, cholesterol, triglycerides and LDL and increased the serum concentrations of insulin and HDL-cholesterol. In another streptozotocin study, rats were fed with N. gaditana (10%) for two months [N. gaditana supplementation decreased concentrations of glucose and HbA1c and improved renal and hepatic functions while attenuating the oxidative stress and inflammation in diabetic rats. The marine-water microalga N. oculata and its extract minimised the pancreatic tissue damage and maintained the integrity of the genomic DNA [N. oculata is a good source of omega-3 fatty acids, specifically EPA. Intervention with N. oculata suspension  for 14 days had no effect on body weight, which is similar to the current study [N. oceanica were present at 1940 mg/kg of the microalgal biomass which then gives a dose of approximately 4 mg/kg/day when mixed in the food. For comparison, a much higher dose of astaxanthin (200 mg/kg/day) given to Spontaneously Hypertensive Rats for 11 weeks reduced blood pressure [N. oceanica algal biomass. Further, this study tested N. oceanica without disrupting the cell structure, which is likely to further reduce the bioavailability of these components as ball-mill disruption enabled the protein and fatty acids to become bioavailable to mice [N. oceanica biomass, when used after processes such as ball milling [No studies have reported the effects of lycaemia . Diabetio months . N. gadiomic DNA . N. oculnt study . Using tnt study  improvedpressure . These c to mice . It can  milling , may impCaulerpa lentillifera showed correlations between gut microbiota and 15 physiological variables, especially oral glucose tolerance, liver weight and abdominal fat pads [N. oceanica intervention.The gut microbiota plays an important role in health and disease . Dietaryfat pads . The curNannochloropsis species also contains high-value products such as other fatty acids, sterols and carotenoids with applications in food, cosmetic and pharmaceutical industries [Nannochloropsis biomass is a good source of protein and carbohydrates which may have health benefits in addition to the increased EPA and carotenoids if the cell wall is broken [Marine fatty fish such as salmon, mullet and mackerel are the main sources of EPA and DHA for human consumption . Howeverdustries . Defattes broken . Furthers broken .Nannochloropsis species varies from 63 to 119 nm due to the distinct genetic traits in each strain, with N. oceanica having one of the thickest cell walls [N. oceanica may be necessary for optimal biological activity [Nannochloropsis using microwave, super-critical, ultrasound and enzyme-assisted extractions at industrial scales [An advantage of this study was that the cell wall was not disrupted, and therefore, the effectiveness of microalgal components other than cell wall-bound EPA and carotenoids could be determined. A key feature of microalgae is the rigidity of the cell wall, which can limit the bioavailability of nutrients; hence, other studies have used several cell disruption methods such as mechanical, physical, chemical and enzymatic approaches  or solvell walls . Cell meactivity . Health l scales . Our stuThere may be a role for microalgal protein in providing a sustainable source of protein to augment diets that maintain weight loss. Typically, people regain weight after weight loss, with only diets with increased protein content having a beneficial effect in maintaining the reduced weight . Animal The dose of 5% of diet in rats corresponds to approximately 30 g per day intake in adult humans . This isN. oceanica intervention increased lean mass in rats, possibly due to the increased protein intake and decreased fat mass in obese rats, but this intervention did not change cardiovascular, liver and metabolic parameters or gut structure. As N. oceanica biomass can be produced sustainably in large quantities, it could be a source of essential amino acids and prebiotics that may improve health in chronic diseases such as metabolic syndrome. These are additional effects to the production of EPA and carotenoids by Nannochloropsis as a biorefinery. Further, the industrial usefulness of Nannochloropsis biomass for biofuels and animal feed supplements means that production of these amino acids and prebiotics can be undertaken using existing processes.


Pseudomonas aeruginosa is known for a high adaptive capacity due to the ability to synthesize several compounds that give advantages for competing with other microorganisms in the environment. The LV strain synthesizes bioactive compounds, mainly by secondary metabolism, with antitumor and antimicrobial activities against microbial pathogens. Pseudomonas aeruginosa is known for a high adaptive capacity due to the ability to synthesize several compounds that give advantages for competing with other microorganisms in the environment. The LV strain synthesizes bioactive compounds, mainly by secondary metabolism, with antitumor and antimicrobial activities against microbial pathogens. Pseudomonas genus allows them to live in large populations in the most diverse environments, in which they can interact with plants, animals, and humans. The plasticity of Pseudomonas strains makes them important agents for use in biotechnological applications , in Astorga, Brazil   was used for genomic DNA extraction according to the manufacturer’s standard protocols. The LV strain genome was sequenced on the MiSeq platform at Embrapa Soja in Londrina, Brazil. The library was assembled using the Nextera XT DNA library preparation kit  according to the manufacturer's specifications. Paired-end reads obtained by shotgun sequencing yielded 3,378,198 sequences, with genome coverage of 150-fold. A de novo genome assembly was constructed with SPAdes v3.11.1  v2.0 server (http://rast.nmpdr.org) (https://antismash.secondarymetabolites.org) (https://cge.cbs.dtu.dk/services/ResFinder) (crpP), β-lactams , aminoglycosides [aph(3′)-llb], phenicols (catB7), and fosfomycin (fosA). With CRISPRfinder (https://crispr.i2bc.paris-saclay.fr)  . The RASsFinder) ; they weclay.fr) , we founCP058323 . The version described in this paper is the first version.This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession no.


MTHFR gene, especially C677T, have been associated with various diseases, including cardiovascular diseases (CVDs), cancer, inflammatory conditions, diabetes, and vascular disorders. The C677T MTHFR polymorphism is thought to be the most common cause of elevated Hcy levels, which is considered an independent risk factor for CVD. This polymorphism results in an amino acid change from alanine to valine, which prevents optimal functioning of the enzyme at temperatures above 37 °C. Many studies have been conducted to determine whether there is an association between the C677T polymorphism and increased risk for CVD. There is much evidence in favour of this association, while several studies have concluded that the polymorphism cannot be used to predict CVD development or progression. This review discusses current research regarding the C677T polymorphism and its relationship with CVD, inflammation, diabetes, and epigenetic regulation and compares the evidence provided for and against the association with CVD.The 5-10-methylenetetrahydrofolate reductase (MTHFR) enzyme is vital for cellular homeostasis due to its key functions in the one-carbon cycle, which include methionine and folate metabolism and protein, DNA, and RNA synthesis. The enzyme is responsible for maintaining methionine and homocysteine (Hcy) balance to prevent cellular dysfunction. Polymorphisms in the  MTHFR gene is a 77 kDa protein, with a second 70 kDa isoform found in humans [5-10-Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in folate and homocysteine (Hcy) metabolism. The gene for this enzyme is located on the short arm of chromosome 1 1p36.3) and encodes for dimeric proteins [n humans . MTHFR cn humans ,3. In to6.3 and en humans . For exan humans . Mudd etMTHFR polymorphism (rs 1801133). This polymorphism involves the substitution of cytosine with thymine at position 677, resulting in an amino acid change from alanine to valine in the enzyme [MTHFR gene mutation affects Hcy levels and is thought to contribute to hyperhomocysteinemia, reduced folate levels, and several CVD-associated diseases [MTHFR gene and involves the substitution of adenine with cytosine, resulting in an amino acid change from glutamate to alanine in the enzyme [The most common cause of elevated Hcy levels is thought to be the C677T phism rs 801133. Tdiseases . The C67diseases . This prdiseases . Anothere enzyme . This mue enzyme .MTHFR gene polymorphism in new-borns across 16 regions of Europe, Asia, Australia, the Middle East, and the Americas reported that the TT genotype was more frequently observed in China, Mexico, and Southern Italy, with a particularly low frequency observed in new-borns of African ancestry [MTHFR gene polymorphisms, especially C677T and A1298C (rs 1801131), and several disease states, including kidney disease, CVD, neural tube defects, cancer, and liver disease [MTHFR gene polymorphisms and CVD.Researchers investigating the ethnic and geographical distribution of the C677T ancestry . Furtherancestry . Therefoancestry . The MTHancestry . ResearcCRP gene [Inflammation is mediated primarily by the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) pathway, which involves several related transcription factors. The NF-κB protein is sequestered and rendered inactive in the cytoplasm during normal conditions by the inhibitor of nuclear factor kappa B alpha (IκBα) protein . NF-κB iCRP gene . DiabeteCRP gene .MTHFR C677T polymorphism and inflammation, Dedoussis et al. reported an association between the homozygous mutant genotype (TT) and increased inflammatory markers [MTHFR C677T genotype association in 574 CVD-free subjects from the Attica region in Greece and reported that 11% of subjects presented with the homozygous TT genotype, 48% with the heterozygous CT genotype, and 41% with the homozygous CC genotype . Inflammatory markers, including CRP, white blood cell counts, and amyloid-A levels, appeared elevated in the TT genotype subjects compared to the CC and CT genotype individuals despite controlling for confounding variables [MTHFR polymorphism and miR-149 on NAFLD. Results showed that miR-149 was upregulated in TT genotyped hepatocytes treated with long-chain fatty acid (FFA), while MTHFR was downregulated in these hepatocytes [MTHFR gene, was upregulated in response to FFA in TT genotyped hepatocytes and in turn decreased MTHFR expression [MTHFR genotype affects susceptibility to NAFLD.Regarding the  markers . The stuariables . Mild MTariables . Researcariables . An estiariables , which iariables . Researcariables ,28,29,30ariables . An et aatocytes . Howeverpression . This stMTHFR C677T polymorphism was shown to be associated with inflammatory diseases, such as psoriasis (Caucasian Czech population) [MTHFR gene polymorphisms contributed to systemic inflammation in a cohort of 292 patients by determining two markers of systemic inflammation, namely neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) [MTHFR gene polymorphisms.The ulation)  and influlation) , while bulation) ,34. Systulation) , and insulation) . Furtherulation) . Khalighio (PLR) . This stio (PLR) . The aboB vitamins play important roles in the one-carbon cycle , which iThe C677T and A1298C polymorphisms are thought to be primary contributors to hyperhomocysteinemia, resulting in an increased risk of developing thrombosis . Xuan etMTHFR polymorphism, while only 37% of women with normal BMD presented with the mutation. Taken together, these results suggest an association between the C677T MTHFR polymorphism and elevated Hcy levels and inflammation, which may further influence osteoporosis onset [MTHFR gene may contribute to disrupted oestrogen metabolism and in turn influence oestrogen-associated diseases [Research has reported an association between hyperhomocysteinemia and low bone mineral density (BMD) as well as CRP, vitamin D, vitamin B12, and folate levels in postmenopausal women . Two hunis onset . The MTHis onset . Catechois onset , and mayis onset ,50. Resediseases .Vitamin B12 deficiency is thought to contribute to increased Hcy levels. Patients with low folic acid levels often present with low levels of B12 and other B vitamins . In a stMTHFR gene polymorphisms, increased Hcy levels, and cardiovascular complications. A meta-analysis by Kang et al. reported an association between increased plasma Hcy and increased risk for haemorrhagic stroke [MTHFR gene polymorphism is associated with a three-fold increased risk for the premature development of CVD [Many studies have reported associations between c stroke . Klerk ec stroke , and Yanc stroke , while Fc stroke . Kluijtmt of CVD . Howevert of CVD .MTHFR C677T gene polymorphism in CVD development in diabetes patients. Patients with T2DM and severe CVD (107) but without nephropathy or retinopathy were recruited for the study [MTHFR C677T polymorphisms and reported 31, 62, and 14 subjects with the CC, CT, and TT genotypes, respectively. The results reported no correlation between MTHFR genotypes and several measured parameters, including visceral fat area, diabetes duration, cholesterol and triglyceride profiles, vitamin B12, Hcy, and HbA1c levels, and fasting plasma glucose [MTHFR C677T polymorphism cannot be used to assess CVD risk in diabetes patients [Bahadir et al. assessed the implications of the he study . The res glucose . This stpatients . HoweverMTHFR polymorphism contributes to thrombosis [Incidence of thrombosis further aggravates CVDs and associated disorders. Hyperhomocysteinemia has been associated with increased incidence of venous thrombosis. However, research has provided conflicting reports as to whether the C677T rombosis . Some rerombosis ,63, whilrombosis ,65,66,67MTHFR gene variants may contribute to disruptions in the Hcy pathway, which in turn exacerbates T2DM-associated complications [MTHFR gene polymorphism and MI risk, ultimately producing conflicting results.Several studies have suggested that ications ,69. Highications . Xuan etications ,45. MI iications . MI is cications . Therefoications . Howeverications . FurtherMTHFR polymorphism in relation to hypertension in a Japanese population and found no significant relationship between the two [MTHFR polymorphism is associated with T2DM susceptibility in Asian population but not in African or Caucasian populations [The C677T polymorphism is thought to be an independent risk factor for hypertension. Nishio et al. conducted a study in 1996 investigating the frequency of the C677T  the two . However the two . Furtherulations . FurtherDNA methylation, which is vital for normal gene regulation, involves the addition of a methyl group to cytosine nucleotides and is catalysed by DNA methyltransferases . DNA metMTHFR gene polymorphisms [MTHFR C677T polymorphism but was instead associated with the NOS3 G894T polymorphism in this population. The NOS3 gene has been linked with diabetes and nitric oxide (NO) bioavailability, which influences endothelial function and contributes to vascular complications in diabetes [Type 2 diabetes mellitus (T2DM) develops as a result of many interacting factors, including environmental and genetic aspects. Several studies have shown that DNA methylation patterns appear altered in diabetes patients ,106,107,orphisms . Matsha orphisms . Furtherdiabetes . This reMTHFR polymorphisms [MTHFR gene polymorphism and the severity or progression of DR in a Brazilian population while excluding the influence of African ancestry from the data analysis. The authors suggest that the C677T MTHFR polymorphism is not a useful predictive marker for diabetes or DR [Several studies have suggested that hyperhomocysteinemia contributes to the development of diabetic nephropathy (DN) and retinopathy (DR) in patients with diabetes, especially those with orphisms ,110. Diaorphisms . Nitric orphisms . One-carorphisms . Diabetiorphisms . The patorphisms . The MTHorphisms . The C67orphisms . In contes or DR .MTHFR gene promoter region on diabetes complications, such as DR and DN [MTHFR gene promoter region was associated with DR, in addition to biochemical, oxidative stress, and inflammatory parameters related to T2DM and its associated complications [Dos Santos Nunes et al. recruited 105 individuals to study the influence of methylation of the R and DN . In totaications . The CpGications . This epMTHFR gene polymorphism, as this polymorphism has been linked with increased incidence of neural tube defects related to low folate status [MTHFR deficiency [Vitamin B supplementation is particularly important during pregnancy if either parent is homozygous for the C677T e status ,118. Bete status ,120. Froficiency .Interestingly, folate stabilises enzyme activity in individuals with the C677T polymorphism, effectively neutralising effects of the polymorphism . JacquesMTHFR gene may play a role in the development of CVDs and diabetes-associated disorders, such as retinopathy and nephropathy (MTHFR with CVD and diabetes development. However, too many studies have reported on positive associations to ignore the potential use of this genetic polymorphism as a screening tool to determine the development and progression of several disease states. Further studies should be conducted with larger cohorts in various geographic regions and with various ethnic groups to successfully conclude the relationship between this gene and cardiovascular risk.Polymorphisms in the hropathy . Researc


Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), continues to be a global health burden. We have reported that patients with marked deficiency in the production of glutathione (GSH) had impaired granulomatous effector responses against M. tb infection, which were restored when supplementing patients with liposomal GSH (lGSH). However, the effects of GSH deficiency in the lung parenchyma in altering granuloma formation and effector responses against M. tb infection remain unexplored. We aim to elucidate the effects of diethyl maleate (DEM)-induced GSH deficiency during an active M. tb infection in an in vivo mouse model. We assessed for total and reduced GSH levels, malondialdehyde (MDA) levels, cytokine profiles, granuloma formation and M. tb burden. DEM administration significantly diminished total and reduced GSH levels in the lungs and plasma and increased MDA levels in infected mice compared to sham-treated controls. DEM treatment was also associated with an increase in IL-6, TNF-α and ill-formed granulomas in infected mice. Furthermore, M. tb survival was significantly increased along with a higher pulmonary and extrapulmonary bacterial load following DEM treatment. Overall, GSH deficiency led to increased oxidative stress, impaired granuloma response, and increased M. tb survival in infected mice. These findings can provide insight into how GSH deficiency can interfere with the control of M. tb infection and avenues for novel therapeutic approaches. However, virulence factors of M. tb prevent phagocytic degradation of the bacteria and ensure its survival in the macrophage, leading to a primary infection [M. tb of nutrients required for its replication, thus suppressing active infection, and localizing the pathogen to the lungs [M. tb persists in a quiescent state in the granuloma, leading to latent tuberculosis infection (LTBI) [Tuberculosis (TB) is one of the oldest documented infectious diseases in the world. The etiological agent, nfection . Infectenfection . The grahe lungs ,6. If thn (LTBI) –11.M. tb and can serve as an important effector molecule mediating macrophage control of M. tb [M. tb [M. tb infection [Glutathione (GSH) is an endogenous antioxidant tripeptide composed of glutamate, cysteine, and glycine. The two forms of GSH are reduced GSH (rGSH) and oxidized GSH (GSSG). rGSH is the functional form, containing antioxidant characteristics which prevent cellular damage from reactive oxygen species (ROS). rGSH reacts with ROS, producing GSSG and water, mitigating the ROS and any corresponding potential damage ,13. We hof M. tb . Furtherof M. tb ,10. Immub [M. tb . Howevernfection ,12.M. tb compared to isolates derived from healthy patients [M. tb survival and growth in infected PBMCs from T2DM patients is significantly increased compared to healthy controls [M. tb, and enhanced in vitro granuloma formation and M. tb control [M. tb growth in vitro.Patients with T2DM exhibit diminished synthesis of GSH-synthesizing enzymes, including the catalytic subunit of glutamine-cysteine ligase (GCLC), and thus produce lower levels of total GSH ,13. T2DMpatients ,17. In a control ,17–19. AM. tb infection have yet to be explored. We therefore aim to develop an in vivo mouse model to determine the effects of GSH deficiency on the effector and granulomatous responses against M. tb infection in the lung parenchyma of C57BL/6 mice and explore cytokine production in the respiratory tract when GSH deficiency is induced. Diethyl maleate (DEM) is a known GSH-decreasing agent and was used to induce GSH-deficient conditions in M. tb infected mice [M. tb in the lungs and spleen of the mice.However, the effects of GSH deficiency in the lung parenchyma and its impact on the innate and adaptive immune response during an active ted mice . GSH dimted mice ,21. The 2.M. tb infection and disease progression, mice were aerogenically infected with M. tb H37Rv and placed into either untreated or DEM-treated groups. Six mice from each group were sacrificed at specific time points: 2 weeks, 4 weeks, and 8 weeks post-infection, and samples were analyzed using various assays with hematoxylin and eosin staining, which were conducted for microscopic evaluation of tissue morphology.To evaluate the role of GSH in controlling 2.1.Mycobacterium tuberculosis H37Rv (M. tb) strain was obtained from Dr. Thomas Shinnick (Center for Disease Control (CDC), Atlanta, GA, USA). The bacteria were grown to OD600 = 0.7 in Middlebrook 7H9 medium  supplemented with 10% ADC  enrichment, aliquoted and stored frozen at −80 °C. To prepare inoculum for mice aerosol infection, stock vials were thawed and used as described in previous research [research .2.2.For animal studies, 8–10 weeks old C57BL/6 mice  were purchased from the Jackson Laboratory . Bacterial stocks were generated as described previously . Mice we2.3.Portions of lungs were fixed in 10% neutral formalin solution, paraffin-embedded and cut into 5 μm sections for staining with hematoxylin-eosin (H&E) to visualize the organization and distribution of leukocytes. The stained sections were analyzed using a Nikon Microphot DXM 1200C microscope and photographed using NIS-Elements software  as described previously . Morphom2.4.M. tb-infected mice were measured spectrophotometrically using an assay kit procured from Arbor Assays (Cat. # K006-H). Levels of IL-6 and TNF-α in the lung tissue homogenates and plasma from M. tb-infected mice were measured using enzyme-linked immunosorbent assay (ELISA) kits procured from Thermofisher (Cat. # BMS603-2 and BMS607-3). MDA levels in the lung tissue lysates and plasma were measured spectrophotometrically using an assay kit procured from Cayman Chemicals (Item # 10009055).In order to determine the effects of GSH depletion caused by DEM treatment, the levels of the total and reduced forms of GSH in the lung tissue homogenates and plasma from 2.5.p-values of <0.05 considered significant. Any placement of an asterisk (*) denotes a direct comparison to the previous category. When two asterisks are represented (**), a p-value below 0.005 is implied. When three asterisks are represented (***), a p-value below 0.0001 is implied.GraphPad Prism Software 8 was utilized for statistical analysis. An unpaired t-test was performed when comparing only two categories with Welch corrections applied. All values reported represent the means for each respective category with 3.3.1.We observed a significant decrease in total GSH levels in the plasma of DEM-treated mice compared to the sham-treated control group at both 2 weeks post-infection and 4 weeks post-infection ,B. Furth3.2.M. tb-infected mice treated with DEM had significantly higher levels of IL-6 at both 4 weeks  on TB patients. Furthermore, treating mice with the GSH-decreasing agent, DEM, while comparing them to untreated mice substantiates the positive effects of GSH supplementation observed in our previous studies. DEM has been known to reduce GSH levels for many years. A study by Mitchell et al. suggests that DEM depletes cellular GSH to less than 5% of the GSH in untreated cells , deemingM. tb-infected C57BL/6 (WT) mice with DEM (50 mM) led to effects similar to those seen in individuals with low GSH levels [The treatment of H levels ,13. For H levels .M. tb. The significantly increased survival of M. tb in both the lungs and the spleen of the DEM-treated mice suggests the importance of GSH in controlling M. tb infection  with lGSH and DEM to better understand the effects of GSH supplementation with TB and T2DM comorbidity.Limitations of our study include using a mouse model of TB, which may lead to minor differences in results compared to humans. In further studies, our lab plans to treat diabetic mice (infected with