Pteris vittata, which involves transforming arsenate As(V) to an As(V) phosphoglycerate derivative by a glyceraldehyde 3-phosphate dehydrogenase and transporting this complex by an efflux transporter. In the present review, we summarize the evolution of these arsenic resistance mechanisms from prokaryotes to eukaryotes and discuss future approaches that could be utilized to better understand and improve As resistance mechanisms in plants. Arsenic (As) is a toxic metalloid for all living organisms and can cause serious harm to humans. Arsenic is also toxic to plants. To alleviate As toxicity, all living organisms (from prokaryotes to higher plants) have evolved comprehensive mechanisms to reduce cytosolic As concentration through the set of As transporters localized at the plasma and tonoplast membranes, which operate either in arsenite As(III) extrusion out of cells  or by sequestering arsenic into vacuoles (by ABC transporters). In addition, a special arsenate resistance mechanism found in some bacterial systems has evolved in an As hyperaccumulating fern  Arsenic (As) is a toxic metalloid that possesses properties intermediate between metals and non-metals. Arsenic is highly toxic to humans, with low and non-cytotoxic concentrations of As greatly enhancing the carcinogenicity in mammalian systems . Accordi+ antiporter (ACR3) or sequestered into vacuoles by ABC transporters [Living organisms, from bacteria to higher plants, have evolved elaborate resistance mechanisms to deal with the toxic effects of As. In ancient times when arsenite (As(III)) was the major form of As, prokaryotic cells developed mechanisms to export As(III) by ArsB transporter ,8. Aftersporters . Additiosporters  and As exists mainly in the form of arsenite (As(III)) and arsenate(As(V)). Under anaerobic conditions, As mainly exists in a reduced form, As(III), and it is taken into living organisms via aquaporins or hexose transporters . Under aArsenate reductases exist in almost all living organisms, including prokaryotes, yeast, and higher plants. The major pathways for arsenate reduction are mediated by arsenate reductases, including ArsC, ACR2, and HACs, which arose independently . The mecStaphylococcus aureus plasmid pI258–encoded ArsC gene product [3− intermediate, (2) Cys82 attacks Cys10 with the formation of a Cys10-Cys82 disulfide intermediate, (3) Cys89 attacks Cys82, forming a Cys82-Cys89 disulfide, and (4) reduced ArsC is regenerated by thioredoxin (Trx) reduction of the oxidized Cys82-Cys89 disulfide [Escherichia coli plasmid R773, which exhibits arsenate reductase activity dependent on the presence of reduced GSH and Grx without forming disulfide [5R, while the active site of Grx-linked ArsC is HX3CX3R [Prokaryote arsenate reductases can be divided into two different classes, thioredoxin (Trx)-linked and glutaredoxin (Grx)-linked ArsC classes . The Trx product , which misulfide . The proisulfide . There i HX3CX3R . The pro HX3CX3R . However HX3CX3R . Thus, tSaccharomyces cerevisiae [arsC classes, and it is a homolog of the Cdc25a cell-cycle protein tyrosine phosphatase (PTPase) [5R with the bacterial Trx-linked ArsC family [Arabidopsis thaliana ACR2 has an arsenate reductase activity in vitro, and the knocking down of AtACR2 results in As(V) sensitivity and As accumulation in A. thaliana seedlings [AtACR2 does not affect As redox status and As accumulation in A. thaliana [Pteris vittata ACR2p (PvACR2), a homolog of AtACR2, also exhibits arsenate reductase activity in vitro [P. vittata yet. A novel arsenate reductase ARQ1/HAC1 had been identified in A. thaliana [P. vittata. The PvHAC1 was mainly expressed in the rhizomes, while PvHAC2 was predominantly expressed in the fronds of P. vittata. It is suggested that multiple arsenate reductases may work together in a tissue-specific manner and play a role in As-hyperaccumulation [Eukaryotes, including yeasts and plants, do not have homologs of prokaryotes arsenate reductase. The eukaryotic arsenate reductases can be divided into two major groups, ACR2p-like proteins and rhodanese-like proteins . The firrevisiae ,25. ACR2(PTPase) . SimilarC family ,26. HoweC family . Althougeedlings . Howeverthaliana . Pteris in vitro , but thethaliana ,13, and thaliana ,31. The thaliana . Recentlmulation . 4R, whereas ACR2 arsenate reductases contain the HCX5R active site  and As(V) enter the cell via aquaporins and phosphate and hexose transporters, respectively ,38. SincArsR and ArsB. The ArsR protein is a member of the SmtB/ArsR family of metalloregulatory proteins [arsB encodes a membrane protein extruding As(III) out of the cytoplasm, thus diminishing As(III) accumulation [Bacillus subtilis, with homology to ACR3, a protein encoded by the yeast S. cerevisiae, which also confers As resistance [ArsB gene or an ACR3 gene [ArsB genes exist mainly in Firmicutes and Gamma proteobacteria, but ACR3 genes are predominantly found in Actinobacteria and Alpha proteobacteria [ArsA and ArsD [ArsA, could bind to ArsB and convert the As(III) carrier protein into a primary ATP-driven As(III) pump [ArsRDABC operon are more resistant to As(V) and As(III) than those expressing only the ArsRBC genes [E. coli, a glycerol facilitator, GlpF, which was first identified as an antimonite (SbIII) channel, is the major uptake pathway for As(III) [Sinorhizobium meliloti, ArsB is replaced by another protein, aqpS, which belongs to the major intrinsic protein of the aquaporin superfamily, and functions as an efflux channel for As(III) [An extrusion of As out of the cells is a major mechanism for As resistance in prokaryotic cells . During proteins . ArsR biproteins . The arsmulation . After omulation . In addimulation  reportedsistance . Later iCR3 gene . The Arsbacteria . Furtherand ArsD . ArsA, aII) pump . The ArsII) pump ,43. ArsDII) pump . Cells eBC genes . In E. c As(III) . In micr As(III) . However As(III) . Since a As(III) .+ antiporters, ArsB has 12 membrane-spanning helixes [P. vittata, ACR3 homologs exist but have different functions [P. vittata and mediates the detoxification of As by compartment As(III) in the vacuole. PvACR3 is highly expressed in both sporophyte roots and gametophytes. A knockdown of PvACR3 in the gametophyte results in an As(III)-sensitive phenotype [A. thaliana, PvACR3 was located at the plasma membrane and mediated As(III) efflux from the roots and translocation to the shoot [A. thaliana roots [ACR3 genes  were identified in P. vittata, which were mainly expressed in fronds of P. vittata [ACR3 genes suggests that they are critical for the As tolerance and hyperaccumulation in P. vittata. Until now, the ACR3 protein family was only identified in bacteria, fungi and non-angiosperm plants [ACR3 genes in angiosperms and their presence in non-angiosperms, such as Picea sitchensis and Welwitschia mirabilis, suggests that ACR3 was lost in the angiosperms shortly after diverging from the gymnosperms [ACR3 in higher plants also suggests that they may use different transporters for As efflux. In most cases, ArsB is sufficient for bacterial arsenical resistance. In eukaryotic cells, the As extrusion mechanism is more complicated. No eukaryotic ArsB orthologs have been identified yet. In yeast, As(III) is extruded out of the cell via ScACR3 transporter . Althoug helixes , whereas helixes . In someunctions . PvACR3 henotype . Howeverhe shoot ,51. Unlina roots ,53. Rece vittata . PvACR3; vittata . The dupm plants . Accordinosperms . The losE. coli and yeast contain only two and four members of aquaporins, and the human genome has 11 members, while plants developed a larger aquaporin family [A. thaliana and rice contain 35 and 34 members of aquaporins, respectively [S. cerevisiae FPS1, an ortholog of E. coli glycerol channel GlpF, mediates the uptake and efflux of glycerol from yeast cells [S. cerevisiae genome contains four aquaporin genes, FPS1, AQY1, AQY2, and AQY3 [Aquaporins play critical roles in As uptake as well as extrusion, especially in eukaryotic cells. Although aquaporins are present in almost all living organisms, the number of aquaporins varies among organisms  Figure . E. colin family ,57. For ectively . The S. st cells ,46. The st cells . The S. and AQY3 . FPS1 anand AQY3 . The functions of aquaporin proteins for As transport have been extensively studied in plant species. Based on their protein similarity and subcellular localization, aquaporins are divided into five major subgroups in higher plants, including nodulin 26-like intrinsic membrane proteins (NIPs), plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), small basic intrinsic proteins (SIPs), and uncategorized intrinsic proteins (XIPs) . MultiplA. thaliana, AtNIP1;1, AtNIP1;2, AtNIP3;1, AtNIP5;1, AtNIP6;1, and AtNIP7;1 have been demonstrated as As(III) transporters [atnip1;1 mutant exhibited 30% lower As accumulation than wild-type plants [AtNIP5;1 [AtNIP5;1 in root epidermal, cortical, and endodermal cells suggested that it might also be involved in As(III) uptake [AtNIP7;1 shows increased tolerance to As(III) and As(V), reduces the total As concentrations in plants, which indicates that AtNIP7;1 functions as an As(III) uptake transporter [A. thaliana. AtNIP3;1 also plays an important role in the xylem loading of As(III) since it is also expressed in root pericycle cells [AtNIP6;1 also showed reduced As content in the seeds [In sporters ,64,65,66e plants . AtNIP5;) uptake ,68. The nsporter . AtNIP1;nsporter . The trale cells ,71. Phlole cells . AtNIP6;le cells . The knohe seeds , which sXenopus laevis oocytes [In rice, As(III) uptake is mainly mediated by OsNIP2;1, also known as a rice low-silicon transporter (OsLsi1), which is localized in a polar manner at the distal side of the plasma membrane of the exodermis and endodermis cells . Beside, oocytes ,75,77,78P. vittata, which encodes a plasma membrane-localized protein, is mainly expressed in the roots and mediates As(III) transport [PvTIP4;1 in A. thaliana enhances As accumulation and induces As sensitivity [In addition, aquaporins in other plant species have also been reported to play a role in As(III) uptake and translocation. It has been found that PvTIP4;1 aquaporin in ransport . The hetsitivity . A. thaliana [A. thaliana genome, other transporter families, including aquaglyceroporins, might be involved in this process. This issue needs to be addressed in further studies. In rice, certain aquaporins such as OsNIP2;1, OsPIP2;4, OsPIP2;6, and OsPIP2;7 have been found to be able to mediate As(III) efflux when the cellular As(III) concentration is higher than outside since they mediate the As(III) transport across the membrane according to the concentration gradient [Arsenic efflux is also a major arsenic resistance mechanism in plants. Until now, only a few studies dealt with As(III) efflux transporters in thaliana . Since ngradient . Comparegradient ,80. Whengradient . This prgradient . However3, which is then sequestered into the vacuole by a C-type ABC transporter, ScYCF1 (yeast cadmium factor 1), a homolog of the human MRP. ScYCF1 was first reported as a vacuolar glutathione S-conjugate pump that conferred cadmium resistance in S. cerevisiae [73As(III) in the presence of MgATP and glutathione, while those of Δycf1 do not have 73As(GS)3 transport activity. Plants and fungi contain a specialized subcellular organelle, vacuole, to store nutrients, metabolites, as well as xenobiotics. Besides extruding As(III) out of the cells, yeast and plants develop their own As tolerance mechanism using the vacuolar compartmentation of As(III). In yeast, As(III) could be chelated with glutathione to form As(GS)revisiae . Ghosh erevisiae  found thA. thaliana phytochelatin synthase mutant (cad1) is 10–20 times more sensitive to As(V) than the wild-type [γ-ECS) [A. thaliana PC synthase gene (AtPCS1) enhances As(V) tolerance [A. thaliana [AtABCC1 and AtABCC2 exhibits severe growth retardation upon As treatment. Rice OsABCC1 plays an important role in As resistance as well as As accumulation in grain [OsABCC7 is highly expressed in the roots and encodes a plasma membrane-localized protein. OsABCC7 is preferentially localized in the xylem parenchyma cells in the stele region of the primary and lateral roots, with the transport activity for both As(III)-phytochelatin and As(III)-glutathione complexes. The knockout mutants of OsABCC7 showed significantly reduced As concentrations in xylem sap and shoots [In plants, As(III) is also detoxified by being compartmentalized into the vacuoles, followed by complexing with thiol compounds, such as PCs (phytochelatins) ,83. PCs ild-type . Overexp [γ-ECS) ,88 or tholerance ,90. The thaliana . A doublin grain . OsABCC1in grain . OsABCC7d shoots . These r3 or As(III)-PCs complexes out of the cytosol. Both ScYCF1 and HsMRP1 transport As(GS)3, but plant ABCC1 proteins transport As(III)-PCs. The different substrate preferences of these proteins suggest that they may have some divergence during evolution. It would be worth studying what sequential or structural differences determine this substrate recognition. ScYCF1, HsMRP1, and plant ABCC1 proteins can detoxify arsenic by transporting As(GS)Pseudomonas aeruginosa strain DK2 has a specific pathway for As resistance, involving two novel genes conferring resistance to As(V), a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and an MFS transporter, ArsJ. Under this scenario, As(V) could be transformed to As(V) phosphoglycerate derivative, 1-arseno-3-phosphoglycerate (1As3PGA) by GAPDH, followed by its extrusion through ArsJ permease [permease . P. vittata, which consists of three essential genes that are significantly induced by As(V), namely glyceraldehyde 3-phosphate dehydrogenase (PvGAPC1), organic cation transporter 4 (PvOCT4), and glutathione S-transferase (PvGSTF1) [Pseudomonas aeruginosa strain DK2 and P. vittata, it is highly possible that this As resistance pathway in the P. vittata evolved from Pseudomonas aeruginosa strain DK2. The systematic analysis of the genome sequences would be helpful for proving it. It should be noted, however, that this mechanism has not been found in angiosperms. A similar system has been found in PvGSTF1) . PvGAPC1(1)How did the arsenic resistance trait evolve from prokaryotes to higher plants? For example, the arsenate reductases belong to different protein families in different organisms, such as ArsC protein in prokaryotes, ACR2 in yeast and fern, and HAC1 in higher plants. Although the ACR2 protein exists in higher plants, no clear evidence demonstrates its function as an arsenate reductase. It is interesting to know whether these proteins come from the same ancestor or have evolved separately.(2)P. vittata [P. vittata? In addition, the present review focuses on the tolerance mechanisms for inorganic arsenic species, while organic arsenic species are also present in the soil and should be dealt with by living organisms.Are there any new mechanisms for arsenic resistance in higher plants? In prokaryotes, a rare case of mechanism for the detoxification of arsenate by converting arsenate to As(V) phosphoglycerate and transporting it out of the cell was reported . A simil vittata . However(3)ACR2 and ACR3 [We know that arsenate reductase, aquaglyceroporins, and ABC transporters participate directly in the transport of arsenic or arsenic-containing complexes. However, their regulation is still understood poorly, especially in higher plants. In bacteria arsRDABC operons, ArsR protein acts as a trans-acting transcriptional repressor that regulates the transcription of the operon . Yeasts and ACR3 ,94. The and ACR3 . The phoand ACR3 . The mosand ACR3 . HoweverDespite the significant efforts that have been made to reveal the mechanistic basis of arsenic resistance in different organisms, there is still a long way to go for a comprehensive understanding of this process, let alone the application of this knowledge for reducing arsenic accumulation in crops. In our view, future studies need to be focused on the following issues:P. vittata, and higher plants (Foods contaminated with arsenic can potentially threaten human health. Reducing As content in the edible parts of crops needs a comprehensive understanding of arsenic resistance mechanisms, which include arsenate reduction, arsenic efflux, and arsenic sequestration. In this review, we summarized key As resistance mechanisms in various living systems, including prokaryotes, yeast, an As hyperaccumulator r plants , as wellr plants . Underst


We investigated the interactions between the PNPLA3 rs738409 genotype and nutritional factors and their possible effect on the risk of NAFLD development in 2950 patients with NAFLD and 12,907 controls. In the PNPLA3 risk allele group, high protein, fat, sodium, phosphorus, niacin, and vitamin B6 intakes were associated with a decreased risk of NAFLD. In the non-risk allele group, only high fat intake was associated with a decreased risk of NAFLD. Among these nutrients, high sodium intake had a significant protective interaction with the PNPLA3 genotype against NAFLD (p = 0.002). Among salty foods, only kimchi had a significant protective effect against the PNPLA3 genotype (p = 0.012). Thus, the PNPLA3 genotype is differentially associated with nutritional factors. In particular, it interacts with kimchi, a fermented vegetable dish. Therefore, fermented vegetables may serve as a tailored therapeutic food for people with the PNPLA3 risk allele.Genetic and nutritional factors contribute to the development of non-alcoholic fatty liver disease (NAFLD); however, gene–diet interactions in NAFLD development are poorly understood. In this case–control study, a large dataset from the Korean Genome and Epidemiology Study cohort ( Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide, and its prevalence continues to increase . As a spPNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), sorting and assembly machinery component 50 (SAMM-50), farnesyl diphosphate farnesyl transferase I (FDFT1), collagen type XIII alpha 1 (COL13A1), neurocan (NCAN), glucokinase regulatory protein (GCKR), membrane-bound O-acyltransferase domain-containing protein 7 (MBOAT7), apolipoprotein C3 (APOC3), sterol regulatory element binding transcription factor 2 (SREBF2) rs133291, membrane-bound O-acyltransferase domain-containing 7 transmembrane channel-like 4 (MBOAT7-TMC4) rs641738, 17β-hydroxysteroid dehydrogenase type 13 (HSD17B13), and serpin family member 1 (SERPINA1) [PNPLA3 and NAFLD risk is the most robustly observed [PNPLA3 rs738409 C > G SNP, which leads to the replacement of isoleucine with methionine at position 148 (I148M), contributes to the development of NAFLD by promoting triglyceride synthesis and accumulation in hepatocytes [PNPLA3 genotype-dependent manner [Traditionally, a sedentary lifestyle with a high calorie intake but low energy output has been considered the major cause of hepatic fat accumulation. Recent investigations have provided new insights into the pathogenesis of NAFLD. Genome-wide association studies (GWAS) have discovered that some single nucleotide polymorphisms (SNPs) increase the risk of NAFLD, including patatin-like phospholipase domain-containing 3  from KBP [In this case–control study, we received a dataset from the KoGES City cohort  ,24,25 cop < 1 × 10−5. After quality control filtering, the experimental phenotypes were used to analyze the genotype datasets from the 1000 Genome Phase 1 and 2 Asian panels. GWAS identified 7,975,321 SNPs on chromosomes 1–22. The SNP genotype (PNPLA3 rs738409) of participants was extracted from the Korea Biobank array (referred to as KoreanChip), which was optimized for the Korean population to demonstrate the findings of blood biochemical traits through GWAS [The genotype data were provided by the Center for Genome Science, Korea National Institute of Health and were processed using the Korea Biobank Array . The experimental results of the Korea Biobank Array were filtered using the following quality control criteria: call rate > 97%, minor allele frequency > 1%, and Hardy–Weinberg equilibrium, ugh GWAS .n = 72,299) to identify the genetic indicators of NAFLD. The genetic risk of the PNPLA3 rs738409 SNP for NAFLD development was tested using additive, dominant, and recessive models. We compared the baseline characteristics between participants with and without NAFLD using the Student’s t-test for continuous variables and Pearson’s chi-squared test for categorical variables. Furthermore, the association between the PNPLA3 rs738409 SNP and NAFLD was assessed using Pearson’s chi-squared test and logistic regression analysis. The association was adjusted for age, sex, body mass index, smoking status, and alcohol intake. The influence of dietary factors on the PNPLA3 rs738409 SNP risk allele and non-risk allele groups was investigated, as was the interaction between dietary factors and the PNPLA3 rs738409 SNP. All genetic association tests were conducted using PLINK version 1.9 (https://www.cog-ge-nom-ics.org/plink) [p values < 0.05.We performed GWAS in our KoGES City cohort (g/plink) , and theg/plink) . Differen = 72,299), the PNPLA3 rs738409 G allele frequency was 0.41. Among the subjects , those with missing data  were excluded. In a review of medical records, subjects with fatty liver disease but without viral hepatitis were screened (n = 3568). Among them, participants with alcohol consumption exceeding the predefined criteria were excluded (n = 618). Among the controls , subjects with underlying diseases were excluded . Additionally, we excluded subjects with missing nutrient intake data in both the control group (n = 105) and in patients with NAFLD (n = 27). Finally, 2950 patients with NAFLD and 12,907 healthy controls were included in the analyses (p < 0.001) than in the NAFLD group. In the KoGES City cohort  had a higher proportion of NAFLD cases than the non-risk allele group    (The frequency of the × 10−10) . After a× 10−10) . p < 0.001) and fat (p < 0.001) intakes (p = 0.067)  . p = 0.003), phosphorus (p < 0.001), zinc (p = 0.04), vitamin B1 (p < 0.001), vitamin B2 (p < 0.047), niacin (p < 0.001), and vitamin B6 (p < 0.001) than those in the NAFLD group , fat , sodium , phosphorus , zinc , vitamin B1 , vitamin B2 , niacin , and vitamin B6  significantly decreased the risk of NAFLD.Subsequently, the proportions of NAFLD cases were compared between the high- and low-nutrient-intake groups . High inPNPLA3 genotype, the population was divided into two groups , high intakes of protein , fat , sodium , phosphorus , niacin , vitamin B6 , and ash  were associated with a decreased risk of NAFLD (CC), a high fat intake was associated with a decreased risk of NAFLD , but other nutrients did not show a significant association  (PNPLA3 risk group (p < 0.0001) (PNPLA3 non-risk group (p = 0.851)  . The dat 0.0001) , while n= 0.851) . PNPLA3 genotype groups , white rice, baechu kimchi, leaf mustard or scallion kimchi, green pepper, orange, coffee, sugar (for tea/coffee), and cream (for tea/coffee) were protective against NAFLD , while multigrain rice, yogurt, nuts, pickled radish, vegetable salad, and fried food increased the risk of NAFLD development . In the PNPLA3 non-risk allele group , white rice, baechu kimchi, green pepper, orange, strawberry, pear, coffee, sugar (for tea/coffee), and cream (for tea/coffee) were protective , whereas multigrain rice, yogurt, green tea, and fried food increased the NAFLD risk .To determine which food the sodium originated from, the association between food-intake frequency and NAFLD was investigated in the e groups . In the PNPLA3 genotype against NAFLD development (p = 0.012)  . This inPNPLA3 genotype, and an interaction exists between nutrients or foods and the PNPLA3 genotype in the risk of NAFLD. A high fat intake decreased the risk of NAFLD, regardless of the PNPLA3 genotype. High intakes of protein, phosphorus, sodium, niacin, vitamin B6, and carotene were associated with a decreased risk of NAFLD in the PNPLA3 risk allele group. Among these protective nutrients, only sodium showed a significant interaction with the PNPLA3 genotype. To identify the source of the sodium, the association between food intake frequency and the PNPLA3 genotype was investigated. Only baechu kimchi, a salty food, had a significant protective interaction with the PNPLA3 genotype with respect to high sodium intake: a high intake of baechu kimchi significantly decreased the risk of NAFLD, with a greater magnitude in the PNPLA3 risk allele group than in the PNPLA3 non-risk allele group. Therefore, the preventive effect of a high sodium intake in the PNPLA3 risk allele group appears to be derived from high intake of baechu kimchi. Using the KoGES database, we discovered that nutrients and foods differentially affect the risk of NAFLD depending on the In the current study, a high sodium intake was inversely associated with NAFLD. This observation was a conundrum. It is possible that a high-sodium diet may indeed have a preventive effect on NAFLD. A recent animal study demonstrated that mice fed a high-sodium and high-fat diet developed less hepatic steatosis, metabolic syndrome, and insulin resistance compared with those fed a normal or low-sodium and high-fat diet . The redKimchi, a representative Korean food, is a salted and fermented vegetable usually prepared from winter cabbage . The heaLactobacillus casei) combined with plant extracts reduced the markers of NAFLD and increased the concentration of butyric acid in a mouse model [PNPLA3 risk genotypes indicates that fermented vegetables, such as kimchi, may be chosen as tailored foods for those affected. The key factor linking kimchi and its protective effect against NAFLD may be the short-chain fatty acids (SCFAs) produced by the gut microbiome . Among Sse model . As a fese model  and, in Streptococcus and serum folate concentrations [PNPLA3 genotype, whereas a high intake of protein is more beneficial in the PNPLA3 risk allele group. Nevertheless, caution should be exercised, as a ketogenic diet can elevate liver enzymes and worsen lipid profiles in patients with NAFLD [In the present study, a high fat and protein intake had a preventive effect against NAFLD. A high-fat and low-carbohydrate diet, often known as a ketogenic diet, is a popular weight-reduction method. Many clinical trials have demonstrated that a ketogenic diet is effective in improving NAFLD ,60,61,62trations . Furthertrations . Our stuth NAFLD ,65. Addith NAFLD ; its recPNPLA3 encodes a calcium-independent triacylglycerol lipase that contains a phospholipase domain. Upon liver injury, PNPLA3 expression is induced in hepatic stellate cells (HSCs) [PNPLA3 I148M, HSCs become more proinflammatory and profibrogenic and produce less retinol, which potentially mediates the transition of HSCs into a myofibroblast-like phenotype [PNPLA3 risk genotypes warrants further investigation. s (HSCs) . In the henotype ,68. Our This study had several limitations. First, there may have been a recall bias in collecting data on food intake habits because of the self-reporting nature of the food-frequency questionnaire. Second, the identification of fatty liver depended solely on the medical records of the subjects. The nationwide database did not include the results of imaging studies such as sonography, computed tomography, or magnetic resonance imaging. Thus, a detailed evaluation of NAFLD severity was not possible. Third, the present study could not count the various beneficial nutrients such as antioxidants, polyunsaturated fatty acids, or amino acids. As these beneficial nutrients may directly attenuate the oxidative stress that results in hepatic fibrosis ,69, futuPNPLA3 genotype, and nutritional and dietary factors were investigated. The genetic risk of NAFLD conferred by PNPLA3 in the Korean population was verified. In addition, we discovered some nutritional and dietary factors that can significantly influence the development of NAFLD, and these factors may interact with the PNPLA3 genotype. Both increasing the proportion of fat and protein relative to that of carbohydrate and consuming fermented vegetables may help reduce NAFLD, regardless of the PNPLA3 genotype. In PNPLA3 risk genotypes, the role of a high-protein diet appears to be more important, as does meeting the daily requirement of vitamins and minerals. Furthermore, kimchi, a fermented vegetable, may induce an added protective effect in PNPLA3 risk genotypes because of its gene–diet interaction. These results suggest that planning a therapeutic diet with high protein and fermented vegetables may be a valid strategy for those with PNPLA3 risk genotypes and NAFLD, and warrants future interventional studies. Furthermore, in the near future, studies are needed on the mechanisms of interactions between various beneficial nutrients, PNPLA3, and NAFLD development. In the present study, the associations and interactions between NAFLD, the PNPLA3 genotype is differentially associated with nutritional and dietary factors. In particular, it interacts with kimchi, a fermented vegetable. This indicates that tailored nutritional therapy based on an individual’s genetic background may be a good strategy to prevent NAFLD, and fermented vegetables may serve as a therapeutic food for those with the PNPLA3 risk allele. The


Glioblastoma is a complex and aggressive primary brain tumour that is rapidly fatal. Timely and accurate diagnosis is therefore crucial. Here, we explore the newly emerging field of epitranscriptomics to understand the modifications that occur on RNA molecules in the healthy and diseased brain, focusing on glioblastoma. RNA modifications are modulated by various regulators and are diverse, specific, reversible, and involved in many aspects of brain tumour biology. Epitranscriptomic biomarkers may therefore be ideal candidates for clinical diagnostic workflows. This review summarises the current understanding of epitranscriptomics and its clinical relevance in brain cancer diagnostics.6A). The m6A mark, similar to many other RNA modifications, is strictly regulated by so-called ‘writer’, ‘reader’, and ‘eraser’ protein species. The abundance of genes coding for the expression of these regulator proteins and m6A levels shows great potential as diagnostic and predictive tools across several cancer fields. This review explores our current understanding of RNA modifications in glioma biology and the potential of epitranscriptomics to develop new diagnostic and predictive classification tools that can stratify these highly complex and heterogeneous brain tumours.RNA modifications are diverse, dynamic, and reversible transcript alterations rapidly gaining attention due to their newly defined RNA regulatory roles in cellular pathways and pathogenic mechanisms. The exciting emerging field of ‘epitranscriptomics’ is predominantly centred on studying the most abundant mRNA modification, N6-methyladenine (m I. I6A mRN6A methylation [6A binding sites, which led to the identification of specific tumour-suppressive RRACH binding miRNAs that facilitate FTO-dependent, transcript-specific demethylation [Indeed, GSCs and differentiated GBM cells have distinct patterns of mhylation . Howeverhylation . Taken thylation .6A ‘eraser’, ALKBH5, has also been linked to GSC self-renewal and tumourigenesis [The other migenesis  through igenesis  and is oigenesis . ALKBH5 igenesis .6A methylation and increased GSC tumourigenicity [6A modification are essential for GSC maintenance and dedifferentiation, with the preservation of a stem-like phenotype mediated by SOX2 mRNA stabilisation via the recruitment of ELAV1 to m6A-modified SOX2 [Contrary to reports demonstrating positive associations between reduced mgenicity ,87,88,89genicity ,91,92,936A-modified RNA species analysed, as well as the intra- and intertumoural heterogeneity of GBM. Future experimental work must be context- and compartment-dependent to explain these inconsistencies and further our understanding of m6A RNA methylation and the interplay among regulator proteins in GSCs [Discrepancies between different studies might be explained by the diversity of cell types and m in GSCs .6A RNA methylation levels and m6A regulator expression reported in GBM [6A levels and low survival, and a specific inhibitor of METTL3 slowed tumour progression in a medulloblastoma mouse model [6A RNA methylation explored above [6A RNA methylation levels relative to normal brain tissue, with decreased METTL3 and increased FTO levels in glioma tissues offered as a causative link [6A levels and reduced cell proliferation and migration in vitro, perhaps related to a disruption in HSP90 chaperone activity and enhanced apoptosis [6A writer, METTL4, was shown to promote glioma cell proliferation and migration [6A-dependent depletion of MYC levels [6A ‘eraser’, ALKBH5, have been tested and show efficient inhibition of GBM cell proliferation [ALKBH5 was reported as a significant negative prognostic factor for GBM patients [ALKBH5 expression levels in GBM tissue are inconsistent [RNA modifications play increasingly important roles in the tumourigenesis and development of GBM; several ‘writers’, ‘erasers’, and even ‘readers’ of RNA modifications are proposed as potential diagnostic biomarkers and novel targets for treatment . Yet, thd in GBM ,19. In pse model . In lineed above , Li et aive link . Here, Mpoptosis . Other spoptosis ,96. Meanigration ,97. ReceC levels . MoreoveC levels . PromisiC levels . While sC levels ,101. Simferation  enhancedferation , and impferation . While Apatients ,106, repnsistent ,107.6A ‘reader’ and member of the YTH protein family, YTHDF1. Relative to normal brain tissue, YTHDF1 has the highest expression in GBM, and is stabilised by musashi RNA-binding protein 1 (MSI1). GBM cell proliferation and migration are enhanced by the MSI1 stabilisation of YTHDF1, and high expression of these genes in GBM is associated with reduced survival [MYC (YTHDF2-MYC) and targeting IGFBP3. Interestingly, YTHDF2 expressing GSC have an increased sensitivity to cell killing by linsitinib, an IGF1/IGF1R inhibitor. As NSC cell viability is unaffected by linsitinib treatment, this drug is proposed as a potentially specific anti-GBM treatment [Another approach to sensitise glioma cells to treatment was explored by modulating msurvival . Notablysurvival ,109. Increatment .6A can also have a role in treatment response. Unfortunately, there are several inconsistencies in the literature regarding the expression levels of the regulators and global m6A levels, and more in-depth studies are required to decipher the intricate web of RNA modifications in glioblastoma.Epitranscriptomic regulators and RNA modifications are extensively involved in essential functions of the healthy brain and play various roles in brain disease. This is well shown in glioblastoma, where m6A methylation, other forms of RNA methylation also play important roles in GBM biology. For instance, the downregulation of m6Am writer, PCIF1, promotes glioma cell proliferation and tumour growth in mice [5C methylated mature miRNAs are linked to poor GBM patient outcomes through a mechanism where this modification inhibits miRNA gene silencing [5C methylation was found to have a significant role in GBM formation through a mechanism involving ‘eraser’ TET1, while TET2 and TET3 downregulation is linked to GBM tumourigenesis [Beyond m in mice . m5C metilencing . hm5C meigenesis ,114,115.igenesis ; ADAR2 wigenesis ,118. Uprigenesis ,120. Likigenesis .The field of epitranscriptomics is rapidly expanding and is anticipated to impact clinical diagnostics. The search for rapid, simple, and reliable methods to explore and detect new disease classification and therapeutic targets are essential steps for precision medicine. Currently, several RNA methylation detection methods exist, with diverse sensitivity, sample input requirements, and data analytical processes. Epitranscriptomics platforms may be antibody-based or employ mass spectrometry, polymerase chain reaction (PCR), next-generation sequencing (NGS), or nanopore direct RNA sequencing .6A or m5C enzyme-linked immunoassay (ELISA). ELISA kits are useful for assessing global m6A/m5C methylation transcriptome-wide but are unable to distinguish m6A from m6Am or detect a methylation mark at single-nucleotide or even at gene resolution. The detection of modifications at a single RNA species level can be measured by Dot-blot, another antibody-based method. This is also a relatively inexpensive method, with 100 ng to 1 μg required starting material, and can be used to detect RNA modifications in diverse RNA species [Probably, the simplest, most accessible, and affordable methods are antibody-based, i.e., m species ,124.Mass-spectrometry-based methods, i.e., liquid chromatography–coupled tandem mass spectrometry (LC–MS/MS), are more sensitive than antibody-based approaches. LC–MS/MS allows the absolute quantification of modified nucleosides, and, when coupled with nucleic acid isotope labelling (NAIL–MS), can assess the temporal dynamics of RNA modifications . A drawb6A-reverse transcription (RT)-quantitative PCR, single-base-elongation- and ligation-based PCR amplification method (SELECT), and primer-extension-based method [PCR-based approaches are locus-specific RNA modification detection methods based on the premise that the queried modification impedes reverse transcription. These methods have high sensitivity and specificity; thus, they can be used for various RNA species. Some of the most well-known PCR-based approaches are the md method ,127.6A-seq or methylated RNA immunoprecipitation sequencing (MeRIP-seq) [6A individual-nucleotide resolution crosslinking and immunoprecipitation sequencing (miCLIP-seq), m6A-label-seq, m6A-sensitive RNA-endoribonuclease-facilitated sequencing (m6A-REF-seq), MazF RNase-assisted sequencing (MAZTER-seq), and deamination adjacent to RNA modification targets (DART-seq) [6A-selective allyl chemical labelling and sequencing (m6A-SAC-seq) [6A-SAC-seq requires very little starting material (2 ng of poly A+ RNA) but is a particularly laborious process.NGS-based detection methods are diverse and frequently used for whole transcriptome profiling at single-nucleotide resolution. Some approaches require antibody coupling, such as mRIP-seq) . Other sART-seq) ,130,131.SAC-seq) ,134,135.6A, inosine, or pseudouridine [One of the most promising epitranscriptomic approaches is Oxford Nanopore Technologies (ONT) sequencing, which can detect modifications in naïve RNA, such as mouridine ,137. Comouridine . Algoritouridine ,140,141.6A mark, other detection and data analytical methods facilitate the exploration of m5C, m7G, inosine, pseudouridine, and many other modifications on RNA. Although RNA modification detection methodology and bioinformatics tools are advancing rapidly, these techniques will require further adaptation before their implementation in diagnostic clinical service laboratories where there are real sample quantity and time constraints for analytical processes and data interpretation.Beyond the m6A levels are reported as potential diagnostic markers in the peripheral blood of small-cell lung (NSCLC) and breast and gastric cancer patients relative to healthy and/or benign cancer controls [6A levels correlated to tumour-staging parameters were depleted following surgery, and accompanying upregulations in ‘writer’ and/or downregulations in ‘eraser’ genes were observed [6A levels in rheumatoid arthritis (RA) patients’ and ischemic stroke patients’ blood were elevated relative to healthy controls, along with decreased ‘eraser’ ALKBH5 and FTO levels and increased YTHDF2 ‘reader’ levels in RA samples [6A levels in the peripheral blood of individuals with Type 2 diabetes mellitus (T2DM) are lower than in controls, with concomitantly high m6A ‘eraser’ FTO gene expression [6A methylation status and gene expression of m6A regulators as diagnostic tools across a range of pathologies are presented in The potential use of epitranscriptomic regulators as biomarkers has been explored across multiple cancer types; however, only a handful of studies have attempted to measure RNA modifications in body fluids directly. Of note, high global mcontrols ,143,144.observed ,143,144. samples ,146. In pression . Similarpression ,149,150.6A regulators with significant prognostic value in a diverse range of cancer types are presented in In recent epitranscriptomics studies exploring new diagnostic classification markers, investigations often stem from in silico analyses of public sequencing datasets  to identify the predictive and prognostic significance of the diverse regulators of RNA modifications. For instance, in lung adenocarcinoma, high HNRNPC and IGF2BP3 levels correlate with reduced overall survival ,157, andIDH1/2 mutations, 1p/19q codeletion, and MGMT promoter methylation, has improved the accuracy of diagnostics, prognostication, and prediction of treatment response for glioma patients, there has been no tangible improvement in clinical management and patient outcomes. Epitranscriptomics of known glioma sub-entities may yield further advances in tumour stratification and help guide treatment selection for precision medicine, as was shown in preliminary studies of colon cancer [Like other cancer types, the value of epitranscriptomics as a tool for brain cancer diagnostics is still in its early days. The WHO classification system for primary brain tumours has integrated molecular diagnostics with traditional histopathology . While tn cancer 6A RNA methylation regulators in public primary brain cancer sequencing datasets, and these key findings are summarised in Several studies explored m6A ‘readers’ include YTHDF2 overexpression, induced through the EGFR/SRC/ERK pathway to promote tumourigenesis, invasiveness, and cell proliferation independently associated with poor prognosis [6A ‘reader’ eIF3 was performed in independent glioma cohorts as several eIF3 subunits are localised to chromosomes 1p and 19q [6A regulators IGF2BP2, IGF2BP3, HNRNPC, and YTHDF2 and m5C-related long noncoding RNAs CIRBP-AS1, GDNF-AS1, LINC00265, and ZBTB20-AS4 have prognostic significance in LGG [Key findings in glioma patients in relation to mrognosis . Similarrognosis . A syste and 19q ,200, reg and 19q . All 13  and 19q . Among o and 19q . m6A rege in LGG ,204.6A ‘writer’ METTL3 was positively associated with a higher malignant grade and poorer prognosis of IDH-wildtype but not IDH-mutant gliomas [6A regulator expression when comparing glioma cohorts comprising different IDH mutational and 1p/19q codeletion states. While these cohorts represent different glioma sub-entities with distinct molecular pathophysiology, the average survival times are vastly different, particularly between oligodendroglioma  and GBM , and thus divergent epitranscriptomic regulator expression patterns may relate to the very different biology of these distinct pathological entities [6A regulators, and a risk signature  was significantly associated with prognosis, the immune microenvironment and treatment efficacy [ALKBH5, IGF2BP2, IGF2BP3, HNRNPA2B1, YTHDF1, YTHDF2, RBM15, and WTAP) was shown to be prognostic for glioma patients, and the expression of IGF2BP2 and IGF2BP3 was linked to tumour occurrence, development, and progression [ALKBH5, which is upregulated in glioma and associated with immune signalling, and inflammatory and metabolic pathways genes [ related to m6A regulators, all with high expression in high-grade glioma, and further correlation to IDH status and transcriptome subtype [6A regulators function in the tumour microenvironment, tumour cell stemness, and infiltration have also been established [6A RNA methylation regulators and clinicopathological features and treatment sensitivity of gliomas are outlined in Expression of the m gliomas . Anotherentities . In thisefficacy . A similgression . A recenys genes . Another subtype . Links bablished . Likewisablished . Further6A regulator expression levels among the different brain tumour entities and non-tumour brain tissue, including ALKBH5, WTAP, METTL14, and YTHDC2 [6A RNA methylation regulating gene expression levels may help to explain the opposing reports of high and low global m6A in brain tumour tissue relative to ‘normal’ brains [6A regulator genes, which are summarised in 6A ‘erasers’ FTO, which is frequently observed to be downregulated in GBM relative to other tumours and control tissue [6A levels.Again, there are several conflicting reports of md YTHDC2 ,107,213.’ brains . There il tissue ,101. If 6A, m5C, and other RNA methylation profiles have helped to identify epitranscriptomic regulators and their involvement in cancer biology. While the direct assessments of RNA modifications in diagnostic laboratory settings are technologically challenging, there is a wealth of evidence supporting the significant relationships between epitranscriptomic regulator gene expressions and clinicopathological parameters in cancer, including their use as diagnostic, prognostic and/or predictive biomarkers. With distinct roles defined in stem cell biology and neural development along with high levels detected in brain tissue, RNA modifications are, theoretically, highly relevant to multiple aspects of gliomagenesis and biology. Indeed, multiple studies have reported epitranscriptome regulator signatures that are highly predictive of glioma pathology and patient outcomes.Epitranscriptome profiling studies, particularly in cancer research, are becoming more common. Studies investigating global m6A methylation levels in highly annotated cohorts of discrete tumour types as well as biofluids are warranted.Further technological advances are needed to adapt epitranscriptomic approaches for clinical diagnostics and will likely uncover entirely novel targets for further stratifying tumour molecular phenotypes, directing treatment(s) and guiding patient management. In the interim, the detection of regulator genes and global m


Ca2+ homeostasis is vital for several physiological processes, and alterations in the patterns of expressions of the proteins and molecules that modulate it have recently become a cancer hallmark. Store-operated Ca2+ entry is a major mechanism for Ca2+ entry from the extracellular medium in non-excitable cells that leads to increases in the cytosolic Ca2+ concentration required for several processes, including cancer stem cell properties. Here, we focus on the participation of STIM, Orai, and TRPC proteins, the store-operated Ca2+ entry key components, in cancer stem cell biology and tumorigenesis.Tumors are composed by a heterogeneous population of cells. Among them, a sub-population of cells, termed cancer stem cells, exhibit stemness features, such as self-renewal capabilities, disposition to differentiate to a more proliferative state, and chemotherapy resistance, processes that are all mediated by Ca Normal stem cells are undifferentiated or partially differentiated cells that are characterized by their ability to self-renew, the process of bringing about indefinitely more cells of the same type, as well as to differentiate in more specialized mature cells. The term “stem cell” was coined by Ernst Haeckel in 1868 to describe the ancestor unicellular organism from which all multicellular organisms were supposed to evolve .Cancer stem cells (CSC), also known as tumor-initiating cells, share features of both cancer and stem cells. These cells constitute a sub-population of tumor-resident malignant cells responsible for recurrence, metastasis formation, and chemoresistance. Experimental evidence indicates that CSC exhibit “stemness” properties, that is, the ability of cells to perpetuate their lineage, to bring about differentiated cells and to interact with their microenvironment to maintain a balance between quiescence, proliferation, and regeneration . AccordiCSC have been reported to derive from “normal” tissue resident stem cells or from differentiated cells undergoing transformation . Further+/CD24−/low) together with expression of ALDH1 is a feature of breast cancer stem cells as compared to non-stem breast cancer cells [CSC show similar surface markers as normal stem cells in a given tissue, but a number of cell surface and intracellular biomarkers are commonly used to identify CSC among differentiated tumor cells and to isolate them. These markers include the clusters of differentiation (CD) CD44, CD24, or CD133, among others, the epithelial cell adhesion molecule (EpCAM) or the intracellular markers aldehyde dehydrogenase-1 (ALDH1) and the Notch, Wnt/β-catenin, Nanog, Sox2 pathways (for a more extensive review of CSC biomarkers in different tumors please see ). CD44 ier cells . Neverther cells . ALDH1 her cells . Intereser cells . The funer cells ; furtherer cells .Different signaling pathways and transcription factors have been reported to play an essential role in the state of cell stemness. Among them, developmental signaling pathways such as Notch, Wnt/β-catenin or Hedgehog play important roles in normal stem cell function. Notch signaling pathway is activated by the interaction of ligands of the DSL family with the receptor protein Notch, a single-pass transmembrane protein. The interaction of the ligand with the Notch extracellular domain (NECD) leads to the cleavage and release of the Notch intracellular domain (NICD) that acts as a transcription factor and interacts with the transcription factors of the CSL family and Mastermind . The CSLThe Hedgehog (Hh) pathway also plays a major role in normal stem cell and CSC biology and tumorigenesis. Hh signaling begins with the interaction of Hh ligands  with Patched-1, and to a lesser extent Patched-2, a twelve-pass transmembrane protein receptor. This process internalizes the Patched receptor and relieves the constitutive repression of the G-protein-coupled receptor Smoothened, which, in turn, leads to the nuclear translocation or the transcription factor Glioma-associated oncogene (Gli) resulting in the transcription of Hh target genes  Figure . The Hh 2+ pathway. The interaction of Wnts with Frizzled, a family of G-protein coupled receptors, leads to the activation of phospholipase C and, thus, IP3 -dependent Ca2+ release from the intracellular stores and subsequent Ca2+ influx across the plasma membrane [The Wnt/β-catenin signaling pathway promotes CSC self-renewal while reducing the differentiation of CSC to proliferating tumor cells . The Wntmembrane . The intmembrane . A recipmembrane .2+ concentration [2+ participates in several pathological conditions, including cancer. Resistance to apoptosis and chemotherapy, high proliferation rate or the ability to migrate and to invade different tissues, have been considered key features in all cancer types for years [2+ [2+ homeostasis has been included as a cancer hallmark.Calcium ion modulates a myriad of physiological processes, such as muscle contraction, secretion or gene transcription, through a sophisticated and well-orchestrated machinery that deftly tunes cytosolic Cantration . In addior years . As stator years ,5. All tears [2+ , thus, i2+ concentration are required to trigger several Ca2+-dependent downstream effectors that modulate cellular pathways, such as calmodulin, which is essential for cell cycle and proliferation [2+ concentration is achieved by the cells either by releasing Ca2+ from intracellular reservoirs, or by the opening of Ca2+-permeable channels in the plasma membrane (PM), which ensures an unlimited source of Ca2+ influx from the extracellular medium. Recent reports have demonstrated that CSC exhibit altered function in those mechanism  membrane, IP3- and ryanodine (Ry)- receptors, are vital for CSC stemness, proliferation, and metastasis in different cancer types, such as glioblastoma [Concerning the Cablastoma , melanomblastoma , and breblastoma .2+ entry, CSC present channels in the plasma membrane that are permeable to Ca2+ and could be gated by a variety of stimuli, such as voltage, second messengers or depletion of intracellular Ca2+ stores. For instance, over-expression of L- and T-type voltage-dependent Ca2+ channels is involved in tumorigenesis, proliferation, migration, and resistance to drugs in ovarian and glioblastoma CSC [2+ channel α2δ1 subunit, which modulates Ca2+ oscillation amplitude and the expression of different genes by keeping transcription factors in the nucleus, has started to be considered a tumoral marker in many cancers, such as lung [2+ entry and have a key role in the CSC physiopathology of different cancers. For instance, TRPC3, which is over-expressed in triple-negative breast cancer cells, is activated by lysophosphatidic acid, promoting the process of self-renewal in CSC [Regarding Catoma CSC ,52,53,54 as lung ,56, brea as lung , and liv as lung  or laryn as lung , with a l in CSC . Enhancel in CSC , glioblal in CSC , or neurl in CSC  and linkl in CSC  in the sl in CSC . Conversl in CSC ,66,67 anl in CSC . This inl in CSC .2+ reuptake, and the proteins involved, such as Ca2+-ATPases, Ca2+ exchangers or mitochondrial uniporter [2+ ATPase (SERCA) presents an important antiapoptotic function in breast CSC, by reducing Ca2+-dependent apoptosis during glucose deprivation. This process is mediated by CaMK2α, which triggers the activation of NF-κB, and, in turn, SERCA over-expression [2+ uniporter and the Na+/Ca2+ exchanger, located in mitochondria and in the plasma membrane, respectively, are highly expressed in glioblastoma CSCs [Caniporter ,71,72 mipression . Anotheroma CSCs ; however2+ influx from the extracellular medium into excitable and non-excitable cells, is physiologically triggered by the activation of phospholipase C (PLC) and the production of IP3, which subsequently leads to the release of Ca2+ from intracellular stores, mainly the ER, resulting in the activation of store-operated calcium channels in the plasma membrane and a rapid increase in cytosolic Ca2+ concentration [2+ homeostasis, which might contribute to tumor development, poor prognosis, and chemotherapeutic drug resistance [Store-Operated Calcium Entry (SOCE), a major mechanism for Cantration ,76. SOCEsistance .2+ concentration in the intracellular stores: (1) as the Ca2+ sensors of intracellular organelles, mediated by EF-hand Ca2+-binding domains in the N-terminus; and (2) as the transmitters of the filling state of intracellular Ca2+ stores to, and the activators of, Ca2+ channels in the plasma membrane. The latter is achieved by direct interaction between different domains within the STIM cytosolic C-region and the store-operated Ca2+ channels  family, STIM1 and STIM2, and their splice variants, possess a single transmembrane domain, with the N-region located either in the ER lumen or the extracellular medium, and a long cytosolic C-region ,79. Bothiewed in ,81,82).2+ Release-Activated Ca2+ (CRAC) channels that exhibit high Ca2+ selectivity and an inwardly rectifying current, termed ICRAC, which its exclusively conducted by members of the Orai family [2+ (SOC) channels, responsible to mediate a non-selective cation current denominated ISOC, formed by both, Orai1 and TRPC1, the first identified member of the canonical Transient Receptor Potential (TRPC) channel subfamily [SOCE could be mediated by two types of channels with different biophysical properties: (1) the Cai family ; and (2)ubfamily ,85.2+ channels, with four transmembrane domains spanning the PM and both, N- and C-terminus, facing the cytoplasm [Drosophila melanogaster Orai1 (dOrai1) presented a hexamer configuration, with the ion pore formed by the first transmembrane domain of the Orai subunits and located in the center of the complex surrounded by the remaining Orai plasma membrane domains [2+ influx, each of them with different biophysical properties that are extensively discussed here [2+ influx forming heteromers, such as the arachidonate-regulated Ca2+ channels (ARC), where three Orai1 and two Orai3 subunits form a pentamer [2+ influx [Orai1 was initially characterized as the main component of CRAC channel during a RNAi screening in 2006, when it was found that the Orai1 R91W mutation was responsible for abrogated CRAC channel function, critical for T-cell activation, in immunodeficient patients . Orai1 aytoplasm . Originaytoplasm ; however domains . The thrsed here ,91. Somesed here . In addipentamer , or inte+ influx .ISOC currents [+, Ca2+, and Cs+ [2+ than Orai1 and allow a massive ion influx from the extracellular medium, required for the maintenance of SOCE and store replenishment [TRPC1 belongs to the TRP channel superfamily, whose members ubiquitously mediate ion fluxes across the whole animal kingdom in a cell type-dependent manner . All TRPcurrents ,100,101. and Cs+ , are lesnishment .2+ stores depletion that will be sensed by STIM proteins  modulating each other, in a reciprocal dependency, in hypoxic hepatocarcinoma cells (HCCs). HIF-1α up-regulates STIM1 transcription, which in turn, induces higher SOCE, activating the CaMKII and P300 pathways, which are required for the accumulation of HIF-1α in HCCs .CDKN1A and G0S2 and the down-regulation of CCNB1 genes [Even less is known about the role of TRPC1 in CSC, since some of the inhibitors used to block SOCE, act over both Orai1 and TRPC1 channels. For instance, treatment with SKF96365, a SOCE inhibitor, impairs CSC proliferation in the glioblastoma stem-like cell line, TG1, triggering these cells to adopt a quiescent state by up-regulation of B1 genes . SimilarB1 genes . Even wh2+ current modulators due to their lower Ca2+ conductivity and greater fast Ca2+-dependent inactivation as compared to Orai1 [2+ influx pathways that are independent of the filling state of intracellular Ca2+ stores [2+ current mediated by a Orai1/3 channel [2+ influx mediated by the physical interaction between Orai1 and secretory pathway Ca2+-ATPase-2 [2+ influx mediated by the physical and functional interaction of Orai1 with the small conductance Ca2+-activated K+ channel 3 [+ channels by Orai1-mediated Ca2+ entry, which in turn leads to plasma membrane hyperpolarization, thus maintaining the driving force for Ca2+ influx and Ca2+ entry through Orai1 channels [As described above, native CRAC channels are hexameric structures comprised by the heteromeric association of Orai1, Orai2, and Orai3. Although all Orai family members can conform the channel, Orai2 and Orai3 also act as Cato Orai1 ,118. Sevto Orai1 ,120, livto Orai1 , and breto Orai1 ,123, Orato Orai1 , breast to Orai1 , oral [1to Orai1 , while Oto Orai1 , as wellto Orai1 , pancreato Orai1 , and proto Orai1  + stores . These p channel ,136,137,ATPase-2 ,139,140,hannel 3 ,142 or whannel 3 ,144. In channels ,145,146.2+ current, promoted a decrease in Sox2 expression, effect that was associated with reduced spheres formation and with the inhibition of their proliferation and self-renewal capacities [The role of Orai family proteins has also been described in the induction of CSC phenotype in a variety of cancers, such as glioblastoma, lung, and OSCC cancer cells. This CSC phenotype includes self-renewal capacity, tumor spheres formation, drug resistance, increased migration ability, and enhanced expression of stemness-related transcription factors and CSC-related markers ,147,148.pacities . Similarpacities . Converspacities .Regarding the role of Orai3 in the CSC phenotype acquisition in cancer cells, it has been demonstrated that Orai3 overexpression is correlated with tumoral aggressiveness and chemoresistance acquisition in breast cancer cells ,147. OraAltogether, the presented data support an essential role of SOCE mediators-STIMs, Orais, and TRPC proteins in the induction of CSC phenotype. However, our current understanding about the role of these proteins in cancer stemness is incomplete since the existing studies do not take in consideration their participation in other pathways, such as the store independent function of STIM1, Orai1, and Orai3, while the possible implications of Orai2 or TRPC1 in the stemness properties of CSC remain unclear.


Oxidative stress plays a role in carcinogenesis. This study explores the roles of oxidative stress-related genes (OSRGs) in lung adenocarcinoma (LAC). Besides, we construct a risk score model of OSRGs that evaluates the prognosis of LAC patients.  OSRGs were downloaded from the Gene Set Enrichment Analysis (GSEA) website. The expression levels of OSRGs were confirmed in LAC tissues of the TCGA database. GO and KEGG analyses were used to evaluate the roles and mechanisms of oxidative stress-related differentially expressed genes (DEGs). Survival, ROC, Cox analysis, and AIC method were used to screen the prognostic DEGs in LAC patients. Subsequently, we constructed a risk score model of OSRGs and a nomogram. Further, this work investigated the values of the risk score model in LAC progression and the relationship between the risk score model and immune infiltration. CCNA2, CDC25C, ERO1A, CDK1, PLK1, ITGB4, and GJB2 were 0.970, 0.984, 0.984, 0.945, 0.984, 0.771, and 0.959, respectively. This indicates that these OSRGs have diagnosis values of LAC and are significantly related to the overall survival of LAC patients. ERO1A, CDC25C, and ITGB4 overexpressions were independent risk factors for the poor prognosis of LAC patients and were associated with risk scores in the risk model. High-risk score levels affected the poor prognosis of LAC patients. Notably, a high-risk score may be implicated in LAC progression via cell cycle, DNA replication, mismatch repair, and other mechanisms. Further, ERO1A, CDC25C, and ITGB4 expression levels were related to the immune infiltrating cells of LAC, including mast cells, NK cells, and CD8 T cells.  We discovered 163 oxidative stress-related DEGs in LAC, involving cellular response to oxidative stress and reactive oxygen species. Besides, the areas under the curve of ERO1A, CDC25C, and ITGB4 of OSRGs are associated with poor prognosis of LAC patients. We confirmed that the risk model based on the ERO1A, CDC25C, and ITGB4 is expected to assess the prognosis of LAC patients. In summary,  Increasing the neutrophil infiltration and oxidative intermediates by oxidative stress contributes to disease occurrence. Current studies indicate that oxidative stress regulates cancer progression –3. For iss in BC .TWIST2) modulates tumorigenesis, tumor progression, and epithelial-mesenchymal transformation (EMT). TWIST2 is substantially downregulated in lung cancer tissues and cells. TWIST2 overexpression causes apoptosis, promotes the expression of E-cadherin protein, and inhibits the expression of N-cadherin, vimentin, and slug proteins. Besides, TWIST2 causes oxidative stress in lung cancer cells and inhibits lung cancer progression by modulating the FGF21-mediated AMPK/mTOR signaling pathway [Nrf2), is a hub transcription factor for cell adaptation and defense against oxidative stress. Oxidative stress reduces Nrf2 SUMOylation and promotes LAK cell invasion and migration. SUMOylation of Nrf2 increases its antioxidant capacity and reduces the level of ROS in LAC cells. Decreased SUMOylation of Nrf2 and increased ROS stimulate the JNK/c-Jun signaling axis to enhance cell migration and cell adhesion, as well as promote LAC cell invasion [Oxidative stress plays a significant role in cancer progression –8. Twist pathway . The nucinvasion . At presinvasion –11. Herehttp://www.gsea-msigdb.org/gsea/index.jsp) [The OSRGs were searched on the online GSEA website (dex.jsp) . The inphttps://portal.gdc.cancer.gov/) database. Of these, 59 were normal lung samples, whereas 535 were LAC samples. The expression data of OSRGs in 594 samples were retrieved. The expression of OSRGs in LAC tissues was identified by the limma package. The inclusion criteria were |logFC| = 1 and false discovery rate (FDR) < 0.05, which were defined as the oxidative stress-related DEGs.The gene expression data of 594 LAC patients with FPKM type were downloaded from the official website of TCGA  annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis were used to analyze the roles and mechanisms of multiple genes , 14. Thehttps://string-db.org/) was used to observe the interaction between multiple genes [The online STRING (version: 11.5) website  of patients with LAC were investigated by K-M survival analysis. The screening standard was set at ROC analysis was used to evaluate the diagnostic values of gene expression levels in cancer tissues. The diagnostic values were better when the area under the curve (AUC) was closer to 1 , 15. In CCNA2, ERO1A, CDK1, PLK1, CDC25C, ITGB4, and GJB2) and the prognosis of LAC patients. The screening standard was P < 0.05. Multivariate Cox regression analysis and AIC criteria were performed to screen the oxidative stress-related DEGs that independently influence the prognosis of patients with LAC. Subsequently, we constructed a risk score model [Univariate Cox regression analysis was performed to evaluate the relationship between the oxidative stress-related DEGs  and the risk score model. The expression levels of ERO1A, CDC25C, and ITGB4, and their relationship with the clinicopathological characteristics of patients with LAC in the high- and low-risk groups were explored and observed by scatter diagram and heat map. K-M survival and Cox regression analyses were performed to evaluate the relationship between the risk model and the OS of patients with LAC. The risk model-related nomogram was constructed based on multivariate COX regression analysis results.Correlation analysis was performed to investigate the relationship between the expression levels of risk model genes  software platform was used to analyze the BP, MF, CC, and signaling pathways of the DEGs. The gene expression data of 535 LAC patients in the TCGA database were grouped via the risk score and recorded as the high- and low-risk groups. The impact of the high- and low-risk groups on each gene set on the GSEA platform was explored to understand the signaling pathways involved in the risk score model. The running process was performed 1000-fold , 19. NomERO1A, CDC25C, and ITGB4) and the immune cell infiltration levels. Thereafter, the expression levels of LAC immune infiltrating cells in the high- and low-expression groups of ERO1A, CDC25C, and ITGB4 were analyzed by the median expression values of ERO1A, CDC25C, and ITGB4.ssGSEA analysis method was used to calculate the immune cell infiltration levels in the tissues with LAC. Spearman correlation analysis was used to explore the correlation between the expression levels of oxidative stress-related DEGs ; 5′-CATGCTTGGTCCACTGAAGA-3′ (reverse); CDC25C 5′-TGGTCACCTGGATTCTTC-3′ (forward); 5′-ACCATTCGGAGTGCTACA-3′ (reverse); and ITGB4 5′-TTCAATGTCGTCTCCTCCAC-3′ (forward); 5′-CAATAGGTCGGTTGTCATCG-3′ (reverse).In April 2022, we extracted the cancer tissues and adjacent normal tissues from 8 patients who underwent surgical treatment in our hospital and were diagnosed with LAC. All patients signed the informed consent. The study was reviewed and approved by the ethics committee of Taihe Hospital. The expression levels of R assays . The prit-test. Survival and ROC analyses were performed to analyze the LAC prognosis and diagnostic value of the oxidative stress-related DEGs, as well as the roles of the risk model in the prognosis of LAC patients. Correlation analysis was conducted to explore the relationship between the expression of ERO1A, CDC25C, and ITGB4 and LAC immune infiltration. P < 0.05 was considered statistically significant.The oxidative stress-related DEGs in LAC were analyzed by limma package or A total of 32 gene sets related to oxidative stress were searched on the GSEA platform. These 32 gene sets comprised 784 OSRGs. The OSRGs in normal lung and LAC tissues were corrected and extracted from the TCGA database. Differential expression analysis showed 163 DEGs in LAC tissues compared to normal lung tissues . Among tFOXO signaling pathway, non-small-cell lung cancer, TNF signaling pathway, ferroptosis, transcriptional misregulation in cancer, HIF-1, IL-17, p53, and among other signaling pathways  showed abnormal and statistically significant expression of mast cells, iDC, eosinophils, DC, NK cd56dim cells, NK cells, Tfh, Tgd, Th2 cells, macrophages, CD8 T cells, pDC, T helper cells, Th17 cells, Tcm, neutrophils, and Tem in the high- and low-expression groups of CDC25C  is strongly upregulated during hypoxia in vitro and is associated with hypoxia markers and poor prognosis in LAC. NLUCAT1 downregulation inhibits the proliferation and invasion of LAC cells and increases oxidative stress and sensitivity to cisplatin [Lung adenocarcinoma has a high incidence and mortality rates , 15, 20.isplatin . SeveralCCNA2, CDC25C, ERO1A, CDK1, PLK1, ITGB4, and GJB2 could influence the cancer progression [ERO1A, also known as ERO1L, promotes IL6R secretion by targeting disulfide bond formation. IL-6R binds to IL-6, resulting in the activation of the NF-κB signaling pathway. NF-κB, in turn, binds to the promoter of MUC16, causing its overexpression. ERO1L may trigger CA125 secretion via the IL-6 signaling pathway, form a positive feedback loop, and promote lung cancer development [CCNA2, CDC25C, ERO1A, CDK1, PLK1, ITGB4, and GJB2 significantly correlated with overexpression levels and poor prognosis of patients with LAC and exhibited diagnosis values of LAC. Bioinformatics analysis and PCR identification showed overexpressed oxidative stress-related DEGs ERO1A, CDC25C, and ITGB4 in LAC tissues and were independent risk factors for poor prognosis in patients with LAC. The risk model based on ERO1A, CDC25C, and ITGB4 is an independent risk factor for poor prognosis in patients with LAC. In the risk model-related nomogram, the risk score demonstrated the greatest impact on the prognosis of LAC patients. This indicates that our risk score model evaluates the prognosis of LAC patients.The expression levels of gression –26. For elopment . ThroughCCNB1) is an important gene in mitosis and is upregulated in LAC tissues. CCNB1 overexpression contributes to the advanced tumor stage and short OS. A negative correlation has been discovered between miR-139-5p and CCNB1 expression levels. Through negative CCNB1 regulation, miR-139-5p inhibits cell proliferation and migration [CASC2 is downregulated in LAC. Its overexpression inhibits the proliferation of LAC cells and improves apoptosis. It also directly inhibits miR-21 expression and upregulates p53 protein expression to mediate cell proliferation and apoptosis in LAC [ERO1A, CDC25C, and ITGB4 is closely related to the signaling mechanisms of cancer progression, preliminarily confirming that our risk model is closely associated with LAC progression.Cell cycle, homologous recombination, and p53 signaling pathway are associated with cancer progression –31. Cycligration . lncRNA PD-1 and PD-L1 blockers have been approved as standard therapy for non-small-cell lung cancer. In contrast with chemotherapy or radiotherapy, PD-1/PD-L1 blocking therapy improves the remission rate. It prolongs the survival time, with fewer side effects in patients with advanced non-small-cell lung cancer treated with a single drug or combined therapy [PD-1 on the surface of NK cells. In contrast with peripheral NK cells, the role of NK cells in tumor is poor, and this dysfunction is associated with the expression level of PD-1. PD-1 blocking therapy reverses the PD-L1-mediated inhibition of PD-1 NK cells [ERO1A, CDC25C, and ITGB4 expression levels and the immune microenvironment. As a result, the expression levels of ERO1A, CDC25C, and ITGB4 significantly correlated with the levels of NK cells, mast cells, Tfh, NK cd56dim cells, iDC, neutrophils, and NK cd56bright cells. Nonetheless, additional future studies are necessary to confirm the roles of the OSRGs ERO1A, CDC25C, and ITGB4 in the LAC immune microenvironment.In recent years, immunotherapy has been a crucial treatment option for patients with LAC –35. Addi therapy , 33. NK ERO1A, CDC25C, and ITGB4 expressions were significantly upregulated in the tissues from our hospital. Nevertheless, large amounts of tissues and patient prognostic data are necessary to verify the risk score model. Therefore, future studies should collect additional clinical tissue samples to detect the expression levels of CDC25C, ERO1A, and ITGB4 and investigate their roles in the prognosis of LAC. Moreover, other research should explore the roles and mechanisms of CDC25C, ERO1A, and ITGB4 in the immunity and progression of LAC at the cellular level.Our study applies bioinformatics analysis to investigate the roles of the OSRGs in the progression of LAC. The strengths of this study include large sample size, long follow-up time, and comprehensive prognostic data in the TCGA database. Besides, we provide novel candidate markers for LAC treatment and a risk model that evaluates the prognosis of LAC patients. Through PCR detection, CCNA2, CDC25C, ERO1A, CDK1, PLK1, ITGB4, and GJB2 of OSRGs have diagnosis values of LAC and are associated with the prognosis of patients with LAC. ERO1A, CDC25C, and ITGB4 overexpressions are independent risk factors for poor prognosis in patients with LAC. A high-risk score is an independent factor affecting the poor prognosis of LAC patients. ERO1A, CDC25C, and ITGB4 expressions of risk score model genes significantly correlate with the levels of mast cells, IDC, NK cells, and CD8 T cells of LAC immune infiltrating cells. Therefore, the risk score model based on the ERO1A, CDC25C, and ITGB4 is expected to predict the prognosis of patients with LAC.In conclusion,


Background: Parental non-responsive feeding practices and child eating behaviors both play significant roles in childhood obesity. However, their longitudinal relationships are less clear. This systematic review aimed to examine their bidirectional associations. Methods: A systematic search of five databases was conducted from inception to February 2022. Data synthesis was performed using a semi-quantitative and quantitative approach. Results: A total of 14 studies with 15348 respondents were included. A total of 94 longitudinal effects from 14 studies of parental non-responsive feeding practices on child eating behaviors were investigated, and 19 statistically significant effects were discovered. Seventy-seven longitudinal effects from nine studies of child eating behaviors on parental feeding practices were examined, with fifteen being statistically significant. The pooled results of meta-analysis showed five statistically significant associations: parental restrictive feeding positively predicted child enjoyment of food ; use of food as a reward positively predicted child emotional eating ; child food responsiveness positively predicted restrictive feeding ; use food as a reward . In addition, the pooled effects showed that child satiety responsiveness negatively predicted restrictive feeding . Conclusions: The bidirectional relationships between parental non-responsive feeding practices and child eating behaviors are inconsistent and a few showed statistical significance. Theory-driven longitudinal studies using validated instruments and controlling for potential confounders are needed to unveil their relationships and provide evidence for obesity prevention interventions. Childhood overweight and obesity is a worldwide health issue that affected 39 million children under the age of 5 in 2020 . In ChinObesity in young children is a multi-faceted problem  ,8, of whn = 4987) in the Netherlands showed that parental restrictions of food and pressure to eat were both positively correlated with four-year-old children’s food responsiveness, emotional overeating, and satiety responsiveness [n = 977) in Australia indicated that parental higher overt restriction was associated with more preschool children’s food responsiveness [Some cross-sectional studies have reported the relationships between parental non-responsive feeding practices and child eating behaviors ,26,27,28siveness . Venturasiveness  also pron = 797) showed that greater parental use of food as a reward positively predicted child emotional eating and food responsiveness with a 2-year follow-up, while child eating behaviors did not predict parental feeding practices  [n = 229) showed that mothers’ restriction of food amount at 21 months of the child was a negative predictor for child eating in the absence of hunger (EAH) at 27 months, but EAH did not prospectively predict maternal food restriction [Recent longitudinal studies provided empirical support for bidirectional relationships ,30,31. Fciation) . Similartriction . Lumeng triction  did not Overall, current findings of the bidirectional relationships between parental non-responsive feeding practices and child eating behaviors have been inconsistent. Given the increasing prevalence of overweight and obesity among young children and progressive evidence linking parental non-responsive feeding practices to child eating behaviors, the aim of this systematic review was to identify and summarize the relationships of interest reported previously. Findings from this review may enhance our understanding of their bidirectional correlations, provide recommendations for future research, and guide future interventions for the prevention of childhood obesity.The systematic review and meta-analysis were guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines  and the http://bvsalud.org/en/, accessed on 12 February 2022), NARCIS , Grey literature report , and Open grey EU . The search was limited to publications in English. Free text and the Medical Subject Headings (MeSH) terms were used for the search. The following terms were used with Boolean operators: ‘child’ AND ‘feeding’ AND (‘eating’ OR ‘diet’ OR ‘food intake’) AND . A manual search of the bibliography of included studies was performed to identify additional studies.A systematic search (from inception to February 2022) was carried out on PubMed, Embase, PsycINFO, ScienceDirect, and Cochrane Library. We also searched for grey literature on Virtual Health Library ((1)Both parental feeding practices and child eating behaviors were reported.(2)Children aged 1–6 years at baseline .All studies examining the longitudinal relationships between parental non-responsive feeding practices and child eating behaviors were included if they met the following criteria:(1)Were reviews, editorials, commentaries, letters, or methodological papers;(2)Were non-English papers;(3)Did not report the bidirectional relationships between caregivers’ non-responsive feeding practices and children’s eating behaviors;(4)Focused on children with diseases that might influence their eating;(5)Included primary caregivers who were not the child’s parents;(6)Used observation for the assessment of feeding practices or eating behaviors. Observational assessment may be subject to the constraints of specific occasions and, thus, cannot represent the meaning of behavior completely, while self-reports tend to refer to general practices ,40. AddiStudies were excluded if they possessed the following features:The PRISMA flow chart was followed during the screening stage . One inv(1)Restriction means that the caregivers enforce strict limitations on the child’s access to food or opportunities to consume specific food . Typical(2)Pressure to eat means that caregivers insist, demand, or physically struggle with the child in order to get the child to eat more food ,46,47,48(3)Emotional feeding represents caregivers using food as a method to manage or calm the child when he/she is upset, fussy, angry, hurt, or bored ,49. For (4)Use food as a reward, also called instrumental feeding, involves using threats and bribes or pushing children to eat more ,50. BaseReward for behavior means that caregivers threaten to take something away for misbehavior or promise/offer something to the child in return for desired behavior ,50.Reward for eating means that threats and bribes can be used to manage child’s behavior for the purposes of general obedience or behaviors specific to eating ,50.Nonfood incentives to eat means that threats and bribes around child eating behaviors  may be nonfood incentives  .Based on the conceptual analysis of food parenting practices ,43,44, n(1)Food fussiness refers to being highly selective about the range of food that are accepted . It is a(2)Satiety responsiveness refers to the amount of food the child eats in a meal . This is(3)Food responsiveness refers to the desire to eat food when they see or smell food or are supplied with food . It is a(4)Emotional eating refers to eating in response to emotions  . It usua(5)Enjoyment of food refers to the extent of enjoying all kinds of food and desire to eat ,54. The Child eating behaviors were categorized into five types, which are commonly used in previous studies ,51,52,53The Joanna Briggs Institute (JBI) Critical Appraisal Checklist for Cohort Studies was used for quality appraisal . This toStatistical parameters representing the associations between parental non-responsive feeding practices and child eating behaviors were extracted by regression coefficient (β) with their 95% confidence interval (CI) or standard error (adjusted for covariates). We contacted the authors if their studies did not report these statistics. The studies eventually without available standard errors or 95% CIs of regression coefficients were excluded from the meta-analysis. We thus used a semi-quantitative approach for data synthesis of all included articles to describe their associations across the time points, as adopted by recent reviews ,59.I2 statistic, in which I2 > 30% was considered to indicate moderate heterogeneity and I2 > 50% was considered to indicate substantial heterogeneity. A p value < 0.05 from the noncentral chi-squared test for heterogeneity was considered to indicate statistically significant heterogeneity [The studies with necessary information were included in the meta-analysis. For associations evaluated multiple time points in one study, they were first synthesized within the study, and the summarized data were used for meta-analysis. In examining the longitudinal associations between specific feeding practices and child eating behaviors, the pooled regression coefficients with corresponding 95% CI across studies were calculated and presented using forest plots. Random-effects models were used when heterogeneity among the studies was presented . The degogeneity . The pubA total of 2483 articles were identified. The removal of duplicates resulted in 1982 articles for initial screening, and 249 articles were retrieved. After screening the full texts of 249 articles, 14 prospective studies were included. The PRISMA flow chart is shown in Eleven studies were rated high quality. Three studies were rated moderate quality see .n = 3) [n = 2) [n = 2) [n = 1) [n = 6) [n = 9847; 10 studies) and parents . Nine studies used voluntary (response) sampling and the response rate ranged from 59.83% [Characteristics of the studies are shown in n = 3) ,62,63, N [n = 2) ,53, Norw [n = 2) ,64, Port [n = 1) , and Aus [n = 6) ,52,66,67 [n = 6)  to 4845  [n = 6) . The carm 59.83% ,66 to 10Seven studies collected data at two time points ,53,65,67n = 7) [n = 3) [n = 2) [n = 1) [n = 1) [Tools used for the assessment of parental feeding practices are shown in n = 7) ,62,65,67n = 7) , which wn = 7)  (n = 3)  [n = 3) ,63,64, t [n = 3)  (n = 2)  [n = 2) ,66, the  [n = 2)  (n = 1)  [n = 1) , and the [n = 1)  (n = 1)  [n = 1)  conducteTools used for the assessment of child eating behaviors are also shown in n = 10) [n = 7) [n = 4) [n = 3) [n = 5) [n = 5) [n = 3) [n = 6) [n = 2) [The longitudinal associations are shown in n = 10) ,65,66,67 [n = 7) ,63,64,65 [n = 4) ,32,53,63 [n = 3) ,32,53, a [n = 5) ,52,63,67 [n = 3) ,34,65, u [n = 2) ,63.n = 10) [(n = 7) [n = 6) [n = 7) [n = 10) ,65,66,67[(n = 7) ,62,65,67 [n = 6) ,63,64,66 [n = 7) ,62,65,67 [n = 7) ,65.Specifically, 10 studies ,65,66,67n = 6) [n = 5) [n = 4) [n = 4) [n = 6) ,63,65,66 [n = 5) ,53,63,65 [n = 4) ,32,53,63 [n = 4) ,32,53,63Six studies ,63,65,66I2 between 29.7% and 55.7%).However, all five synthesized results were only based on available data from no more than three studies and heterogeneity between studies was also considerable  was positively associated with parental non-responsive feeding practices. The results of meta-analysis showed that children’s food responsiveness positively predicted parental use of food as a reward and restrictive feeding. The explanation for our finding is that, if a child likes to eat or eats more, food is an obvious reward for parents to give  because Theoretically, feeding and eating behaviors interact with each other; however, current findings from longitudinal studies do not provide strong evidence. There may be several reasons. First, age may play a role in the non-significant findings. For example, Bauer et al.  found thWe also found inconsistent findings about the longitudinal associations between specific feeding practices and eating behaviors. The reasons are as follows. Child sex may be a contributing factor for the inconsistency ,83. MothTo the best of our knowledge, this review was among the first that comprehensively synthesized longitudinal data on the prospective relationships between parental non-responsive feeding practices and child eating behaviors. However, there are several limitations to this systematic review. First, although meta-analysis was conducted, the quantitative synthesis was mainly based on two or three studies because some studies did not report standard error or 95% CI, which precluded us from pooling all extracted data and from examining their bidirectional associations. Thus, the results from the meta-analysis should be interpreted with caution due to the limited number of studies included. Second, variability in the assessment of parental feeding practices  and child eating behaviors  may make it difficult to compare findings; therefore, results should be interpreted with caution. Third, all included studies employed an array of self-reported questionnaires to assess our interest variables, which may be subject to recall bias. Furthermore, the included studies were mainly conducted in western countries such as Australia and the US. Findings from this review may not be extrapolated to other populations .More longitudinal studies are needed to further examine the bidirectional relationships between parental non-responsive feeding practices and child eating behaviors. Such studies should be adequately powered and include a representative sample. Second, validated instruments should be used consistently across studies for future comparison between different studies. Third, more potential confounding factors  should be taken into consideration. In addition, empirical studies should be derived from theoretical frameworks, such as Ecological System Theory , which cThere is mixed evidence for the bidirectional associations between parental non-responsive feeding practices and child eating behaviors. Only weak correlations were found between certain feeding practices and eating behaviors. Parents may be more likely to adopt restrictive feeding and food as a reward with children who have higher food responsiveness, whereas parents may be less likely to use restriction with children who have higher satiety responsiveness. Children may be more likely to have emotional eating if their parents use food as a reward more frequently. Future prospective, theory-driven studies using validated instruments and representative sampling while controlling for potential confounders are needed to provide more evidence. It is also important to understand the nature of their relationships, which may help to develop more personalized interventions to prevent childhood obesity. In addition, it is necessary to conduct relevant research in Asian and low-middle income countries, as all included studies were in western and high-income countries.


Escherichia coli have become widespread among food producing animals. These strains serve as a reservoir of antibiotic resistance genes (ARGs) and act as a possible source of infection to humans as transmission can occur by direct or indirect contact.Extended spectrum beta-lactamase (ESBL) producing E. coli in poultry over a 2-year period (2017-2019) from Zimbabwe. A total of 21 ESBL positive isolates from poultry cloacal specimens were selected for whole genome sequencing from animal E. coli isolates bio-banked at the National Microbiology Reference laboratory using phenotypic susceptibility testing results from the National Escherichia coli Surveillance Program to provide representation of different geographical regions and year of isolation. Cloacal swabs were collected from 3000 broiler live birds from farm 1 and from farm 2, 40 backyard chickens and 10 ducks were sampled. Antimicrobial susceptibility and ESBL testing were performed as per Clinical Laboratory Standards Institute guidelines. Whole genome sequencing of ESBL producing isolates was used to determine sequence types (STs), ARGs, and phylogroups.This study investigated the faecal carriage of ESBL producing and colistin resistant E. coli isolates were confirmed as ESBL producers. Three defined sequence type clonal complexes (CCs) were identified , with ST10CC associated with the most antibiotic resistant profile. The ESBL phenotype was linked to the presence of either cefotaximase-Munich-14 (CTX-M-14) or CTX-M-79. Plasmid mediated quinolone resistant determinants identified were qnrB19 and qnrS1 and one ST10CC isolate from farm 1 broiler chickens harbored a mobile colistin resistance gene (mcr-1). Phylogenetic groups most identified were B1, A and unknown.Twenty-one of the included E. coli belonged to a diverse group of strains. The detection of several ARGs highlights the importance of implementing enhanced control measures to limit the spread in animals, environment, and humans. This is the first report of mcr-1 in Zimbabwe, which further underscores the importance of the One Health approach to control the spread and development of AMR.The avian ESBL producing  Escherichia coli is a global priority to curb an ever-increasing range of infections caused by bacteria that are no longer susceptible to the common or first –line antibiotics that are used to treat them. The spread of these resistant E. coli strains is a serious threat to public health worldwide. The World Health Organization (WHO) has recognised the need for an improved and coordinated global effort to contain AMR  surveillance on tain AMR . The higtain AMR . The magtain AMR .E. coli between these two host species. There are several antimicrobials that are used to raise poultry in several countries  in humans, animals, and the environment, ever since its first detection in November 2015 in China]. To date, nine different plasmid-mediated mcr variants have been described, designated as mcr-1-9, isolated from humans, animals, and the environment  and Harare 13 broiler chickens for analysis. All these ESBL positive carriage isolates displayed MDR phenotypes. The most common MDR phenotype was resistance to ampicillin, cefotaxime, ceftriaxone, ciprofloxacin, nalidixic acid, and tetracycline (AMP-CTX-CRO-CIP-NAL-TET) that was observed in 11 isolates as shown in Twenty-one isolates had a combined length of contigs of assembled genomes ranging from ~4.7 Mbp to 9.8 Mbp, with a minimum contig length required to cover 50% of the genome (N50), ranging between 84 kbp to 340 kbp. The SNP matrix output table with sequenced isolates aligned and compared to reference genomes were used to build a phylogenetic tree as shown in E. coli isolates included in this study presented with diverse ESBL genes, where six harboured the cefotaximase-Munich (CTX-M)-14 gene, one harboured the CTX-M-15 gene, two harboured CTX-M-55, two harboured CTX-M-79 and other Temoneira (TEM) and OXA gene variants. No sulfhydryl variable (SHV) variants were detected as shown in qnrS19 (13), qnrB1 (12), qnrD1 (2) and aac (6’)-lb-cr genes respectively. One isolate was positive for the mcr-1 gene. Phylogroups identified were as follows: group unknown (8), group A (5); group B1 (4); group D (2); group B2 (1) and group G (1) respectively.The 21 avian ESBL Multilocus sequence typing identified three diverse ST clonal complexes (CCs), which included the following: ST10CC; ST155CC and ST23. Among the ST10CC the STs included ST10, ST48 (2), ST215, ST761, ST2461, while ST155CC included ST155 and ST23CC had ST88. Most of the isolates  belonged to one of these defined CCs. The remaining proportion (42%) of isolates in this study represents the undefined clonal complexes referred to as “unknown CCs”. These ST unknown CCs are represented by ST1140, ST1196, ST2197, ST127, ST937, ST5123, ST117, ST2732 and ST2107 Table 1.The most predominant plasmids were Col156, Col440I, ColMG828 and Col pHAD28 that were widely distributed among the ST10 and ST2197 strains. All strains characterised in this study harboured incompatibility group F (IncF) plasmids. The other replicon types detected in this study included IncY, Incl1, Incl2, IncH, IncQ, Inc X, p0111. The plasmid replicons represented as IncFlB were sixteen (n=16) followed by IncFll (n =11) and IncFlA (n = 5).A total of nine isolates [three from human isolates  and six from poultry isolates  in Zimbabwe. The isolates from this study revealed the prevalence of faecal carriage of ESBL E. coli in poultry to be 11%, which is comparable to similar studies that were done in León, Nicaragua (13%)   and Lebaua (13%) . Extensiua (13%)  accordinua (13%) . Notablyua (13%) .in silico MLST was used to determine the circulating clones of importance in the faecal carriage ESBL E. coli from Zimbabwean avian species. The most common STs determined were ST10CC, ST155CC and ST88CC, which are known to be interspecies clones  play an important role in the transmission of virulence and resistance genes of ESBL producing isolates . The dife tissue .IncF plasmids, which agrees with other epidemiological studies that revealed an association of blaTEM, blaOXA, and blaCTX-M genes to IncF plasmids (qnr), tetracyclines (tet), and trimethoprim have been observed to co-exist with blaTEM, blaOXA, and blaCTX-M variant genes in this study. The co-existence of two or more beta-lactamases in the same strain was observed in ST10 and ST155 isolates as reported in other studies  in Zimbabwe. The mcr 1.1 positive E. coli isolate belongs to the ST10 clone. Furthermore, our findings suggest that the diversity among the ST10CC in terms of harboring several AMR determinants, is responsible for the high resistance profiles found in this study. The presence of other AMR determinants on the same plasmid as the one harboring mcr-1 gene may pose a huge public health threat due to the existing risk of spread of resistance genes e.g., mcr-1 among bacterial strains, to other food producing animals, environment, and humans  This selection provided representation of different geographical region and years of isolation . Figure 1Escherichia coli ATCC 25922 was used as quality control strain. Additional data included on collection of each isolate were the year, location of isolation, and city .All the selected isolates were sub-cultured on MacConkey or eosin methylene blue (EMB) , incubated at 37°C  for 18 to 24 hours and then stored in 20% glycerol broth at -80°C. Antimicrobial susceptibility testing was determined by the Kirby Bauer disc diffusion method using the Clinical Laboratory Standards Institute (CLSI) guidelines . The antE. coli was extracted using the Wizard® Genomic DNA Purification Kit  according to the manufacturer’s instructions (http://www.basespace.illumina.com) and then converted to FASTQ files.Genomic DNA (gDNA) of avian ructions  and storDe novo assembly was performed with SPAdes 3.11 (E. coli strain K12 sub-strain MG1655 was used as reference genome (NCBI accession: NC_000913.3). After the core-genome alignment, a reconstruction of maximum likelihood phylogeny with 1000 bootstrap replicates with RAxML v8.2.4 based on a general time-reversible nucleotide substitution model was performed (E. fergusonii as an outgroup (NCBI accession: GCA_000026225.1). The phylogenetic tree was visualised in Figtree v1.4.4  STs  ; (ii) phThe data presented in this study are deposited in the NCBI BioProject Number PRJNA 799483 with accession numbers listed in the Ethical clearance was obtained from the Faculty of Health Sciences Research Ethics Committee, University of Pretoria (Ethics Reference Number: 782/2018) and the Medical Research Council of Zimbabwe .All authors provided critical input and contributed to the manuscript writing and approved its final version. Conception or design of the work FT, JP, MK, SZ, TM, LM. Methodology FT, JP, MK, JM, SM, LWM, AT. Formal analysis and interpretation FT, JP, MK, RK, GT, AG, GP, DG, TM. Writing – original draft preparation FT, JP, MK. Writing – review and editing of the article FT, JP, MK, ME, AT, TM. Final approval of the version to be published FT, JP, MK, SZ, TM, LWM, GT, AG, RK, GP, JM, SM, LM, DG, AT, ME. All authors contributed to the article and approved the submitted version.