This is partly due to the inherent difficulty of detecting these compounds without intentionally seeking them. Inconsistencies in product descriptions and terminology have led to additional confusion regarding cranberry product composition and the possible presence of oligosaccharides. This review will present our current understanding of cranberry oligosaccharides and will discuss their occurrence, structures, ADME, biological properties, and possible prebiotic effects for both gut and urinary tract microbiota. Our hope is that future investigators will consider these compounds as possible significant contributors to the observed biological effects of cranberry.Cranberry is a well-known functional food, but the compounds directly responsible for many of its reported health benefits remain unidentified. Complex carbohydrates, specifically xyloglucan and pectic oligosaccharides, are the newest recognized class of biologically active compounds identified in cranberry materials. Cranberry oligosaccharides have shown similar biological properties as other dietary oligosaccharides, including effects on bacterial adhesion, biofilm formation, and microbial growth. Immunomodulatory and anti-inflammatory activity has also been observed. Oligosaccharides may therefore be significant contributors to many of the health benefits associated with cranberry products. Soluble oligosaccharides are present at relatively high concentrations (~20%  Vaccinium macrocarpon, Ait. Ericaceae), and numerous detailed reviews have discussed the chemistry and biological properties of previously identified compounds from various cranberry materials ,,28,2) ans (3) 1 ,26. The . The 2 aantities ,29,30  that is then used to produce sweetened (CJK) or blended (CJB) fruit juices ,35,36,37Cranberry products are also significant in the dietary supplement market . These pThe multiple forms of processed cranberry materials  correspoDifferent types of cranberry processing physically and chemically separate components of fresh whole berries (FWB) based on solubility and other component characteristics. Juice extraction involves the separation of solid and liquid fruit components by pressing or decanting, followed by centrifugation and filtering, and the liquid may be further filtered in stages to remove suspended solid particulates ,58,59. JProcessing steps may also change the chemical composition and chemical characteristics of cranberry products in ways similar to those observed for other fruits ,55,65,67Chemical and biological reactions that occur at each processing step also influence the final chemical composition of each cranberry product. Treatments with pectinase and other enzymes cleave polymeric pectins, cellulose, and hemicelluloses found in the whole berries into lower molecular weight (MW) compounds. These relatively low MW plant cell wall components may end up in both pomace and juice based on solubility and interactions with other components of both materials, as has been observed for other fruit juices ,76,77. DWhile all cranberry products may have health benefits, CHEP and juices made from CJS are two of the most commonly studied products in terms of UT health. The bioactive compounds present in both materials must therefore survive many of the processing steps described above as well as those unique to the production of both CJS and CHEP. Such compounds would also need to be originally present in whole berry materials (such as WBP) rather than be the products of chemical reactions resulting from specific processing steps. The effects of processing on the chemical composition of the final product are well recognized for flavonoids and other polyphenolic compounds ,2,3 and Vaccinium products also suggests that a core set of similar compounds may be present in all such materials. Multiple species within the Vaccinium genus have been reported to have biological properties similar to those of V. macrocarpon. Among the many examples that exist, two include the anti-biofilm properties for lingonberry extracts (V. vitis-idaea) +, is believed to indicate a xyloglucan octasaccharide with a Hex4Pent4 structure ..18 sorbew/w of CCA, w/w of CJA) and SNA (SNA Oligos: 53% w/w of SNA) fractions. The cause for these distinct overall percentages of soluble oligosaccharides in different source materials has yet to be established but it may be due to both product formulation and processing  as the 15% methanol fraction. Yields were not given for the other fractions obtained from the separation. The A2 fraction was partially characterized as containing arabinoxyloglucans with small amounts of phenolic pigments, and was further separated using Sephadex LH-20 with water to remove these phenolic compounds and yield the A6 material. The A6 fraction was initially described as containing "neutral, sugar-rich, low-viscosity, low-MW polysaccharides"  and andw/w (w/w oligosaccharides (see w/w (20 g/100 g).General chemical analysis of CHEP provides relative percentages for multiple classes of compounds, but the values given account for only 85% total content ,149, leaides see . As lowew/w of CCA) than in comparable fractions from CHEP or WBP ,” but this assumption cannot be definitively confirmed with the limited information provided in most studies. The phrase “cranberry extract” is additionally ambiguous if methods are not reported, as it could describe material obtained from a juice (water) extraction from solid berry material or an organic solvent-based extraction from any type of cranberry material. Descriptions of material as “cranberry juice powder” may refer to either CHEP or dried juice products though the latter scenario is less likely. The drying methods used to obtain an actual juice powder must also be reported as these may affect composition. Descriptions such as “cranberry powder” do not specify whether the powder was obtained from a specialty production process (CHEP), whole berries (WBP), or some other type of extraction and powdering process. Compositional differences between CJS, CHEP and WBP would be significant, with neutral and pectic polysaccharides and higher DP oligosaccharides being present in whole berry materials, and differences for all three materials with regards to relative concentrations of lower MW oligosaccharides resulting from different enzymatic cleavage processes. Descriptions of material as “freeze-dried cranberry powder” are doubly ambiguous as they may refer to lyophilized whole berries that have been ground, or may be inaccurate descriptions of CHEP . In addition to confusion over product origin and initial processing, distinction between the two drying methods is significant as each would have different effects on product chemical profiles see .1H-NMR spectroscopy, and which may contain polar compounds, are therefore highly likely to contain oligosaccharides.Unrecognized oligosaccharides may be present in a variety of cranberry fractions generated from this range of ambiguously described starting materials and may therefore influence observed study results. Numerous chromatographic separation methods may inadvertently enrich for oligosaccharides in conjunction with other components without the awareness of the researchers. Many standard chromatographic separations used for both detection and isolation employ UV or LCMS-based detection methods, both of which are largely insensitive to the presence of cranberry xyloglucans and other neutral oligosaccharides see  151]. E. E151]. w/w) of most available cranberry materials [w/w; 20 g/100 g)  see  are at lThe PAC-enriched cranberry materials used in various clinical studies are also likely to contain significant amounts of oligosaccharides and other cranberry compounds. For example, the 37 mg of PACs reported by Howell et al. ,156 is pThe chemical nature of oligosaccharides and their tendency to remain undetected requires that minimum standards be established for both describing and reporting the oligosaccharide content of various cranberry materials. The following are some suggestions based on our observations of the current cranberry oligosaccharide literature. Many researchers investigating cranberry materials may not be familiar with the detailed chemical analysis of complex carbohydrates or may not have access to advanced structural characterization resources such as MALDI-TOF MS or NMR instruments. Fortunately, such familiarity and resources would not be required to confirm the presence or absence of this class of compounds. Those seeking additional knowledge regarding the chromatographic and spectroscopic methods used by complex carbohydrate experts for oligosaccharide structural elucidation are referred to other publications on the topic ,120,157.(A)(1)(a)Monosaccharide composition analysis suitable for neutral or pectic polysaccharides and oligosaccharides to give relative molar percentages of monosaccharide constituents.(b)1H-NMR spectrum (D2O) showing distinct anomeric signals corresponding to oligosaccharides previously characterized from cranberry materials.Identification of possible monosaccharide composition using one of the following methods:(2)(a)Detailed description of the cranberry source material using standardized terminology  and addi(b)Inclusion of sufficient separation details (including yields and accurate sorbent descriptions) necessary for another researcher to repeat the methods used.Full description of the source material and separation methods(3)(a)A PDAD max plot UV spectrum will show the presence or absence of trace amounts of organic acids and phenolic compounds, such as flavonoids and PACs. A UV spectrum would also indicate the presence of uronic acids such as those present in pectic oligosaccharides.(b)Visual description of the dried material. Trace amounts of anthocyanins can be visually detected as a slight pinkish or purplish color when the material is dried.Reported presence or absence of additional possible constituents(4)(a)Characterization by MALDI-TOF MS or other MS-based methods customized for oligosaccharide detection using neutral oligosaccharide standards of at least DP 3–10 (include the specific identity of the standards used for comparison).(b)Glycosyl linkage analysis using chemical derivatization with appropriate methods and standards.(c)2D NMR spectra showing anomeric region correlationsAdditional characterization if possible:Minimum requirements (1–3) for oligosaccharide characterization and content descriptions with additional (4) characterization if possible:(B)1.1H-NMR spectrum (D2O) showing the absence of anomeric signals corresponding to oligosaccharides.2.UV and LCMS-based detection with negative results may be acceptable if oligosaccharide-appropriate derivatization and separation methods are applied.3.The application of a standard monosaccharide analysis method suitable for oligosaccharides and other complex carbohydrates yields 0% monosaccharides.Minimum requirements for eliminating oligosaccharides as possible components of a given cranberry material. Any one of the following three recommendations would be acceptable.Complex carbohydrate phytoconstituents such as cellulose, hemicellulose, lignins, and polyphenolic compounds are often described as dietary fibers in studies investigating nutritional or general health effects. These compounds are classically considered to be high MW polymers and insoluble structural components, but the definition of dietary fiber has recently been modified to include lower MW, soluble oligosaccharides (DP 3–10 or 3–20) ,110. VarCranberry complex carbohydrates are likely to have similar absorption, distribution, metabolism, and excretion (ADME) profiles as other polymeric dietary fibers ,159,160.Most dietary fibers are too large (high MW) to be directly absorbed and therefore remain in the GI tract ,159. HumComplex carbohydrate components that are not degraded by gut microbiota may be excreted unchanged in the feces but also contribute to beneficial health effects ,111,166.The presence of a complex profile of neutral arabinoxyloglucan oligosaccharides in porcine urine after cranberry consumption indicates that these compounds can be absorbed from the GI tract, circulated systemically, and filtered from the blood by the kidneys to be excreted into urine after ingestion ,25,26. TSchistosoma parasitic worms [13C labeled breast milk oligosaccharides indicate that urine contains oligosaccharides that are directly absorbed upon ingestion as well as those that may be derived from the degradation of breast milk polysaccharides by gut bacteria [The presence of multiple types of oligosaccharides in mammalian urine from both dietary and other sources indicates that the kidneys are able to filter this class of compounds from blood into the urine ,180,181.ic worms , indicatic worms ,179,180.bacteria . Animal bacteria ,180,181.While studies investigating urinary oligosaccharides often hypothesize that endogenous oligosaccharides, such as those derived from glycoproteins, are the source of unknown urinary oligosaccharides , these cSome of the unknowns regarding the presence and concentrations of dietary oligosaccharides in adult human urine may be attributed to the difficulty of isolating or detecting these compounds from in vivo systems ,172. Uri13C-labeled oligosaccharides [The detection of soluble oligosaccharides in urine is further complicated by structural features and solution behaviors that require the application of specialized detection techniques such as those used previously for this chemical class ,180,184.charides ,176. MetCurrent evidence indicates that cranberry complex carbohydrates share many of the biological properties and beneficial health effects as dietary fibers from other sources ,188,189.E. coli adhesion is of particular interest as this bacterium is commonly associated with symptomatic UTIs [E. coli [The inhibition of bacterial binding to surfaces is believed to be significant for human health as it prevents pathogenic bacteria from subsequently invading host cells ,198,199.[E. coli  and adhe[E. coli .Oligosaccharides from multiple dietary sources have anti-adhesion properties ,201,202.E. coli strains , though additional E. coli strains have also been used. Multiple anti-adhesion assays have been used to assess the anti-adhesion properties of cranberry fractions containing oligosaccharides . These iE. coli may express multiple and diverse adhesion phenotypes that provide fitness advantages in specific environments [E. coli [In vivo, bacteria such as ronments ,206. Bacronments ,199,207.ronments ,208, but[E. coli ,212,213.[E. coli ,25,26.E. coli (CTF073: ATCC 700928) have since been developed. The labeling of E. coli facilitated the quantification of bacterial cells adhered to confluent layers of epithelial cells after a test sample treatment [E. coli (UTI89) [E. coli .Improved quantitative anti-adhesion assays using human bladder epithelial cells (T-24: ATCC HTB-4) and either radiolabeled  or fluorreatment ,215. Thereatment  and CJS reatment ,29. The reatment  were furE. coli and was able to inhibit the adhesion of P-fimbriated E. coli in a concentration-dependent manner. Low concentrations (0.0001–0.1 mg/mL) of the A6 fraction were observed to block the binding of E. coli O157:H7 to colonic epithelial cells while higher concentrations were not able to inhibit adhesion. CHEP had the highest anti-adhesive effect on the adhesion of P-fimbriated E. coli to bladder epithelial cells but this effect was only slightly greater than that of the A6 fraction. As the A6 fraction contained all neutral oligosaccharide components present in the CHEP material, further interpretations could not be made regarding the relative contributions of different oligosaccharide components to the observed anti-adhesion effects.Both CHEP and the A6 xyloglucan fraction were found to inhibit bacterial adhesion to different relative levels . The A6 1H-NMR profiles with previously obtained fractions  made it possible to determine that SNA-02 also contained arabinoxyloglucan oligosaccharides [2 and 3 , however, and the true effects of each material on biofilm formation may be concentration dependent, as was subsequently observed for the enriched oligosaccharide subfraction Cranf1b-F2. Within a specific concentration range (0.156–1.25 mg/mL) Cranf1b-F2 promoted the formation of biofilm by the non-pathogenic E. coli strain MG1655, while inhibiting biofilm formation by the pathogenic E. coli CFT073 strain. This effect was reversed for the two strains at higher concentrations (2.5–10 mg/mL), with Cranf1b-F2 inhibiting the formation of biofilm by MG1655 to increasing degrees while minimally affecting CFT073 biofilm formation [The contrasting biofilm formation profiles observed by Sun et al.  for the ormation . In vivoFusobacterium nucleatum (Fn) was mixed with a test sample and incubated prior to the addition of either Streptococcus sanguinis SS34 (Ss) or Porphyromonas gingivalis ATCC 33,277 (Pg). Bacterial co-aggregates were visually scored and the lowest sample concentration at which no co-aggregation occurred was reported as the MIC. A similar pattern of results for each fraction was observed for both pair-wise bacterial combinations (Fn+Ss and Fn+Pg) and indicated that the polyphenol constituents of the NDM material were the components primarily responsible for preventing bacterial co-aggregation. The authors [Neto et al.  assessed authors  also obs authors  did not Streptococcus mutans (UA159), Staphylococcus aureus (MRSA), and Staphylococcus epidermidis (RP62A) in monoculture. For all three bacteria, NDMAc significantly inhibited biofilm formation at 0.08 mg/mL while NDM had significant effects at 0.32 mg/mL. In additional results for the inhibition of Streptococcus mutans biofilm formation, MIC values were reported as 0.5 mg/mL for NDM, >0.5 mg/mL for NDMEt, 0.25 mg/mL for NDMAc, and 0.125 mg/mL for NDMAc-MCI-3. The NDMAc-MCI-3 subfraction (30% w/w of NDMAc) contained both polyphenol and xyloglucan oligosaccharide components similar to those present in NDMEt, indicating that both chemical classes may contribute to the effects of cranberry materials on biofilm formation [Neto et al.  also exaormation . The autormation . These dCranberry oligosaccharide and xyloglucan fractions and purified cranberry xyloglucan compounds have shown a distinct lack of antimicrobial properties or cytotoxic effects on numerous bacterial and human cell lines. Cranberry xyloglucan fractions have been screened by antimicrobial activity assays ,28,132 aE. coli CFT073 under different culture conditions. Low concentrations of cranberry pectic oligosaccharide fractions Cranf1b-cPOS, HDP-cPOS, uG3m2, and uG4m3  and the cidal β-lactam antibiotic ampicillin, formed a significantly reduced quantity of persister cells. This result indicated that the presence of cranberry pectic oligosaccharides was able to induce the active growth and subsequent death of bacteria in the presence of the antibiotic. The molecular mechanisms for these apparent growth promoting effects of cranberry pectic oligosaccharides on E. coli are currently unknown but may lead to significant in vivo effects for E. coli survival in the presence of antibacterial drugs or other potentially harmful compounds. Growth promoting effects such as these may also apply for other bacteria with dormant phenotypes and may contribute to the overall effects of cranberry materials on biofilm formation and gut microbiota profiles.In contrast to growth inhibitory effects, cranberry oligosaccharides may have growth promoting effects independent of the use of these compounds by bacteria as growth substrates. Sun et al.  reporteduG4m3 8,  were abl E. coli . The use E. coli  also fouCranberry materials have been attributed with antidiabetic, anti-inflammatory, anti-oxidant, and anticancer effects ,17,235. 50 0.68 mg/mL) than that of the positive control, the commercial anti-oxidant, butylated hydroxytoluene (IC50 2.2 mg/mL). The methylglyoxal trapping capacity of Cranf1b-CL was also assessed to establish if the material was able to trap this reactive carbonyl species but the fraction was inactive, indicating that alternate mechanisms may be involved in the anti-glycation properties of the oligosaccharide fraction. The authors [The formation of advanced glycation end-products (AGEs) is a process known to be associated with the harmful effects of diabetes . The oli authors  proposedE. coli to assess how much active material became bound to bacterial cell surfaces. Assay results were based on the quenching of luminol-dependent chemiluminescence by compounds with anti-oxidant properties. They found that the NDM and NDMAc samples yielded results consistent with the total phenolic contents of these materials, supporting the role of phenolics for this type of anti-oxidant activity. In contrast, E. coli treated with NDMEt did not exhibit anti-oxidant activity in the assay, consistent with the low phenolic content reported for NDMEt. These results do not necessarily indicate that NDMEt did not bind to E. coli, but rather that it did not exhibit anti-oxidant properties detectable by the assay used. Cranberry oligosaccharide mixtures may instead exhibit anti-oxidant effects through alternate mechanisms as was observed by Sun et al. [Neto et al.  performen et al. . The oveV. oxycoccus) exhibited anti-inflammatory and immunomodulatory activity in a mouse model of acetic acid-induced inflammatory colitis. Multiple measurements, taken on live animals and after the animals were sacrificed, indicated that the cranberry pectin (at 100 mg/kg) had a protective effect similar to that of prednisolone (at 5 mg/kg). Colonic myeloperoxidase activity was decreased in animals that consumed the pectin, indicating an inhibition of neutrophil accumulation in the colonic mucosa. This reduction in neutrophil levels was correlated to protective effects such as reduced epithelial cell disruptions and erosions [The pectic polysaccharide obtained by Popov et al.  from smaerosions . Reducederosions  are remierosions , who reperosions  may alsoerosions .In the past decade, numerous technological advances have improved our knowledge and understanding of the role that the human microbiota plays in supporting health or causing disease ,243,244.While numerous studies have focused on profiling and investigating the gut microbiome, delayed recognition of the presence of the UT microbiota, with initial reports in 2012, has delayed our understanding of this microbiome ,259,260.Knowledge regarding the effects of cranberry materials and specific cranberry compounds on human microbiota is still minimal. For the few recent studies ,75,150 tIn the gut, cranberry oligosaccharides and higher MW dietary fibers are likely to be degraded and metabolized by pathways used by bacteria for the fermentation of similar compounds from other dietary sources ,269,270.A number of current references discuss the known effects of various dietary oligosaccharides and other complex carbohydrates on both gut microbiota and human health ,192,275.The unique abilities of different microbes to degrade complex oligosaccharides with specific structural features, such as those of xyloglucans, results in the selection for specific sub-populations of bacteria within a given ecological niche, leading to different types of prebiotic effects for different types of compounds ,281,282.Studies investigating the possible prebiotic effects of cranberry oligosaccharide fractions ,132 haveBifidobacterium longum ssp. longum, four strains of Bifidobacterium longum ssp. infantis, Lactobacillus plantarum, and L. johnsonii. Cranberry materials used included an enriched xyloglucan fraction (Cranf1b-XG) prepared by the authors based on previous studies [Ozcan et al.  investig studies  and the  studies .Bifidobacterium longum spp. longum UCD401 and Lactobacillus plantarum ATCC BAA-793 were able to use the Cranf1b-XG mixture as a sole carbon source, and both were observed to ferment xyloglucans more slowly than glucose. B. longum spp. longum UCD401 produced a relatively modest amount of biomass as compared to growth on glucose, while the biomass produced by L. plantarum was comparable to that produced on glucose. Analysis of spent fermentation medium indicated a preference of B. longum spp. longum UCD401 for lower DP (<7) oligosaccharides, as higher DP molecules were found to accumulate. Comparisons of SCFA concentrations relative to those expected for glucose utilization indicated that B. longum spp. longum UCD401 employed metabolic adjustments to its usual pathway to more efficiently harvest energy from cranberry xyloglucans: levels of lactate decreased, acetate increased, and formate increased. The SCFA metabolic end products of L. plantarum fermentation indicated inefficient substrate utilization, as lactate production was decreased and acetate and formate production were increased relative to levels expected for glucose utilization. The authors [Of the nine organisms tested , only Bi authors  proposedB. longum spp. longum UCD401 on the A6 mixed composition xyloglucan fraction indicated the upregulation of genes involved in the fermentation and degradation of arabinose and xylose, including an α-L-arabinofuranosidase gene, two arabinan endo-1,5-α-L-arabinosidase genes, and a β-xylosidase gene. Genes involved in xyloglucan transport were also upregulated, including those for a carbohydrate ABC transporter substrate-binding protein and carbohydrate ABC transporter membrane proteins, indicating the probable uptake of arabinose and lower DP xyloglucans.When the A2 and A6 fractions  were tesB. longum spp. infantis ATCC 15,697 was unable to grow on Cranf1b-XG as a sole carbon source, this strain was able to grow when conditioned supernatants from L. plantarum or L. johnsonii grown on glucose were added to cultures. In contrast, spent supernatants from B. longum spp. longum UCD401 were not able to induce the growth of B. longum spp. infantis ATCC 15,697 on Cranf1b-XG. The growth rate of L. plantarum was increased in the presence of Cranf1b-XG when combined with spent supernatants from B. longum spp. longum UCD401 grown on Cranf1b-XG, but the amount of biomass produced was decreased, indicating possible competition for xyloglucan substrates or the presence of inhibitory substances.Results also indicated that mixed bacterial communities may have an increased capacity to ferment cranberry oligosaccharides, though the mechanisms of such interactions are likely to be complex . AlthougBacteroides species and the Lachnospiraceae [Prevotella and Roseburia with associated increases in propionate production [Multiple possible cross-feeding and other interactions can occur between diverse bacteria in vivo, as additional gut microbes, including piraceae ,165,280,piraceae . β-Glucaoduction . ArabinoThe presence of different ratios of specific cranberry components in different products may lead to different types of effects on different starting microbiota profiles. The results of a study using an in vitro gut simulator model and two donor microbiota populations  indicateE. coli and other bacteria believed to be uropathogens are within the Proteobacteria and Enterobacteriaceae classifications, organisms within this group may also be significant as colonizers of a healthy UT [While the authors  discussealthy UT . The obsalthy UT  provide The results of in vivo studies may be influenced by the composition of the cranberry material used, the composition of the starting microbiota, and the overall health status of the individual . In vivoFor multiple in vivo studies, cranberry consumption has been associated with minimal effects on measured health parameters for relatively healthy individuals ,143,284.Initial host microbiota populations are known to influence individual responses to dietary changes ,248,249.Simplified, standardized diets for animal and human in vivo studies have been used in an effort to remove some of these variables in terms of biosynthetic potential, intentionally selecting for a dominant microbiome abundance profile in order to detect the effects of an added dietary component that is distinct from the composition of the standardized diet . This tyFor microbiome profiling studies of cranberry materials, the level of microbiome classification and the methods of analysis used may have a significant impact on our ability to interpret the results. For a mouse in vivo model with induced colitis , the addWhile it is tempting to compare and generalize the results of these studies and apply them to larger populations of individuals, this must be done cautiously. Together, these initial microbiome profiling studies ,75,150 iMultiple compounds found in cranberry are known to have a variety of effects on different microbes. For example, organic acids, polyphenols, flavonoids, and complex carbohydrates are all known to have different effects on microbial growth, adhesion, and biofilm formation ,298,299.E. coli detected in urine, using highly selective classical culture methods, is unlikely to sufficiently represent an individual patient’s UT health status [The discovery that the UT is not inherently sterile ,305 chanh status ,259. TheThis new recognition of the UT microbiota and its significance for UT health versus disease indicates that studies intending to investigate the ability of cranberry materials to prevent UTIs may actually have been unknowingly investigating the effects of cranberry consumption on UT microbiota populations. This perspective, when combined with diverse host factors and a limited understanding of cranberry material chemistry, readily explains the high degree of inter-individual variability in response to cranberry product administration ,208,213 The presence of dietary oligosaccharides in urine gains new significance from the perspective of the UT microbiota and inspires the proposal of many new hypotheses for how these compounds may affect UT health in vivo. As discussed in this review, in vitro support is already available for some of these hypotheses. Oligosaccharides may differentially affect the bacterial adhesion and biofilm formation of beneficial and harmful microbes, contributing to the ability of human urine to prevent the adhesion of potential uropathogens ,208. GenInvestigation of these and other proposed hypotheses will require recognition and consideration of the UT microbiota as a significant aspect of any future in vivo study of cranberry materials. Consideration must also be given to any effect that cranberry administration has on overall host health that could be reflected in urine composition. As cranberry product composition may directly affect both urine composition and overall health effects, our knowledge regarding the chemical composition of diverse cranberry products must be improved. True understanding of the effects of cranberry as a dietary intervention for promoting UT health and “preventing UTIs” may ultimately require approaches that incorporate concepts of both systems biology and personalized medicine ,287.The hidden occurrence of soluble, neutral oligosaccharides in a wide range of cranberry products casts doubt on the biological properties of mixed composition cranberry materials that have been previously attributed to polyphenols. The limited descriptions and origins given for various cranberry materials and the confusion that exists with regards to material equivalency see  has alsoFor example, one result of this type of bias is that cranberry complex carbohydrates are likely to be unrecognized but significant contributors to the immune-related and cardiometabolic health benefits associated with cranberry materials ,17,309. Many of the general properties of oligosaccharides and other complex carbohydrates have yet to be specifically investigated for cranberry compounds, but even these few examples show the wide range of possible ways in which cranberry complex carbohydrates may influence the reciprocal interactions between microbiota populations and immune responses in vivo. These interactions may subsequently lead to cardiometabolic health effects by a variety of mechanisms. Ultimately, the dynamics of these interactions are complex and poorly understood and multiple types of studies will be required to further elucidate possible mechanisms for the overall health benefits of cranberry materials.w/w) of previously described NDM [Oligosaccharides may also be unrecognized contributors to many of the oral health benefits associated with cranberry materials. Multiple researchers in the field of oral health have worked with cranberry NDM over nearly three decades from 1989–2017  and yet ibed NDM  is a strIt is not possible to review all studies that might be associated with the potential biological effects of cranberry oligosaccharides, as many have not recognized the possible contributions of these compounds to the observed results. Because of the unrecognized presence and significance of oligosaccharides, however, new questions must be considered with regards to all previous cranberry research on mixed composition cranberry materials. Consideration must be given to whether the observed biological effect or health benefit could be due to oligosaccharides or to some other unrecognized component rather than to just polyphenols or other previously identified constituents. The exact contributions of different complex carbohydrates to the biological properties previously attributed to various cranberry products will require focused experiments and detailed chemical examinations of the materials used. Investigators will also need to consider oligosaccharides and other complex carbohydrates as possible significant contributors to the biological activities reported for mixed composition cranberry materials in both past and future studies.E. coli adhesion to uroepithelial cells [The conventional clinical paradigm regarding the use of cranberry materials for UTI prevention is based on the presence of components in human urine as a result of cranberry consumption that prevent infection by preventing al cells ,23,313. al cells  in spiteal cells . Currental cells ,316, in w/w) of most materials described as “cranberry powder” or “cranberry extract powder,” including some of the capsule-based materials used in clinical studies. Highly enriched cranberry oligosaccharide fractions (no phenolics) have been shown to affect the adhesion and biofilm formation of both beneficial and pathogenic microbes. Oligosaccharide fractions have been observed to have prebiotic effects on microbial growth in vitro with individual organisms and using in vitro gut simulators containing human gut microbiota populations. Whole cranberry materials have been used in vivo in both mice and humans for studies of gut microbiome profile changes and results are consistent with the presence of prebiotic and biologically active dietary fibers. Multiple general health benefits attributed to cranberry products are consistent with those observed for other prebiotic oligosaccharides and diets containing high fiber content. Oligosaccharides structurally related to those found in cranberry materials have been detected in and isolated from the anti-adhesive fractions of porcine urine, indicating the urinary bioavailability of these compounds, consistent with the urinary bioavailability of other dietary oligosaccharides in humans. Both the entire oligosaccharide mixture and subfractions containing discrete groups of oligosaccharides have demonstrated in vitro abilities to inhibit E. coli adhesion to uroepithelial cells. Additional properties have been observed for oligosaccharide mixtures in vitro that suggest additional biological and chemical properties in vivo.As described in this review, we now know that oligosaccharides are present in many cranberry products, including juices, and may constitute a significant portion (~20% The recently recognized importance of the human UT microbiota changes the paradigm for studies investigating any aspect of UT health or disease, and has significant implications for all clinical trials investigating the ability of cranberry products to support UT health or prevent UTIs. Future clinical trials must consider the effects of multiple cranberry constituents on beneficial and harmful microbes, and on overall host health parameters that may influence these microbes. Endpoints must account for the presence and function of the UT microbiota, eliminating the use of outdated classical clinical culture methods  and consWhile cranberry oligosaccharide fractions may provide us with a significant new tool for focused investigations of cranberry bioactivity, the use of refined cranberry oligosaccharide constituents in vivo may, however, be both impractical and ineffective. Oligosaccharides are only one class of numerous cranberry constituents that may affect both host systems and the human microbiome, resulting in diverse and compounded effects on overall health. Product cost must also be considered as a significant factor for products that are to be regularly consumed by patients over long term administration periods . MateriaIn summation, the new paradigm for cranberry bioactivity is that oligosaccharides and other complex carbohydrates are significant components of cranberry materials that contribute to cranberry bioactivity through multiple mechanisms affecting both the host and resident gastrointestinal and urinary tract microbiota, thereby promoting beneficial host-microbe interactions and preventing disease symptoms.The process of scientific discovery is never simple, especially for complex biological systems with seemingly innumerable variables. A physician’s ability to diagnose, treat, and prevent a given disease relies directly on our current level of understanding of that disease state and the mechanisms of action for the treatments being used. As our understanding is constantly evolving, often by leaps and bounds as well as through steady progression, it can be difficult for a single physician or researcher to stay abreast of significant new developments in multiple specific research areas. Sometimes, cross-disciplinary developments emerge, or discoveries made in a seemingly unrelated field become significant. When this happens, it becomes even more difficult for researchers and physicians to stay abreast of current knowledge and consider all the factors that may influence their experimental designs or treatment recommendations. While focused, detailed efforts are necessary for steady progress, it is also important to occasionally view the broader picture to be sure that this steady progress is investigating relevant details. Our hope is that this review of cranberry soluble oligosaccharides and other complex carbohydrates will provide a significant new perspective that can help the field of cranberry research progress in a new direction.


Chromosomal translocations are prevalent among soft tissue tumors, including those of the vasculature such as pseudomyogenic hemangioendothelioma (PHE). PHE shows endothelial cell (EC) features and has a tumor-specific t SERPINE1-FOSB translocation, but is difficult to study as no primary tumor cell lines have yet been derived. Here, we engineer the PHE chromosomal translocation into human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 and differentiate these into ECs (hiPSC-ECs) to address this. Comparison of parental with PHE hiPSC-ECs shows (1) elevated expression of FOSB, (2) higher proliferation and more tube formation but lower endothelial barrier function, (3) invasive growth and abnormal vessel formation in mice after transplantation, and (4) specific transcriptome alterations reflecting PHE and indicating PI3K-Akt and MAPK signaling pathways as possible therapeutic targets. The modified hiPSC-ECs thus recapitulate functional features of PHE and demonstrate how these translocation models can be used to understand tumorigenic mechanisms and identify therapeutic targets.  SERPINE1-FOSB translocation in hiPSC to model the vascular tumor PHESERPINE1-FOSBCRISPR/Cas9-mediated gene targeting to engineer hiPSCSERPINE1-FOSB show increased FOSB expressionhiPSC-ECsSERPINE1-FOSBFunctional features of PHE recapitulated by hiPSC-ECs in vitro and in vivo. SERPINE1-FOSB translocated hiPSCs can thus be used to understand tumorigenic mechanisms and identify therapeutic targets.van IJzendoorn et al. introduce a t SERPINE1-FOSB chromosomal translocation into hiPSCs to model the vascular tumor pseudomyogenic hemangioendothelioma (PHE). hiPSC-endothelial cells carrying the translocation recapitulate functional features of PHE  Chromosomal translocations and their corresponding gene fusions are common in neoplasia and are important in the initiation of tumorigenesis.SERPINE1 to FOSB.,FOSB containing the start codon, resulting in a novel start codon in exon 2 of FOSB. The translocation therefore causes the loss of 48 amino acids at the start of the FOSB protein, which then falls consequently under the control of the SERPINE1 promoter.,,Pseudomyogenic hemangioendothelioma (PHE) is a rare soft tissue tumor characterized by a specific recurrent balanced translocation, t, which fuses + cells, and, more recently, human induced PSCs (hiPSCs).in vitro and differentiated into most cell types of the body,Further understanding of the underlying molecular mechanisms is required to rationally design systemic therapy for patients with inoperable disease. However, like many other soft tissue tumors with translocations, PHE is rare and no cell lines have yet been derived from the tumor, confounding understanding of tumorigenesis and the identification of potential therapeutic targets. A possible approach to model translocation-driven tumors is to engineer the complete chromosomal translocation in human pluripotent stem cells (PSCs) and examine the effects on appropriately differentiated derivatives.SERPINE1-FOSB translocation into hiPSCs and thus generated control and modified isogenic hiPSC pairs. We carried out functional analysis of hiPSC-ECs and whole-genome and transcriptome sequencing of isogenic pairs of hiPSC and hiPSC-EC with and without translocation. We showed that hiPSC-ECs with the SERPINE1-FOSB fusion were distinct from their isogenic controls and exhibited phenotypic and transcriptomic characteristics very similar to PHE. More important, in mice, mutant hiPSC-ECs became invasive and formed abnormal vessels. Our hiPSC model thus mimics PHE, but in more general terms, the approach can serve as a blueprint for using CRISPR/Cas9 in hiPSCs to explore the role of fusion genes in the development of specific rare cancer subtypes for which cell lines are lacking, providing deeper understanding of tumorigenesis resulting from gene fusions.We introduced the t SERPINE1 and intron 1 of FOSB, which leads to the same novel start codon as found in PHE tumors from patients (SERPINE1 intron 1 and FOSB intron 1. A repair template was provided for homologous directed recombination (HDR) containing two 1,000-bp homology arms for SERPINE1 and FOSB, separated by an excisable neomycin resistance cassette flanked by Flp-recombinase sequences (FRTs) (in situ hybridization (FISH) revealed that translocations occurred relatively frequently, with 20 of 100 screened cells harboring a split of the FOSB bracketing probes (chromosome 19) and a colocalization of the distal FOSB probe to the SERPINE1 (chromosome 7)   translocation in hiPSCs. We generated a fusion between intron 1 of patients A. Two dos (FRTs) A. A wilds (FRTs) A. Neomycosome 7) B. hiPSC  and G6) B. Sanger and G6) A and S2Aexpected A, while expected A. The nointron 1 B and S2Cintron 1 B. In addquencing B and S2C3 and G6 D, identiSERPINE1 E. NeitheSERPINE1 C.Figure SERPINE1-FOSB) compared to the parental control hiPSCs (hiPSCWT)  compared with the untargeted parental control hiPSCs , insertions or deletions, structural variants, or single-nucleotide variants (SNVs) were found in the coding genome of the targeted hiPSC clones D3 and G6 (hiPSChiPSCWT) D. Furthel hiPSCs D.To verify the pluripotency of the targeted and parental control hiPSC lines, a teratoma assay was performed in mice. Targeted and parental control hiPSCs retained the ability to form tissues derived from all three germ layers , showing that CRISPR-Cas9 targeting has not affected pluripotency E.+ cell selection, expanded, and cryopreserved for further characterization. hiPSC-ECsWT and hiPSC-ECsSERPINE1-FOSB differentiated from two targeted clones (D3 and G6) exhibited typical EC morphology ) and hiPSC-ECs from clone G6 (hiPSC-ECsSERPINE1-FOSB (G6)) compared to the isogenic hiPSC-ECs derived from the parental non-targeted hiPSC line  (pFDR ≤ 0.05) were upregulated and downregulated, respectively, in hiPSC-ECsSERPINE1-FOSB compared to hiPSC-ECsWT -receptor interaction, pathways in cancer, phosphatidylinositol 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), transforming growth factor β (TGF-β), and hypoxia-inducible factor 1 (HIF-1) signaling pathways, and glycolysis/gluconeogenesis  enrichment analysis revealed alterations in the following biological processes in hiPSC-ECsSERPINE1-FOSB: ECM organization, angiogenesis, cell-matrix adhesion, inflammatory response, cell junction organization, regulation of TGF-β receptor signaling pathway, endothelial cell migration and EC proliferation  was next constructed using DEGs upregulated in hiPSC-ECsSERPINE1-FOSB . Gene interaction networks related to cancer, cellular movement and growth, and TGF-β signaling pathway were used to demonstrate interactions between the identified dysregulated genes and FOSB in hiPSC-ECsSERPINE1-FOSB  (1.9-fold increase), followed by basal EC growth medium supplemented with both 1% PPS and vascular endothelial growth factor (VEGF) (1.58- versus 2.27-fold)  and meshes  in hiPSC-ECs SERPINE1-FOSB compared to isogenic control hiPSCs-ECsWT after 48 h (To investigate the effect of the 27-fold) A. No sigter 48 h B.Figure SERPINE1-FOSB fusion was next examined by real-time impedance spectroscopy with an integrated assay of electric wound healing, as demonstrated previously.SERPINE1-FOSB fusion resulted in a significant decrease in barrier function of hiPSC-ECs -1 (SERPINE1-FOSB compared to the hiPSC-ECsWT (SERPINE1-FOSB fusion.Barrier function of hiPSC-ECs with and without the s (ZO)-1 E, the ads (ZO)-1 C, countes (ZO)-1 E and S5CSC-ECsWT E and S5CSERPINE-FOSB translocation were injected in mice in a Matrigel Plug Assay that allows assessment of vasculogenesis, as described previously.WT and hiPSC-ECsSERPINE1-FOSB(D3) formed stable vessels in vivo composed of human ECs evident at 4 (WT and hiPSC-ECsSERPINE1-FOSB(D3) had similar abilities to form vessels in vivo (+ ECs were evident in the Matrigel plugs with hiPSC-ECsSERPINE1-FOSB(D3), but not the Matrigel Plugs with hiPSC-ECsWT (+ hiPSC-ECsSERPINE1-FOSB(D3) also invaded the surrounding mouse soft tissues (the striated muscle) at 16 weeks post-transplantation in 2 of the 3 mice whereas this was not observed in any of the mice with hiPSC-ECsWT transplants  vessels at 16 weeks were disorganized and disarrayed and often contained thrombi   (The hiPSC-ECsd cells) H and 5I.p = 0.1) J.in vitro models to study rare translocation-driven tumors, both to identify the functional consequences of the translocation and to identify potential therapeutic targets. We used CRISPR/Cas9 to induce a tumor-associated translocation in hiPSCs, specifically the SERPINE1-FOSB translocation to model the rare vascular tumor PHE. Two hiPSC clones among 73 screened contained the translocation. In one of the clones (D3), the translocation was introduced via HDR using the donor DNA template, while in the second clone (G6), the translocation occurred via NHEJ. As the breakpoints were in intronic regions of the two involved genes, in both clones the transcribed and spliced RNA resulted in the typical SERPINE1-FOSB chimeric RNA. Other groups have also shown that CRISPR/Cas9 can be used to introduce chromosomal translocations in other cells, notably hMSCs via both NHEJ and HDR using donor DNA templates and additional exposure to low doses of DNA-protein kinase C (PKC) inhibitor (NU7441) to block NHEJ.There is an urgent need for SERPINE1-FOSB translocation.Whole-genome sequencing of both parental and targeted hiPSC lines showed no deleterious structural variations, CNVs, or mutations at the predicted off-target sites for the gRNAs. Any phenotypic changes observed were thus most likely associated with the FOSB in human umbilical vein ECs (HUVECs) recapitulated some features of PHE pathology.SERPINE1 regulatory elements via SERPINE1-FOSB fusion using CRISPR/Cas9-induced translocation in hiPSCs, thereby recreating the fusion with endogenous regulatory elements of SERPINE1. We found that the SERPINE1-FOSB fusion results in the upregulation of FOSB expression specifically in ECs and not in undifferentiated hiPSCs, in line with known high SERPINE1 expression in vascular cells and its function as a direct transcriptional target of the activator protein 1 (AP-1) family of proteins that includes FOSB.,We recently showed that the overexpression of truncated in vivo in mice in which hiPSC-ECsSERPINE1-FOSB (D3) or hiPSC-ECsWT were co-injected with bone marrow stromal cells (BMSCs) supported our in vitro findings and showed most strikingly the infiltrative growth pattern reminiscent of human PHE.SERPINE1-FOSB (D3) were haphazardly arranged compared to hiPSC-ECsWT and contained higher numbers of fibrin thrombi in two of three hiPSC-ECsSERPINE1-FOSB (D3). These results are in line with the in vitro barrier function analysis and suggest that the endothelium is aberrant inducing thrombi formation.Vasculogenesis assays in vitro and in vivo in our model, and recapitulate some aspects of PHE. Moreover, these pathways that we identify here to be regulated by the SERPINE1-FOSB fusion provide rationale to develop targeted treatment strategies for inoperable multifocal PHE patients. In line with our previous report of a patient with a complete clinical remission following the multi-tyrosine kinase inhibitor telatinib, we confirm upregulated MAPK signaling and overexpression of platelet-derived growth factor receptor A (PDGFRA) and -B induced by the fusion in the current model. Moreover, we identify PI3K-Akt signaling which can be targeted using mammalian target of rapamycin (mTOR) inhibitors. Anecdotal responses to mTOR inhibition in patients with PHE have been reported.25Transcriptome analysis of hiPSC-ECs with the fusion revealed DEGs associated with several pathways that are known to be related to cancer, such as TGF-β signaling, adhesion, metabolism, inflammatory response, angiogenesis, and endothelial cell migration. All of these are linked to the phenotypes we observed In summary, we showed that hiPSCs and hiPSC-ECs can be used to model fusion-driven tumors using CRISPR/Cas9 and a donor DNA template to introduce the translocation. The differentiated hiPSC-ECs carrying the pathognomonic translocation gave insights into the tumorigenesis of PHE and elucidated the pathways regulated by the fusion product that may provide rationale to develop targeted treatment strategies for inoperable multifocal PHE. Overall, this approach facilitated the elucidation of the role of specific fusion genes in the development of specific rare cancer subtypes for which cell lines are presently lacking.SERPINE1-FOSB translocation were investigated. The reasons for this were (1) SERPINE1 is expressed in multiple tissues but predominantly in those that are highly vascularized.SERPINE1-FOSB fusion, this remains an option for future studies.One limitation of the present study is the fact that only hiPSC-ECs carrying the in vivo. The tumor cells typically do not form vessels in PHE but instead are spindle shaped and co-express endothelial markers (CD31 and ERG) and keratin AE1AE3, not all of which are recapitulated in our in vivo model. It may be that the 16-week time frame is not enough to develop these features in vivo. Invasion of hiPSC-ECsSERPINE1-FOSB (D3) into surrounding mouse soft tissue was observed at 16 weeks but not at the 4-week time point, which may suggest that development of the phenotype takes time.Another limitation is that some aspects of PHE were not recapitulated v.orlova@lumc.nl).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Valeria V. Orlova .The SeV reprogrammed hiPSC line LUMC0054iCTRL was used . Mice were maintained at the animal facility of Leiden University Medical Center (LUMC). Teratoma and Matrigel plug assays A were peWT and hiPSCSERPINE-FOSB (D3) as reported before.The teratoma assay was performed on the parental hiPSC,WT and three mice with hiPSCSERPINE-FOSB (D3)  A. Each mFor comparison, a representative case of pseudomyogenic hemangioendothelioma was retrieved from the consultation files of one of the authors (JVMGB). The case concerned a 17-year old male with a multifocal tumor presenting in the soft tissues of the right lower leg. Immunohistochemistry was performed during routine diagnostic workup. The tumor sample was anonimized according to the ethical guidelines described in “Code for Proper Secondary Use of Human Tissue in The Netherlands” of the Dutch Federation of Medical Scientific Societies.FOSB sgRNA TCCACTACACCGTGACGCAG and SERPINE1 sgRNA TGAACACTAGGGCAAGGTGC. The repair template was generated by blunt ligation of FOSB and SERPINE1 homology arms (around 1kb each) into a P15 backbone containing a Neomycin resistance cassette surrounded by two flippase recognition target (FRT) sequences . The CAGGs-Flpo-IRES-puro vector which expresses codon-optimized Flp recombinase was used for transient transfection to recombine FRT sitesA dual sgRNA and Cas9-expressing plasmid was generated by introducing a second gRNA scaffold in the SpCas9-2A-Puro V2.0  plasmid using Gibson ligation as described.hiPSCs were transfected at 60%–70% confluence the day after seeding in a 60 mm dish. Transfection was carried out using Lipofectamine® 2000 (Invitrogen). First, 20 μl Lipofectamine® 2000 was diluted in 300 μl Opti-MEM® Medium and incubated at RT for 5 min. In parallel, 8 μg of both the repair template and double guide RNA/Cas9 was diluted in 300 μl Opti-MEM® Medium. Diluted plasmid DNA was added to diluted Lipofectamine® 2000 in a 1:1 ratio and incubated another 5 min at RT before the DNA-lipid complex was added to the cells in a drop-wise manner. Cells were allowed to grow in the incubator for ∼18 hours before the medium was changed. Antibiotic selection with 50 μg/ml G-418 was performed 24 hours post transfection and was continued for 7 days to select for targeted cells. Once recovered, cells were passaged into 6-well plates and transfected the next day with 4 μg Flp recombinase expression vector to excise the neomycin cassette . At 24h post transfection the medium was supplemented with 0.5 μg/ml Puromycin for 48h to enrich for transfected cells. At 80% confluence, the cells were passaged for clonal expansion on 96-well plates using limited dilution.In Situ Hybridization (FISH) was performed using BAC clones (BACPAC Resource Center). Proximal to SERPINE1 BAC clone RP11-395B7 was selected. Proximal and distal to FOSB respectively BAC clone RP11-84C16 and RP11-902P17 were selected. BAC DNA was extracted using the High Pure plasmid isolation kit (Roche). The RP11-395B7, RP11-84C16 and RP11-902P17 were respectively labeled with Cy5-dUTP, Fluorescein-12-dCTP and Cy3-dUTP using a nick translation labeling reaction. FISH was performed as previously described by our group.Three-color Fluorescence SERPINE1 (primers SF and FR), the 3′ homology arm of FOSB (primer F2 and R2), the wild-type SERPINE1 (primer SF and SR) and wild-type FOSB (primer FF and FR) in clonal lines  was performed on metaphase cells as previously described in detail.COmbined Binary Ratio Fluorescence hiPSCs were differentiated to hiPSC-ECs and characterized as previously described.17RNA isolation was performed with the Direct-zol RNA isolation kit (Zymo-research) according to the manufacturer’s protocol. cDNA was synthesized using M-MLV with oligo dT primers (Promega) according to the manufacturer’s protocol. Real-Time qPCR was performed with Sybr Green (Bio-Rad) on a CFX384 thermocycler (Bio-Rad). All real time PCR experiments were performed in triplicate. Primers are listed in Western blotting was performed as previously describedTo quantify proliferation, cells were cultured in a 96-well plate for 24 hours. Presto Blue (ThermoFisher) was subsequently added to the medium and cells were incubated at 37°C for 30 minutes before determining the Relative Fluorescence Units (RFU) using a plate reader (Perkin Elmer).https://github.com/davidvi) for analysis of tube formation.Tube formation assays were performed in 96-well plates coated with 50 μl Matrigel (Corning). hiPSC-ECs were seeded at a density of 15,000 cells per well in 150 μl EC-SFM supplemented with 1% BSA and 50 ng/μl VEGF. Tube formation was analyzed with ImageJ . Tube formation was imaged with the EVOS Cell Imaging System (ThermoFisher). To quantify tube formation a custom plugin in ImageJ  was used. Analysis scripts are available on GitHub . Data was visualized with the circlize R package (v0.4.3).DNA was isolated for whole genome sequencing using the Wizard Genomic DNA Purification Kit (Promega) and sequenced on the BGISEQ-500 platform (BGI). Reads were aligned to the GRCh38/hg19 reference genome using the Burrow-Wheeler Aligner (v0.7.12) and further processed according to the GATK (Broad institute) best practice pipeline. Copy Number Analysis (CNA) was performed using VarScan (v2.2.4) and analyzed using DNAcopy R package (v3.6). Off-target sites for the used gRNAs were determined using an online tool (https://github.com/biopet/biopet), which comprises FASTQ preprocessing, alignment and read quantification. Sickle (v1.2) was used to trim low-quality read ends15. Cutadapt (v1.1) was used for adaptor clipping16, reads were aligned to the human reference genome GRCh38 using GSNAP (gmap-2014-12-23),FDR ≤ 0.05 was considered significant. Normalized RPKM values were log2 transformed and standardized across each gene using z-scores and heatmap was produced with the R package ggplot2 (v2.2.1). KEGG pathway enrichment analysis was carried out using Enrichr,RNA for transcriptome sequencing was isolated using Direct-zol RNA miniprep kit (Zymo Research). After library preparation, sequencing was performed on the BGISEQ-500 platform (BGI). Raw data was processed using the LUMC BIOPET Gentrap pipeline . One-way ANOVA with Tukey’s multiple comparison for the analysis of three or more groups or Mann-Whitney test for analysis of two groups were used. The data are reported as mean ± SD.


In March 2020, the World Health Organization elevated the coronavirus disease (COVID-19) epidemic to a pandemic and called for urgent and aggressive action worldwide. Public health experts have communicated clear and emphatic strategies to prevent the spread of COVID-19. Hygiene rules and social distancing practices have been implemented by entire populations, including ‘stay-at-home’ orders in many countries. The long-term health and economic consequences of the COVID-19 pandemic are not yet known.During this time of crisis, some chiropractors made claims on social media that chiropractic treatment can prevent or impact COVID-19. The rationale for these claims is that spinal manipulation can impact the nervous system and thus improve immunity. These beliefs often stem from nineteenth-century chiropractic concepts. We are aware of no clinically relevant scientific evidence to support such statements.We explored the internet and social media to collect examples of misinformation from Europe, North America, Australia and New Zealand regarding the impact of chiropractic treatment on immune function. We discuss the potential harm resulting from these claims and explore the role of chiropractors, teaching institutions, accrediting agencies, and legislative bodies.Members of the chiropractic profession share a collective responsibility to act in the best interests of patients and public health. We hope that all chiropractic stakeholders will view the COVID-19 pandemic as a call to action to eliminate the unethical and potentially dangerous claims made by chiropractors who practise outside the boundaries of scientific evidence. To. To32]. One chiropractor boldly stated that “experience” shows that chiropractic treatment has a positive effect on the immune system. This was supported by patient testimonials. Testimonials are questioned as a marketing tool in many areas of medicine , 33, 34,Other claims (4%) questioned undisputed and established science, such as the germ theory, and did not use “evidence” to back their claims. An example is shown in Fig. A few (12%) of the statements were not accompanied by any argument at all as seen in Fig. One statement concerned the risk to the unborn child, resulting from prenatal stress caused by COVID-19. A chiropractor claimed that pregnant women should get chiropractic care in order to minimize consequences on the baby , Australia (both), New Zealand (one), the US , and Canada , searching for information on the pandemic or the immune system in general.Many national chiropractic associations clearly supported the WFC statement : The AmeA search of the web-pages for the four CCEs  and the International umbrella organization (I-CCE), did not reveal the use of inappropriate information relating to chiropractic treatment and viral disorders.Some “traditional” chiropractors are making claims that could harm the public. In our opinion, the concept of adjustment- immunity boosting lacks biological plausibility when viewed through the lens of modern scientific knowledge.There are of course situations where the scientific evidence is lacking and the biological plausibility is disputed, where high-quality randomized clinical trials of spinal manipulation may prove relevant. However, infectious disease is not one of them; the risks to the public are too high. Presently there is no generally accepted and clinically relevant evidence that adjustments protect against, nor impact, viral disease. Nevertheless, the “traditional” chiropractic approach, which forms the basis for the treatment to improve the immune system, survives among some chiropractors, despite scientific advancements over the last century. Some studies suggest that there has been a reluctance to denounce the “traditional” faction of the profession because the majority, middle-of-the-road chiropractors and some of their organisations purposely accommodate all perspectives under the title “chiropractic” and avoid internal conflict , 40.The authors of this article, in line with modern-day concepts, assert that contemporary healthcare cannot be based on ideological beliefs and dogmatic contentions. When “traditional” chiropractors note the lack of evidence, we have observed that they often respond with a science type cliché: “absence of evidence is not evidence of absence”. This cannot be used as an excuse to discard biological principles, common sense, and critical appraisal. If claims are extraordinary and contrary to other generally accepted existing knowledge, the evidence provided must be especially convincing , 42.Correcting misinformation is particularly important, because misinformation reaches more people and spreads more rapidly than accurate news . This isIn all professional groups there will be outliers, people who do not fit in with the mainstream. This is probably unavoidable. However, how likely is it that a substantial group of medical practitioners still believe in the practise of bloodletting and still reject the germ theory? In our opinion, we as a profession must take stronger measures to combat the dangerous anti-science sentiment that remains as a small but prominent part of the chiropractic community.Chiropractors should reflect on their personal beliefs and what it means to be a health care professional, entrusted with ensuring public benefit from their services. Those who make unfounded claims breach a fiduciary agreement that allows for professional self-regulation and permits professionals a great deal of autonomy, provided they adhere to the duty of care, the duty of loyalty, and the duty of disclosure. For health care professionals, this means that they must put patient interests ahead of their own, stay up to date with current best practises, and fully inform patients of treatment options, including risks/benefits of any given procedure. Chiropractors who put their personal beliefs ahead of evidence or who selectively choose evidence that supports their belief system fail to reach these standards and breach their professional duties, are legally negligent, and do not act in the best interests of their patients.We would argue that the chiropractic teaching institutions are obligated to set the example for best practice by taking public stances on issues such as the present COVID-19 crisis. Teaching institutions, with their staff of highly educated and clinically experienced experts, are sought out for guidance, particularly at times of controversy and crisis , 48. TheFour CCEs are tasked with accrediting chiropractic programs within their respective geographical region. However, the standards and processes of these CCEs are not internationally homogeneous and concerns have been raised about some accredited chiropractic programs and the subsequent practice patterns demonstrated by their graduates , 30–33. The COVID-19 crisis highlights the need for all CCEs to be agile in times of crisis and adopt the standards of mainstream healthcare and place patient welfare at the forefront of their agenda.The groups entrusted with ensuring public safety must take steps to prevent pseudoscientific claims by chiropractors. A recent meta-analytic study suggested the following procedure for correcting misinformation . First, In this search of public media in Europe, North America, New Zealand, and Australia, we discovered many cases of misinformation. Claims of chiropractic treatment improving immunity conflict with the advice from authorities and the scientific consensus. The science referenced by these claims is missing, flawed or has no clinical relevance. Consequently, their claims about clinical effectiveness are spurious at best and misleading at worst.However, our examples cannot be used to make statements about the magnitude of the problem among practitioners as our samples were not intended to be representative. For that reason, we also did not include an analysis of the arguments provided in the various postings. In view of the seriousness of the topic, it would be relevant to conduct a systematic study on a representative sample of public statements, to better understand these issues.Our search illustrates the possible danger to public health of misinformation posted on social media and the internet. This situation provides an opportunity for growth and maturation for the chiropractic profession. We hope that individual chiropractors will reflect on and improve their communication and practices. Further, we hope that the chiropractic teaching institutions, regulators, and professional organisations will always demonstrate responsible leadership in their respective domains by acting to ensure that all chiropractors understand and uphold their fiduciary duties.


Lifestyle-related diseases, such as stroke, heart disease, and diabetes, are examples of noncommunicable diseases. Noncommunicable diseases are now the leading cause of death in the world, and their major causes are lifestyle related. The number of eHealth interventions is increasing, which is expected to improve individuals’ health literacy on lifestyle-related diseases.This literature review aims to identify existing literature published in the past decade on eHealth interventions aimed at improving health literacy on lifestyle-related diseases among the general population using selected visual methods, such as educational videos, films, and movies.A systematic literature search of the PubMed database was conducted in April 2019 for papers written in English and published from April 2, 2009, through April 2, 2019. A total of 538 papers were identified and screened in accordance with the PRISMA  flow diagram. Finally, 23 papers were included in this review.The 23 papers were characterized according to study characteristics ; the playback time of the educational videos, films, and movies; and the evaluation of the study’s impacts on health literacy. A total of 7 studies compared results using statistical methods. Of these, 5 studies reported significant positive effects of the intervention on health literacy and health-related measures . Although most of the studies included educational content aimed at improving health literacy, only 7 studies measured health literacy. In addition, only 5 studies assessed literacy using health literacy measurement tools.This review found that the provision of educational content was satisfactory in most eHealth studies using selected visual methods, such as videos, films, and movies. These findings suggest that eHealth interventions influence people’s health behaviors and that the need for this intervention is expected to increase. Despite the need to develop eHealth interventions, standardized measurement tools to evaluate health literacy are lacking. Further research is required to clarify acceptable health literacy measurements. Lifestyle-related diseases, such as stroke, heart disease, cancer, diabetes, and chronic respiratory disease, are leading examples of noncommunicable diseases and are now the leading causes of death in the world . Of the Health literacy is defined as “people’s knowledge, motivation and competences to access, understand, appraise, and apply health information in order to make judgments and take decisions in everyday life concerning healthcare, disease prevention and health promotion to maintain or improve quality of life during the life course” . Low heaThe popularity of mobile technologies remains high and the number of users of mobile technologies is increasing . The groEducational content to improve individuals’ health literacy on lifestyle-related diseases can be offered in many ways, such as group learning, questionnaires, internet-based information searches, and downloadable apps -22. AddiThe aims of this review are to identify literature published in the past decade on eHealth interventions that aimed to improve health literacy on lifestyle-related diseases among the general population using selected visual methods, such as videos, films, and movies. Specifically, our review seeks to categorize study characteristics and the evaluation of the studies’ impacts on health literacy. Four main themes are discussed in this review: target age, measurement of health literacy, dietary health behavior, and the future directions of eHealth interventions.This literature review was performed using a systematic search and was conducted to emphasize the integration of studies across broader topics , with reDetails of the search strategy, study selection, and extraction of information can be found in A data-charting form was created to categorize the included studies, the study characteristics ; the playback time of the educational videos, films, and movies; and the evaluation of the study’s impacts on health literacy .The included studies are characterized in In addition, 3 noninterventional studies ,45,46 weThe objectives of the included studies were categorized into 3 groups: (1) health promotion, (2) disease prevention, and (3) disease management related to the target diseases.One study  was condA total of 15 studies ,41-44,48Outcomes were investigated in 18 studies ,38-44,481c [Studies used a number of measured values: body weight ,41,44,481c ,38,42, s1c , and kno1c ,40,42,431c ,34, nutr1c ,35,38,401c ,35,38,411c , medicat1c ,38, spec1c , and usa1c ,36.To assess changes in measured values, individuals’ health literacy was tested in 7 studies ,42,43,48We categorized the included studies into 3 types of platforms: (1) applications (web-based applications or mobile apps), (2) websites, and (3) others.Various apps were used in 22 studies -37,39-48By connecting with a wearable device , sensingWebsites were based on a textual design to provide educational messages but, unlike apps, were unable to include a gaming component to provide some type of feedback-based reward .Another platform  developeIn this review, we aimed to identify and characterize the features of existing literature describing eHealth interventions that aimed to improve health literacy on lifestyle-related diseases among the general population using selected visual methods, such as videos, films, and movies. Through this research, we identified 4 themes, which we discuss here with their strengths and limitations.We found a difference in target age compared with previous studies focused on health literacy. Kim and Xie  found thIn our review, we identified various ideas to facilitate the use of educational content to improve participants’ health literacy and found that the kind of educational content provided was required to change as the age range of the target population widened. The use of icons and avatars facilitated usage for both younger and older individuals with low health literacy ,48. The Measurement tools for health literacy have yet to be established. Indeed, in this review, among the 22 included studies that developed apps for education -37,39-48According to a review  that ideImprovements to lifestyle play an important role in lifestyle-related diseases ,5. AlthoBased on user evaluations of the interventions and opinions on their future development, eHealth interventions using visual methods and interactive approaches improve user motivation to improve lifestyle and health literacy. However, 1 study  reportedTo our knowledge, this study is the first to review the educational content of eHealth aimed at improving individuals’ health literacy on lifestyle-related diseases. Our findings may provide a new perspective on the development of apps that use eHealth to address lifestyle-related diseases and improve people’s health literacy.Several limitations of our study warrant mention. One limitation is the search process: we searched only a single database and included only studies written in English and published in the decade up to April 2019. As discussed in other review papers about eHealth-based interventions ,14, seleOur review provides an overview of the relationships between eHealth-based interventions with selected visual methods, such as videos, films, and movies, and improved health outcomes . Despite the necessity of self-management systems using ICT to control lifestyle-related diseases, relatively few studies have explored educational videos, films, and movies aimed at improving health outcomes.We also found that the concept of literacy and the tools used to measure the outcome of health literacy–related interventions have not been unified. To more accurately evaluate levels of health literacy and the effects of interventions, future studies need to clarify the concept of health literacy and develop health literacy screening tools.


The most nutrient-dense, whole-food sources of these micronutrients include liver, small fish, eggs, ruminant meat, and dark leafy greens. Investment is needed in some countries to collect data on micronutrient biomarkers and dietary intakes. A food systems approach is essential for improving child diets and reducing malnutrition, which affects millions of children, their futures, and society at large across South Asia and beyond.Micronutrient malnutrition is a key driver of morbidity and mortality for millions of children in South Asia. Understanding the specific micronutrients lacking in the diet during the complementary feeding period is essential for addressing undernutrition caused by inadequate diets. A Comprehensive Nutrient Gap Assessment was used to synthesize diverse evidence and estimate the public health significance of complementary-feeding micronutrient gaps and identify evidence gaps in 8 countries in South Asia. There were important gaps across the region in iron, zinc, vitamin A, folate, vitamin B In step 2, data points in the country-specific spreadsheets were reviewed and assigned an implied nutrient gap burden score per the CONGA methodology.In step 4, the implied nutrient gap burden scores and evidence weight scores were used to calculate a quantitatively derived nutrient gap burden rating. The quantitative burden rating was calculated using data only from the aforementioned 5 core evidence types (excluding “other” data) that were collected in 2010 or later and that were for age or sex groups similar to children 6–23 months of age. The data points excluded from this calculation were considered in step 5. A numeric score was calculated for each nutrient in each country, using the weighted mean of the implied nutrient gap burden score (where the evidence weights are the weight scores) and assigned a label of high, moderate, low, or negligible, per CONGA methodology.In step 5, the quantitative nutrient gap burden scores were reviewed alongside the totality of evidence for each nutrient, including data points that did not meet criteria for inclusion in the quantitative burden score calculation and additional available information for each data point  to determine whether the final rating assigned to the nutrient gap should deviate from the quantitatively derived rating. A final qualitative rating of high, moderate, low, or negligible was assigned to each nutrient for each country, and any deviation from the quantitative burden score was documented and explained.The certainty of evidence was rated for each nutrient burden  in step 6 using established CONGA methodology criteria,In step 7, all other coauthors, who are subject matter and contextual knowledge experts, reviewed the final qualitative nutrient gap burden and evidence certainty ratings produced in steps 5 and 6, respectively. Disagreements with final qualitative ratings were discussed and critically re-evaluated. Ratings were finalized only when consensus was achieved and documentation of additional considerations or deviations from quantitative burden scores was added. Table S2 in the Supporting Information online contains a spreadsheet with all data points included in each CONGA, data sources, and nutrient gap and certainty scores.and a certainty-of-evidence rating of at least moderate) and potential micronutrient gaps , the most nutritious, regionally available, whole-food sources of these micronutrients were identified on the basis of food-composition data from Bangladesh,For identified micronutrient gaps . The portion size of each food required to achieve an average of 33.3% of requirements —the equivalent of 100% of requirements for 2 micronutrients or 33.3% of requirements for all 6 micronutrients—was also calculated to demonstrate the ideal foods to fill ≥ 2 important micronutrient gaps simultaneously. Adjustments for differences in bioavailability between plant and animal-source foods were made for iron and zinc. For more details on how micronutrient density of and average share of requirements for identified foods were calculated and how local availability was determined, refer to other articles in this journal supplement.,Because many whole foods are good sources of several micronutrients, these foods were also assessed for how well they met the needs of 6 micronutrients critical for child growth and development and likely to be lacking in South Asian diets: namely, iron, vitamin A, zinc, folate, vitamin Bn = 63) and India (n = 59) had the highest number of data points identified in the literature review, followed by Bangladesh (n = 41), Sri Lanka (n = 40), Nepal (n = 38), Afghanistan (n = 31), the Maldives (n = 30), and Bhutan (n = 19). In several countries, fewer than half of these data points met qualifying criteria for inclusion in the quantitative micronutrient gap burden score .A total of 321 qualifying data points from 50 evidence sources (see Supporting Information online) were identified in the literature review (CONGA step 1), and 137 of these met criteria for inclusion in the quantitative burden score (CONGA steps 3 and 4) . PakistaThe variety of evidence sources differed by country. Pakistan, for example, had a 2018 national nutrition survey, 2017–2018 DHS, a 2018 Fill the Nutrient Gap report, a 2017 Cost of the Diet analysis, and 2018 Optifood analysis, among others. In contrast, Bhutan had a 2015 national nutrition survey with limited relevant data to extract and few other nutrition-specific evidence sources. Recency of data collection for identified evidence varied across countries; however, all countries had at least 1 evidence type collected within the previous 6–7 years. All countries also had relevant nationally representative data available for several micronutrients and/or evidence types. Relevance to the age group of interest varied by evidence type and data source; however, the majority of data reviewed were for children younger than 5 years or 6–59 months old.12 deficiency was available for children aged 6–59 months or 12–59 months in Pakistan and India, and for other age groups in Bangladesh. All countries except Bhutan had at least 1 biochemical or functional marker of iodine deficiency ; however, most estimates were for school-aged children. Sri Lanka was the only country with an estimate of calcium deficiency in children (6–59 months old), and Pakistan assessed calcium deficiency for women of reproductive age. No qualifying biochemical evidence for any age group was identified in the literature search for niacin, thiamine, vitamin C, or vitamin B6—similar to a CONGA in 6 countries in Eastern and Southern Africa.The number of data points varied considerably across micronutrients and evidence types . Biochemother and considered in the qualitative assessment (CONGA step 5). Data on micronutrient adequacy of household diets were identified in Bangladesh only. Data on micronutrient adequacy of national food supplies were identified for all micronutrients considered except iodine, and for all countries except Bhutan. Evidence containing micronutrient-informative food-group intake of individuals or households was available for iron and vitamin A (from consumption of iron- and vitamin A–rich foods in children aged 6–23 months) and iodine . At least 1 data point classified as other evidence  was identified for all micronutrients and all countries.In contrast to the CONGA in Eastern and Southern Africa,Adjustments to quantitative burden ratings involved a critical qualitative assessment of the totality of evidence, including evidence excluded from the quantitative burden rating, “other” evidence, and temporal trends. Table S2 in the Supporting Information online includes all initial and final qualitative micronutrient gap burden and certainty-of-evidence ratings. Although most initial ratings remained the same, 15 of 88 micronutrient gap burden ratings and 6 of 88 certainty ratings were adjusted. Among ratings that changed, burden ratings tended to reduce in severity, whereas certainty ratings tended to increase in certainty, but there were some exceptions.n = 4), all of which reduced in severity , because initial ratings were largely based on high estimates of inadequacy in the food supply, which did not fully account for household production of dairy.12 and 2 vitamin C burden ratings reduced in severity from high to moderate and 2 niacin burden ratings increased in severity from negligible to low. One iron burden rating and 1 vitamin B6 burden rating increased in severity from negligible to low after evidence was considered that was excluded from the quantitative burden estimates.Calcium had the most changes to initial burden ratings . Minimally processed plant and animal-source foods, including breast milk, contain important beneficial bioactive compounds  that can help regulate the immune system, build a healthy gut microbiome, and ultimately may help reduce risk of overweight or obesity and diet-related noncommunicable diseases in middle childhood, adolescence, and adulthood.12, folate, and calcium), as measured by average share of requirements per 100-g portion, are chicken liver, ruminant liver, small fish, eggs, ruminant meat, and dark leafy greens. Small fish are also good sources of vitamin D and long-chain omega-3 fats, which are important for child development and have other health benefits.According to Food and Agriculture Organization Food Balance Sheets, South Asia has the lowest per capita availability of meat (19 g/day) of any region globally, and India the lowest of any country (10 g/day).12, calcium, and vitamin C. Previous literature on regional patterns of micronutrient gaps in South Asia is limited. In accordance with our findings in the present study, a review of iron, iodine, vitamin A, and zinc deficiencies in South Asia found that there was a low burden of iodine deficiency  but a high burden of iron, vitamin A, and zinc deficiencies.Unsurprisingly, there was heterogeneity in micronutrient gap burdens and certainty ratings across countries and micronutrients in South Asia. Although micronutrient gaps varied across countries, most countries had identified or potential gaps in multiple micronutrients. Diets were apparently inadequate in micronutrients, especially iron, zinc, vitamin A, folate, vitamin B12, and especially calcium deficiencies, and no evidence for vitamin C, niacin, thiamine, or vitamin B6 deficiencies. Among these, investment is warranted in collecting biomarkers on folate, vitamin B12, calcium, and vitamin C status where recent evidence does not exist, given that low-certainty evidence suggests there are potential gaps in these micronutrients in several South Asian countries. Although vitamin D gaps were not assessed, deficiency is common in at least parts of South Asia,,,Evidence on micronutrient gaps was most robust in India, Pakistan, and Nepal, because of their recent national micronutrient surveys. In accordance with a CONGA on Eastern and Southern Africa,Assessing micronutrient gaps in young-child diets across South Asia using the CONGA method allowed for a critical assessment of the relevant evidence from different types of data sources that are not usually synthesized . This comprehensive assessment addresses this knowledge gap and provides a more complete understanding of micronutrient gaps in each country than would have been possible if relying on only 1 type of data or a subset of the evidence base. Moreover, evidence sources, even robust sources, often have conflicting findings. Therefore, the use of multiple sources reduced the likelihood of erroneous conclusions. Furthermore, assessing the certainty of the micronutrient gap ratings provided transparency about the evidence behind them, which will help limit inappropriate use of evidence or overconfidence in findings that are not robust, representative, validated, or recent. Finally, because CONGA is a standardized process,This assessment has important limitations. A systematic search was not conducted to identify evidence sources for inclusion in the CONGA. Although it is unlikely that robust, large-scale evidence sources were not identified, it is likely that some qualifying evidence was not. Thus, it is unclear how many additional resources would have been included in a systematic review and the extent to which their inclusion would have influenced ratings. Also, although the CONGA method was designed to be standardized and minimize bias, it has built-in flexibility to allow reviewers to override automated ratings on the basis of their qualitative assessment of the total evidence base, including data points excluded from the automated rating process. As a result, the final ratings, to some extent, are a reflection of the subjective perspective of experts who reviewed the evidence. The impact of this bias was minimized by having 2 experts make the initial ratings and revisions jointly and multiple subject-matter and local experts review and validate the final ratings. Notably, although the CONGA method can be applied subnationally, we did not assess in this analysis subnational differences in nutrient gaps (which can vary considerably within countries) or foods to fill these gaps. In addition, there may be other nutrient gaps that we did not identify in this analysis because there was limited robust evidence for several micronutrients. Finally, differences in ratings across countries may be due, in part, to differences in the date of data collection across countries. Additional limitations of the CONGA method are specified elsewhere.The findings from this CONGA are essential for addressing micronutrient malnutrition and inadequate diets among young children in South Asia. They provide a clear understanding of the specific issues and limitations in data on micronutrient gaps and evidence across the region. The numerous causes of these important micronutrient gaps should be further studied and addressed through a food-systems approach that identifies and removes the largest barriers to adequate intakes, including availability, accessibility, affordability, cultural acceptability, desirability, convenience, knowledge, and feeding practices.Policy and programmatic interventions to address micronutrient gaps should be guided by a systematic analysis of context-specific determinants and by drivers influencing access to and consumption of nutritious and safe whole-food sources, identified in this study, that can fill critical micronutrient gaps during the complementary feeding period. Finally, a multisystem response involving the health, food, water and sanitation, and social protection systems is warranted in countries in the region to collectively create the necessary conditions for micronutrient-adequate diets and adequate feeding practices.nuaa144_Supplementary_DataClick here for additional data file.